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Distribution and localization of a G protein-coupled inwardly rectifying K+ channel in the rat
Abstract
The cellular distribution of the mRNA of the inwardly rectifying K+ channel KGA (GIRK1) was investigated in rat tissue by in situ hybridization. KGA was originally cloned from the heart and represents the first G protein-activated K+ channel identified. It is expressed in peripheral tissue solely in the atrium, but not in the ventricle, skeletal muscle, lung and kidney. In the central nervous system KGA is most prominently expressed in the Ammon's horn and dentate gyrus of the hippocampus, neocortical layers II-VI, cerebellar granular layer, olfactory bulb, anterior pituitary, thalamic nuclei and several distinct nuclei of the lower brainstem. The abundant expression of KGA in many CNS neurons supports its important role as a major target channel for G protein mediated receptor function.
... While the distribution of neuronal GIRK mRNA transcripts and proteins in mouse (Kobayashi et al., 1995;Liao et al., 1996) is similar to that of in rat (DePaoli, Bell, & Stoffel, 1994;Karschin et al., 1996;Karschin & Karschin, 1997;Karschin et al., 1994;Liao et al., 1996), a few species differences have been described in the thalamus and cortex. For example, in the mouse thalamus, GIRK2 immunoreactivity is found in many thalamic nuclei, whereas in rat, GIRK2 is observed only in a few such nuclei (Karschin & Karschin, 1997;Liao et al., 1996). ...
... GIRK4 is expressed in the rodent brain, although at lower levels and in more restricted neuronal populations than the other three GIRK channel subunits (Chen et al., 1997;Iizuka, Tsunenari, Momota, Akiba, & Kono, 1997;Karschin et al., 1996;Karschin & Karschin, 1997;Karschin et al., 1994;Murer et al., 1997;Spauschus et al., 1996). To date, no specific antibodies have been developed that would allow for immunohistochemical techniques. ...
G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels are critical to brain function. They hyperpolarize neurons in response to activation of different G protein-coupled receptors, reducing cell excitability. Molecular cloning has revealed four distinct mammalian genes (GIRK1–4), which, with the exception of GIRK4, are broadly expressed in the central nervous system (CNS) and have been implicated in a variety of neurological disorders. Although the molecular structure and composition of GIRK channels are key determinants of their biophysical properties, their cellular and subcellular localization patterns and densities on the neuronal surface are just as important to nerve function. Current data obtained with high-resolution quantitative localization techniques reveal complex, subcellular compartment-specific distribution patterns of GIRK channel subunits. Recent efforts have focused on determining the associated proteins that form macromolecular complexes with GIRK channels. Demonstration of the precise subcellular compartmentalization of GIRK channels and their associated proteins represents a crucial step in understanding the contribution of these channels to specific aspects of neuronal function under both physiological and pathological conditions. Here, we present an overview of studies aimed at determining the cellular and subcellular localization of GIRK channel subunits in mammalian brain neurons and discuss implications for neuronal physiology.
... Most noteworthy, ventromedial hypothalamic nucleus and substantia nigra pars reticulata exhibit elevated signal, whereas the surrounding areas contain smaller amounts of transcript. These data largely agree with earlier reports and adult mouse brain expression data (Karschin et al. 1994;Karschin et al. 1996;Chen et al. 1997;Lein et al. 2007;Saenz del Burgo et al. 2008). ...
... Radioactive ISH revealed high levels of expression in the hippocampus and cortex but not in substantia nigra, ventromedial hypothalamus or caudate putamen. Note, however, two independent studies also demonstrated weak expression of Kcnj1 in adult rat brain (Karschin et al. 1994;Wu et al. 2004), which is consistent with our results. Again, such apparent discrepancies between radioactive and non-radioactive ISH may simply be due to differences in detection sensitivity and probe composition. ...
The approximately 350 ion channels encoded by the mammalian genome are a main pillar of the nervous system. We have determined the expression pattern of 320 channels in the two-week-old (P14) rat brain by means of non-radioactive robotic in situ hybridization. Optimized methods were developed and implemented to generate stringently coronal brain sections. The use of standardized methods permits a direct comparison of expression patterns across the entire ion channel expression pattern data set and facilitates recognizing ion channel co-expression. All expression data are made publically available at the Genepaint.org database. Inwardly rectifying potassium channels (Kir, encoded by the Kcnj genes) regulate a broad spectrum of physiological processes. Kcnj channel expression patterns generated in the present study were fitted with a deformable subdivision mesh atlas produced for the P14 rat brain. This co-registration, when combined with numerical quantification of expression strengths, allowed for semi-quantitative automated annotation of expression patterns as well as comparisons among and between Kcnj subfamilies. The expression patterns of Kcnj channel were also cross validated against previously published expression patterns of Kcnj channel genes.
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... The distribution pattern of rCKAx P from our RT-PCR tissue studies extends the observations of and is in general agreement with the published data [6] with the exception that we find a strong signal from skeletal muscle for rCKAT P. The distribution pattern of GIRK1 however shows differences to that previously published. For example in situ hybridisation studies have demonstrated abundant expression of GIRK1 (KGA) in rat brain but no signal in lung, skeletal muscle or kidney [16]. Our RT-PCR data indicate that GIRK1 is widespread in rat brain in agreement with the former findings, but that the peripheral tissues examined including lung, skeletal muscle and kidney also display expression of this gene although in many cases at much lower levels. ...
... Analysis of BIR1 mRNA distribution in rat brain by in situ hybridisation revealed it to be present in many brain regions, and is particularly abundant in the granule cell layer of the dentate gyrus, all pyramidal cells in the CA1, CA2 and CA3 fields of Ammon's horn in the hippocampus, olfactory bulb, taenia tecta and the granule cell layer of the cerebellum. Thus there is a remarkable similarity in the distribution of BIR 1 mRNA to that of GIRK1 mRNA in the central nervous system [16]. Clearly the widespread identity of distribution pattern for these inward rectifier sequences in rat brain suggests that they may also be linked functionally. ...
The distribution of mRNA encoding the inwardly rectifying K+ channel, BIR1 [1] was investigated in rat tissues, and a comparison made with the expression of related genes rcKATP and GIRK1 using the reverse transcription-polymerase chain reaction (RT-PCR). This showed BIR1 to be expressed in all areas of the brain examined, in the eye but not in any other peripheral tissue. This pattern was distinct from rcKATP and GIRK1. Additional in situ hybridisation studies of the central expression of BIR1 demonstrated high levels of BIR1 mRNA in the hippocampus, dentate gyrus, taenia tecta and cerebellum and at lower levels in the cortex, habenular nucleus, olfactory bulb, primary olfactory cortex, thalamus, pontine nucleus and amygdaloid nucleus.
... GIRK3 (Kir3.3) is encoded by the KCNJ9 gene in humans and is widely distributed in the rodent brain [48][49][50]. It cannot form functional homotetramers [41], but it is capable of assembling with the other members of the GIRK subfamily. ...
Among the large number of potassium-channel families implicated in the control of neuronal excitability, G-protein-gated inwardly rectifying potassium channels (GIRK/Kir3) have been found to be a main factor in heart control. These channels are activated following the modulation of G-protein-coupled receptors and, although they have been implicated in different neurological diseases in both human and animal studies of the central nervous system, the therapeutic potential of different subtypes of these channel families in cardiac conditions has remained untapped. As they have emerged as a promising potential tool to treat a variety of conditions that disrupt neuronal homeostasis, many studies have started to focus on these channels as mediators of cardiac dynamics, thus leading to research into their implication in cardiovascular conditions. Our aim is to review the latest advances in GIRK modulation in the heart and their role in the cardiovascular system.
... However, the most abundant neuronal G-protein gated inwardly rectifying channel is composed of Kir 3.1 and Kir 3.2 subunits. In situ hybridisation studies report robust expression of mRNAs for Kir3.1 and Kir 3.2 (and Kir 3.3) in most brain regions (Karschin et al., 1994;1996) and ...
The cloned G-protein gated inwardly rectifying K+ channel (a tetramer composed of Kir3.1-3.4 subunits) is activated by direct binding of Gβγ dimers, liberated by receptor activation of the Gi/o subfamily of heterotrimeric guanine nucleotide binding (G)-proteins. The interaction of these three membrane-associated components, G-protein coupled receptor (GPCR), heterotrimeric G-protein and channel, is rapid in native cells, with full channel activation via the GABA-B receptor occurring within a few hundred milliseconds (Sodickson & Bean, 1996 and 1998), and current deactivation occurring with a time constant of 1-2 seconds. Recent discovery of the Regulators of G-protein signalling (RGS) protein family has solved a major discrepancy between the slow deactivation of purified G-proteins and the fast deactivation of G-protein mediated signalling pathways. Their discovery has generated considerable interest in the kinetics of G-protein signalling and the organisation of these signalling components in the cell membrane. For these studies, the GIRK signalling system was reconstituted in mammalian HEK-293 cell lines, stably expressing the cloned neuronal channel subunits (Kir3.1 and Kir3.2A) plus a Gi/o-coupled GPCR (α2A adrenergic, A1 adenosine, D2 dopamine, M4 muscarinic and the heterodimeric GABA-B1b/2 receptors). Chapter 1 provides a general introduction to G-protein signalling and reviews our current understanding of the factors involved in the regulation of GERK channels. In Chapter 2, the methods and experimental protocols used in the study are described. In Chapter 3, I present a systematic analysis of the factors that contribute to the rapid activation of the channel complex, and in Chapter 4 the characteristic fast desensitisation of receptor-activated currents is examined. Factors influencing channel deactivation upon removal of agonist are explored in Chapter 5, and in Chapter 6 I describe the effects of the novel RGS protein family in these cell lines. Conclusions and future directions for this work are presented in Chapter 7.
... Like members of the six TMD superfamily functional inwardly rectifying potassium channels are the result o f tetramerisation (Yang et al, 1995). To date CGNs have been shown to express K irl.l (Horio et al, 1996), Kir2.1 (Horio et al, 1996, Karschin et al, 1996, Kir2.2 (Stonehouse et al, 1999), KirS.l (Karschin et al, 1994(Karschin et al, , 1996, Kir3.2 (Karschin et al, 1996, Signorini et al, 1997, Slesinger et al, 1997, Kir3.3 and Kir3.4 (Karschin g/ a/, 1996). ...
Cultured cerebellar granule neurons (CGNs) possess transient and delayed rectifier type voltage-gated potassium (K+) conductances. An additional component of outward current has been described in these cells which has been termed standing outward current (IKso). This current is outwardly rectifying, non-inactivating and is reversibly and concentration dependently inhibited by muscarine. The aim of this study was to characterise a number of biophysical and pharmacological properties of IKso in an attempt to identify the molecular correlate of the current and elucidate the mechanism of muscarinic modulation. The effect of M2 and M3 muscarinic receptor antagonists on the muscarine concentration response curve was determined. The M2 antagonist (methoctramine) had little effect on the control concentration response curve, while the same concentration of a M3 antagonist (zamifenacin) produced a rightward shift identifying the M3 receptor subtype as mediating the muscarine effect. (Additional concentrations of zamifenacin resulted in an estimated pA2 value of 8.13). Inhibiting a classical downstream product of M3 receptor activation (PLC), only slightly reduced the muscarinic inhibition of IKso, suggesting that M3 receptors may act through a novel pathway to inhibit IKso in CGNs. It has been proposed that the molecular correlate of IKso may be a member of the ether à go go (eag) family of K+ channels, since vat-eag (r-eag) channels when expressed in mammalian cells show similar properties to IKso. A feature of v-eag currents is a dramatic slowing of the activation kinetics on application of external Mg2+ in a concentration and voltage dependent manner. The activation kinetics of IKso were found to be unaffected by external Mg2+, arguing against eag being the molecular correlate of IKso. IKso also shares certain functional properties with members of the two pore domain superfamily of K+ channels (KT). A lack of voltage dependence of activation has been demonstrated for IKso, which is a diagnostic feature of KT channels. Pharmacologically IKso is inhibited by Ba2+, NMDG, external acidification, is weakly inhibited by quinine and quinidine, but is unaffected by arachidonic acid. These properties mean IKso bears closest resemblance to the KT clone TASK-1. The properties of IKso are almost identical to those of TASK-1, and RT-PCR revealed mRNA for TASK-1 is expressed in CGNs. Additionally immunocytochemical experiments confirmed the presence of TASK-1 protein in both the membrane and cytoplasm of the cells. It seems likely therefore that IKso belongs to the KT superfamily of potassium channels.
... Several types of ionic channels/exchangers including K + channels, nonselective cation channels and/or electrogenic NCXs have been reported to be linked to orexin receptors (Lytton, 2007;Kukkonen, 2011;Kukkonen and Leonard, 2014;Ji et al., 2019). In situ hybridization and immunocytochemical studies have revealed the distribution of NCX and inward rectifier K + channel mRNAs in the basal ganglia (Karschin et al., 1994;Murer et al., 1997;Canitano et al., 2002;Jeon et al., 2008). Here, we find that both the NCXs and inward rectifier K + channels are involved in the excitation of STN neurons induced by the activation of orexin receptors. ...
Central orexinergic system deficiency results in cataplexy, a motor deficit characterized with a sudden loss of muscle tone, highlighting a direct modulatory role of orexin in motor control. However, the neural mechanisms underlying the regulation of orexin on motor function are still largely unknown. The subthalamic nucleus (STN), the only excitatory structure of the basal ganglia, holds a key position in the basal ganglia circuitry and motor control. Previous study has revealed a wide distribution of orexinergic fibers as well as orexin receptors in the basal ganglia including the STN. Therefore, in the present study, by using whole-cell patch clamp recording and immunostaining techniques, the direct effect of orexin on the STN neurons in brain slices, especially the underlying receptor and ionic mechanisms, were investigated. Our results show that orexin-A elicits an excitatory effect on STN neurons in rats. Tetrodotoxin (TTX) does not block the orexin-induced excitation on STN neurons, suggesting a direct postsynaptic action of the neuropeptide. The orexin-A-induced inward current on STN neurons is mediated by the activation of both OX1 and OX2 receptors. Immunofluorescence result shows that OX1 and OX2 receptors are co-expressed and co-localized in STN neurons. Furthermore, Na+-Ca2+ exchangers (NCXs) and inward rectifier K+ channels co-mediate the excitatory effect of orexin-A on STN neurons. These results demonstrate a dual receptor in conjunction with the downstream ionic mechanisms underlying the excitatory action of orexin on STN neurons, suggesting a potential modulation of the central orexinergic system on basal ganglia circuitry as well as its related motor control and motor diseases.
... ScieNTific REPORTS | (2019) 9:508 | DOI: 10.1038/s41598-018-36833-y ( Fig. 3B), underscoring the complexity of underlying mechanisms(s). In the following we routinely used YFP-Gγ and Gγ tandem, which produce a better channel activation than the WT Gγ, and YFP tag allows measuring Gγ expression if needed. ...
The tetrameric G protein-gated K+ channels (GIRKs) mediate inhibitory effects of neurotransmitters that activate Gi/o-coupled receptors. GIRKs are activated by binding of the Gβγ dimer, via contacts with Gβ. Gγ underlies membrane targeting of Gβγ, but has not been implicated in channel gating. We observed that, in Xenopus oocytes, expression of Gγ alone activated homotetrameric GIRK1* and heterotetrameric GIRK1/3 channels, without affecting the surface expression of GIRK or Gβ. Gγ and Gβ acted interdependently: the effect of Gγ required the presence of ambient Gβ and was enhanced by low doses of coexpressed Gβ, whereas excess of either Gβ or Gγ imparted suboptimal activation, possibly by sequestering the other subunit “away” from the channel. The unique distal C-terminus of GIRK1, G1-dCT, was important but insufficient for Gγ action. Notably, GIRK2 and GIRK1/2 were not activated by Gγ. Our results suggest that Gγ regulates GIRK1* and GIRK1/3 channel’s gating, aiding Gβ to trigger the channel’s opening. We hypothesize that Gγ helps to relax the inhibitory effect of a gating element (“lock”) encompassed, in part, by the G1-dCT; GIRK2 acts to occlude the effect of Gγ, either by setting in motion the same mechanism as Gγ, or by triggering an opposing gating effect.
... There is a high level of expression of GIRK channels, which are one of the subfamilies of Kir channels, in MGB neurons [52][53][54]. Activation of GIRK channels using the GABA B receptor agonist baclofen suppressed RD (Fig. 7). Since the GABA B receptor is a G proteincoupled receptor, its activation by baclofen triggers a cascade of signals targeting a number of downstream receptors or channels, including the GIRK channel. ...
Rebound depolarization (RD) is a response to the offset from hyperpolarization of the neuronal membrane potential and is an important mechanism for the synaptic processing of inhibitory signals. In the present study, we characterized RD in neurons of the rat medial geniculate body (MGB), a nucleus of the auditory thalamus, using whole-cell patch-clamp and brain slices. RD was proportional in strength to the duration and magnitude of the hyperpolarization; was effectively blocked by Ni2+ or Mibefradil; and was depressed when the resting membrane potential was hyperpolarized by blocking hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with ZD7288 or by activating G-protein-gated inwardly-rectifying K+ (GIRK) channels with baclofen. Our results demonstrated that RD in MGB neurons, which is carried by T-type Ca2+ channels, is critically regulated by HCN channels and likely by GIRK channels.
... In the heart, acetylcholine (ACh) activates GIRK via m2R and eliciting the K + current, I KACh , which mediates the negative chronotropic effect of the vagus (Drici, Diochot, Terrenoire, Romey, & Lazdunski, 2000;Noma & Trautwein, 1978;Tamargo, Caballero, Gomez, Valenzuela, & Delpon, 2004;Voigt et al., 2014;Walsh, 2015;Yamada et al., 1998) and regulates pacemaking (Kovoor et al., 2001;Mesirca et al., 2014Mesirca et al., , 2013. Heart expresses GIRK1 and GIRK4 in sinoatrial node, atrium, and Purkinje fibers, and much less in the ventricle (Beckmann et al., 2008;Calloe, Goodrow, Olesen, Antzelevitch, & Cordeiro, 2013;Gaborit et al., 2007;Karschin et al., 1994;Yamada et al., 1998). Nevertheless, GIRK1's RNA is as abundant as the major G protein-independent inward rectifier, IRK1 (Kir2.1) in the ventricle (Marionneau et al., 2005). ...
G protein-gated K+ (GIRK, or Kir3) channels mediate inhibitory neurotransmission via G protein-coupled receptors (GPCRs) in heart and brain. The signaling cascade involves activation of GPCR by an agonist, activation of a G protein followed by rearrangement or dissociation of activated GαGTP from Gβγ, and activation of GIRK by Gβγ. Gβγ is the main transducer of GPCR activating signal to the GIRK channel. It promotes channel opening by direct binding to GIRK's cytosolic domain formed by the N- and C-terminal segments of the GIRK's four subunits. Gβγ’s interaction with, and activation of, the GIRK channels are well understood and reviewed elsewhere; however, several important details involving distal parts of the cytosolic domain remain incompletely understood. Gαi/o also binds to GIRKs and has been implicated in regulating channel's gating, in concert with Gβγ. Among known functions of Gα, best-described (though not well understood) are selectivity of signaling (only Gi/o proteins normally couple to GIRKs) and regulation of the basal activity of GIRKs (Ibasal). A role for a direct effect of the activated Gαi/oGTP in GIRK gating has also been proposed but remains elusive. This chapter discusses the mechanisms of signaling within the essential cascade, from GPCR to the heterotrimeric G protein and to the channel. The focus is on the role of Gα and on the relationships between Gα and Gβγ in channel regulation, their role in specific signaling from GPCRs to GIRKs, and the role of stoichiometry and cooperativity of G protein–GIRK interactions in channel's function.
... However, the molecular mechanisms underlying the side eects of clozapine are largely unknown. Several studies, in which Northern blot analysis and in situ hybridization histochemistry were used, have revealed that GIRK1 mRNA is present both in the brain and heart, GIRK2 and GIRK3 mRNAs are present mainly in the brain and GIRK4 mRNA is present mainly in the heart (Karschin et al., 1994; Lesage et al., 1994; Kobayashi et al., 1995; Krapivinsky et al., 1995; Spauschus et al., 1996). Activation of the GIRK channel plays an important role in slowing the heartbeat and inhibiting neuronal excitability (North, 1989; Brown & Birnbaumer, 1990). ...
To investigate the effects of clozapine, an atypical antipsychotic, on the cloned μ‐, Δ‐ and κ‐opioid receptors and G‐protein‐activated inwardly rectifying K ⁺ (GIRK) channel, we performed the Xenopus oocyte functional assay with each of the three opioid receptor mRNAs and/or the GIRK1 mRNA.
In the oocytes co‐injected with either the Δ‐ or κ‐opioid receptor mRNA and the GIRK1 mRNA, application of clozapine induced inward currents which were attenuated by naloxone, an opioid‐receptor antagonist, and blocked by Ba ²⁺ , which blocks the GIRK channel. Since the opioid receptors functionally couple to the GIRK channel, these results indicate that clozapine activates the Δ‐ and κ‐opioid receptors and that the inward‐current responses are mediated by the GIRK channel. The action of clozapine at the Δ‐opioid receptor was more potent and efficacious than that at the κ‐opioid receptor. In the oocytes co‐injected with the μ‐opioid receptor and GIRK1 mRNAs, application of clozapine (100 μ M ) did not induce an inward current, suggesting that clozapine could not activate the μ‐opioid receptor.
Application of clozapine caused a reduction of the basal inward current in the oocytes injected with the GIRK1 mRNA alone, but caused no current response in the uninjected oocytes. These results indicate that clozapine blocks the GIRK channel.
To test the antagonism of clozapine for the μ‐ and κ‐opioid receptors, we applied clozapine together with each selective opioid agonist to the oocytes co‐injected with either the μ‐ or κ‐opioid receptor mRNA and the GIRK1 mRNA. Each of the peak currents induced by each selective opioid agonist together with clozapine was almost equal to the responses to a selective opioid agonist alone. These results indicate that clozapine has no significant antagonist effect on the μ‐ and κ‐opioid receptors.
We conclude that clozapine acts as a Δ‐ and κ‐agonist and as a GIRK channel blocker. Our results suggest that the efficacy and side effects of clozapine under clinical conditions may be partly due to activation of the Δ‐opioid receptor and blockade of the GIRK channel.
British Journal of Pharmacology (1998) 123 , 421–426; doi: 10.1038/sj.bjp.0701621
... The overall effect on the cell is a stabilization of the resting membrane potential, e.g. in the case of slight depolarization by excitotoxic stimuli. The G-protein-activated inwardly rectifying K channels (GIRK) are regulated via neurotransmitters, occur in various subtypes and are expressed differently according to the region of the brain involved (Karschin et al., 1994 ). K channels also play an important role in the transmission of pain stimuli. ...
The spectrum of action of flupirtine includes analgesia, muscle relaxation and neuroprotection. N-methyl-D-aspartate (NMDA)
receptor antagonism has been discussed as a possible mechanism of action of this compound with little direct evidence. The
objective of the present study was to develop a plausible model to explain flupirtine's spectrum of action. A four-stage strategy
was selected for this purpose: Firstly, the serum concentration of flupirtine under therapeutic conditions was determined
on the basis of the current literature. The second stage involved assessing the known in-vitro effects in light of the therapeutic
active concentration. Using whole cell patch clamp recordings from cultured rat superior colliculus neurones interactions
between flupirtine and NMDA receptors were assessed. Only very high concentrations of flupirtine antagonized inward currents
to NMDA (200 μM) at −70 mV with an lC50 against steady-state responses of 182.1 ± 12.1 μM. The effects of flupirtine were voltage-independent and not associated
with receptor desensitization making actions within the NMDA receptor channel or at the glycine modulatory site unlikely.
NMDA receptor antagonism probably has little relevance for the clinical efficacy of flupirtine as the concentrations needed
were far higher than those achieved in clinical practice. However, the activation of a G-protein-regulated inwardly rectifying
K+ channel was identified as an interesting molecular target site of flupirtine. In the next stage, the central nervous spectrum
of action of experimental K+ channel openers (PCO) was considered. As far as they have been studied, experimental K+ channel openers display a spectrum of action comparable to that of flupirtine. In the final stage, a global model was developed
in which flupirtine stabilizes the resting membrane potential by activating inwardly rectifying K+ channels, thus indirectly inhibiting the activation of NMDA receptors. The model presented here reconciles the known functional
NMDA receptor antagonism of flupirtine with the activation of K+ channels that occurs at therapeutic concentrations, thus providing an understanding of flupirtine's spectrum of action. This
makes flupirtine the prototype of a clinically applicable substance group with analgesic, muscle-relaxant and neuroprotective
properties.
... The overall effect on the cell is a stabilization of the resting membrane potential, e.g. in the case of slight depolarization by excitotoxic stimuli. The G-protein-activated inwardly rectifying K channels (GIRK) are regulated via neurotransmitters, occur in various subtypes and are expressed differently according to the region of the brain involved (Karschin et al., 1994 ). K channels also play an important role in the transmission of pain stimuli. ...
The spectrum of action of flupirtine includes analgesia, muscle relaxation and neuroprotection. N-methyl-D-aspartate (NMDA) receptor antagonism has been discussed as a possible mechanism of action of this compound with little direct evidence. The objective of the present study was to develop a plausible model to explain flupirtine's spectrum of action. A four- stage strategy was selected for this purpose: Firstly, the serum concentration of flupirtine under therapeutic conditions was determined on the basis of the current literature. The second stage involved assessing the known in-vitro effects in light of the therapeutic active concentration. Using whole cell patch clamp recordings from cultured rat superior colliculus neurones interactions between flupirtine and NMDA receptors were assessed. Only very high con- centrations of flupirtine antagonized inward currents to NMDA (200 ÌM) at 270 mV with an lC50 against steady-state responses of 182.1 6 12.1 ÌM. The effects of flupirtine were voltage-independent and not associated with recep- tor desensitization making actions within the NMDA receptor channel or at the glycine modulatory site unlikely. NMDA receptor antagonism probably has little relevance for the clinical efficacy of flupirtine as the concentrations needed were far higher than those achieved in clinical practice. However, the activation of a G-protein-regulated inwardly rectifying K1 channel was identified as an interesting molecular target site of flupirtine. In the next stage, the central nervous spectrum of action of experimental K1 channel openers (PCO) was considered. As far as they have been studied, experimental K1 channel openers display a spectrum of action comparable to that of flupirtine. In the final stage, a global model was developed in which flupirtine stabilizes the resting membrane potential by activating inwardly rectifying K1 channels,
... In atrial cells, acetylcholine released by stimulation of the vagal nerve causes the opening of a GIRK channel (IKACII) via the activation of the m 2 muscarinic receptor, inducing hyperpolarization and thus slowing the cardiac frequency. A GIRK channel, termed either GIRK1 (Kubo et al., 1993) or K + channel G protein-activated (KGA) (Dascal et al., 1993) encodes a G protein-activated inward rectifier K + channel found in both brain and cardiac tissues (Dascal et al., 1993;Kubo et al., 1993;Ashford et al., 1994;De Paoli et al., 1994;Karschin et al., 1994;Kobayashi et al., 1995). In the atrial myocyte, GIRK1 seems to play a crucial role in regulating the heart beat via increasing the inward K + influx. ...
The G protein‐activated inwardly rectifying K+ channel (GIRK1) was coex‐pressed in Xenopus oocytes along with the 5‐HTia receptor, a 7‐helix receptor known to be coupled to K+ channels in many neural tissues. Thus, the activation of the 5‐HT1A receptor by its agonist leads to the opening of GIRK1. The GIRK1 current was measured using the two electrode voltage clamp technique with bath application of 5‐HT in the presence of various external potassium concentrations [K+]o. GIRK1 showed a strong inward rectification since only hyperpolarizing voltages evoked inward currents. K+ was the major ion carrier as evidenced by about 44 mV voltage shift corresponding to a 10‐fold external [K+] change. 5‐HT induced a concentration‐dependent inward K+ current (EC 50=10.7nM) which was blocked by Ba2+ Pertussis toxin (PTX) pre‐treatment reduced the K+ current by as much as about 70%, suggesting that PTX‐sensitive G protein (Gi or Go type) are involved in the 5‐HT1A receptor‐GIRK1 coupling in Xenopus oocytes
... are part of the family of inwardly rectifying K + channels. The subfamily has five members (Kir3.1-3.5) with a heterotetramer of Kir3.1 and 3.4 constituting the cardiac channel and is present in the atria and conducting tissues but has little if any expression in the ventricle (Karschin et al., 1994;Krapivinsky et al., 1995). The channel is characteristically activated by ACh binding to muscarinic (M2) receptors leading to the common designation as I KACh . ...
Inhibitory heterotrimeric G proteins and the control of heart rate. The activation of cell signaling pathways involving inhibitory heterotrimeric G proteins acts to slow the heart rate via modulation of ion channels. A large number of Regulators of G protein signalings (RGSs) can act as GTPase accelerating proteins to inhibitory G proteins and thus it is important to understand the network of RGS\G-protein interaction. We will review our recent findings on in vivo heart rate control in mice with global genetic deletion of various inhibitory G protein alpha subunits. We will discuss potential central and peripheral contributions to the phenotype and the controversies in the literature.
... The human Kir3.1 gene has been mapped to chromosome 2, band q24.1 [56]. Kir3.1 is expressed in seyeral neurons of the mammalian brain [57,58] where G-protein-activated Kir channels are known to suppress neuronal firing [59,60]. Two new members of the Kir3.0 subfamily recently cloned from mouse brain [13"], Kir3.2 and Kir3.3, may contribute to some of the diversity in neuronal G-protein-activated Kir channels (Table 1). ...
Recent cloning of a family of genes encoding inwardly rectifying K+ channels has provided the opportunity to explain some venerable problems in membrane biology. An expanding number of novel inwardly rectifying K+ channel clones has revealed multiple channel subfamilies that have specialized roles in cell function. The molecular determinants of inward rectification have been largely elucidated with the discovery of endogenous polyamines that act as voltage-dependent intracellular channel blockers, and with the identification of a critical site in the channel that mediates high-affinity block by both polyamines and Mg2+.
... This has been demonstrated by Ponce et al. (1996) using unilateral kainate lesions of the thalamus that resulted in a loss of staining on the side ipsilateral to the lesion in cortical layer IV. In addition, some studies have analyzed the localization of mRNA through in situ hybridization in the rat brain and also in the mouse brain (Karschin et al., 1994; DePaoli et al., 1994; Kobayashi et al., 1995; Karschin et al., 1996). In agreement with these authors, we found a very high density of GIRK1 mRNA in the cerebral cortex, hippocampus (dentate gyrus and CA1, CA2 and CA3 fields of hippocampus), thalamic nuclei and the cerebellar granular layer. ...
G-protein inwardly rectifying potassium (GIRK) channels mediate the synaptic actions of numerous neurotransmitters in the mammalian brain and play an important role in the regulation of neuronal excitability in most brain regions through activation of various G-protein-coupled receptors such as the serotonin 5-HT(1A) receptor. In this report we describe the localization of GIRK1, GIRK2, and GIRK3 subunits and 5-HT(1A) receptor in the rat brain, as assessed by immunohistochemistry and in situ hybridization. We also analyze the co-expression of GIRK subunits with the 5-HT(1A) receptor and cell markers of glutamatergic, gamma-aminobutyric acid (GABA)ergic, cholinergic, and serotonergic neurons in different brain areas by double-label in situ hybridization. The three GIRK subunits are widely distributed throughout the brain, with an overlapping expression in cerebral cortex, hippocampus, paraventricular nucleus, supraoptic nucleus, thalamic nuclei, pontine nuclei, and granular layer of the cerebellum. Double-labeling experiments show that GIRK subunits are present in most of the 5-HT(1A) receptor-expressing cells in hippocampus, cerebral cortex, septum, and dorsal raphe nucleus. Similarly, GIRK mRNA subunits are found in glutamatergic and GABAergic neurons in hippocampus, cerebral cortex, and thalamus, in cholinergic cells in the nucleus of vertical limb of the diagonal band, and in serotonergic cells in the dorsal raphe nucleus. These results provide a deeper knowledge of the distribution of GIRK channels in different cell subtypes in the rat brain and might help to elucidate their physiological roles and to evaluate their potential involvement in human diseases.
... The GIRK1 cDNA (also referred to as KGA; Dascal et al., 1993) encodes a G protein-activated inward rectifier (Dascal et al., 1993;Kubo et al., 1993b) and is found in both brain and cardiac tissues (Dascal et al., 1993;Kubo et al., 1993b;Ashford et al., 1994;DePaoli et al., 1994;Karschin et al., 1994;Kobayashi et al., 1995). The expres-sion of GIRK1 in Xenopus laevis oocytes produces channels that display many properties similar to IK~AC,~, including coupling with the m2 muscarinic receptor, single-channel conductance, mean open times, G protein activation, inward rectification, and slow activation kinetics upon hyperpolarization (Dascal et al., 1993;Kubo et al., 1993b). ...
Chimeras of GIRK1 and IRK1, a G protein-insensitive inward rectifier, are activated by coexpression of G beta gamma if they contain either the N-terminal or part of the C-terminal hydrophilic domain of GIRK1. The N-terminal domain of GIRK1 also facilitates the fast rates of activation and deactivation following m2 muscarinic receptor stimulation. The hydrophobic core of GIRK1 (M1-H5-M2) is important for determining the brief single-channel open times typical of GIRK1 but not important for determining G beta gamma sensitivity. Coexpression with CIR revealed that the gating properties associated with different GIRK1 domains could not have arisen from altered ability to form heteromultimers. These results implicate specific regions of GIRK1 in G protein activation and suggest that GIRK1 may be closely linked to the m2 muscarinic receptor-G protein complex.
... The study reported here provides one example for direct actions of G protein on an ion channel and presents an opportunity for mechanistic analysis of the control of channel activity by protein-protein interactions between the channel protein and subunits of the G protein. Given the wide distribution of GIRK1 mRNA in the mammalian brain (Dascal et al., 1993;Kubo et al., 1993b;DePaoli et al., 1994;Karschin et al., 1994), biochemical and electrophysiological analysis of GIRK1 channel activation by G~-/is of potential significance in our understanding of transmitter actions in the central nervous system as well as the heart. ...
Activation of G protein-gated K+ channels by G protein-coupled receptors contributes to parasympathetic regulation of heart rate in the atrium and inhibitory postsynaptic potentials in the peripheral and central nervous system. Having found that G beta gamma activates the cloned GIRK1 channel, we now report evidence for direct binding of G beta gamma to both the N-terminal hydrophilic domain and amino acids 273-462 of the C-terminal domain of GIRK1. These direct interactions are physiologically important because synthetic peptides derived from either domain reduce the G beta gamma binding as well as the G beta gamma activation of the channel. Moreover, the N-terminal domain may also bind trimeric G alpha beta gamma, raising the possibility that physical association of G protein-coupled receptors, G proteins, and K+ channels partially accounts for their compartmentalization and hence rapid and specific channel activation by receptors.
Molecular cloning together with functional characterization has shown that the newly identified family of inwardly rectifying K ⁺ channels consists of several closely related members encoded by separate genes. In this report we demonstrate the differential mRNA expression and detailed cellular localization in the adult rat brain of seven members of the IRK and GIRK subfamilies. Using both radiolabeled cRNA riboprobes and specific oligonucleotide probes directed to nonconserved regions of both known and newly isolated rat brain cDNAs, in situ hybridization revealed wide distribution with partly overlapping expression of the mRNAs of IRK1–3 and GIRK1–4. Except for the low levels of GIRK4 transcripts observed, the overall distribution patterns of the other GIRK subunits were rather similar, with high levels of expression in the olfactory bulb, hippocampus, cortex, thalamus, and cerebellum. Marked differences in expression levels existed only in some thalamic, brainstem, and midbrain nuclei, e.g., the substantia nigra, superior colliculus, or inferior olive. In contrast, IRK subunits were expressed more differentially: all mRNAs were abundant in dentate gyrus, olfactory bulb, caudate putamen, and piriform cortex. IRK1 and IRK3 were restricted to these regions, but they were absent from most parts of the thalamus, cerebellum, and brainstem, where IRK2 was expressed predominantly. Because channel subunits may assemble as heteromultimers, additional functional characterization based on overlapping expression patterns may help to decipher the native K ⁺ channels in neurons and glial cells.
Functional analysis of the GIRK proteins was performed in the heterologous expression system, Xenopus laevis oocytes. Macroscopic K⁺ currents were examined in oocytes injected with different combinations of Kir 3.0 complementary RNA (cRNA) and G protein subunit (β1γ2) cRNA. The current-voltage relationships demonstrated strong inward rectification for each individual and pairwise combination of GIRK channel subunits. Oocytes coinjected with any pair of GIRK subunit cRNA exhibited significantly larger inward K⁺ currents than oocytes injected with only one GIRK channel subtype. Ligand-dependent activation of only one of the GIRK combinations (GIRK1 and GIRK4) was observed when channel subunits were coexpressed with the D2 receptor in Xenopus oocytes. Dose-response data fit to a Michaelis-Menten equation gave an apparent Kd similar to that for DA binding in anterior pituitary tissue. GIRK1 and GIRK4 proteins were coimmunoprecipitated from anterior pituitary lysates, confirming the presence of native GIRK1/GIRK4 oligomers in this tissue. These data indicate that GIRK1 and GIRK4 are excellent candidate subunits for the D2-activated, G protein-gated channel in pituitary lactotropes, where they play a critical role in excitation-secretion coupling.
The presence of considerable ligand-independent GIRK1/GIRK4 channel activity in both D2l- and D2S-injected oocytes may be due to significant precoupling between the receptors and the GTP-binding proteins. This ligand-independent channel activity was consistently greater in the presence of the D2S receptor form. The two isoforms of the D2 receptor result from alternative splicing of the D2 DA receptor gene with the D2L isoform, including an insertion of 29 amino acids in the putative third cytoplasmic domain (38, 46, 47) that has been shown to be important for the coupling of seven-transmembrane receptors to GTP-binding proteins (48, 49) and differences in the efficiency of the two D2 isoforms to activate various effectors. For example, the D2S isoform has been demonstrated to be more efficient than D2L in inhibiting adenylate cyclase activity in JEG3 cells (50). The difference between the two receptor isoforms has been suggested to confer differential coupling to various G protein subtypes. Thus, the difference in ligand-independent GIRK1/GIRK4 current between D2L- and D2S-injected oocytes may reflect the specific G protein subtypes endogenously expressed in Xenopus oocytes. Studies to elucidate the apparent receptor-G protein precoupling are underway.
Many different types of K+ channels are involved in cardiac electrical activity (Noble, 1965). Various depolarization-activated K+ channels (Kv channels) determine the shape of the cardiac action potential. Cardiac cells also contain Kir channels (Fig. 1) that are open at very negative potentials but show a reduced conductance at positive membrane potentials, a phenomenon termed inward, or anomalous, rectification (Fig. 2A; Katz, 1949). Work over the past 30 years has described three main types of Kir channel in cardiac tissue. (1) The classical inward rectifier, I
K1, present in atrial and ventricular myocytes, shows “strong” inward rectification. Essentially no current flows through these channels at potentials positive to —40 mV (Noble, 1965; Vandenberg, 1994). (2) The KATPchannel is found in ventricular, atrial, and nodal cells. KATP channels display “weak” rectification, allowing substantial outward current to flow at positive potentials (Noma, 1983; Nichols and Lederer, 1991). (3) The muscarinicreceptor-activated K
+channel, K(ACh), is a strong inward rectifier found predominantly in atrial tissue (Bond et al., 1994). K(ACh) channels open in response to acetylcholine released from the vagus nerve and underlie the resultant slowing of the heart rate.
Within the past decade, molecular cloning techniques have confirmed that 5-hydroxytryptamine (5-HT) receptor subtypes, originally predicted from radioligand binding and functional studies (e.g., 5-HT1, 5-HT2, 5-HT3, 5-HT4), represent separate and distinct gene products. This knowledge has had a crucial impact on electrophysiological approaches to the 5-HT system in two important ways: (1) studies on previously recognized 5-HT receptors are now being directed, through the use of in situ mRNA hybridization and immunocytochemical maps, more precisely toward neurons that express these specific 5-HT receptor subtypes and (2) the functional role of previously unrecognized receptors (e.g., 5-HT5, 5-HT6, 5-HT7) can now be explored. Depending on the expression pattern for each type of neuron, the various 5-HT receptor subtypes can interact with their own set of G proteins, second messengers, and ion channels, to give rise to the wide range of electrophysiological actions produced by 5-HT throughout the brain and spinal cord. In addition, it is becoming evident that more than one 5-HT receptor sub-type may be expressed by the same neuron or by different neurons within the same region. Thus, while the following review is organized primarily according to individual 5-HT receptor subtypes, interactions between different receptor subtypes within a single neuron or region are also discussed where appropriate.
The inferior colliculus (IC) is a pivotal nucleus in the central auditory pathway. It receives and integrates ascending afferent projections from almost all of the major auditory nuclei in the lower brain stem (see Chapters 3 and 4). Auditory information that has been encoded in specific temporal or spatial configurations in the lower brain stem nuclei is further analyzed, relayed, or transformed in the IC. The wealth of afferent inputs includes excitatory and inhibitory projections. The cochlear nucleus (CN), medial superior olive (MSO), and contralateral lateral superior olive (LSO) are sources for major excitatory inputs. The dorsal nucleus of the lateral lemniscus (DNLL), ipsilateral LSO, and ventral nucleus of the lateral lemniscus (VNLL) are sources for major inhibitory inputs. The IC also receives descending projections from the auditory cortex (AC) and medial geniculate body (MGB) (see Chapters 7 and 8), and local inputs from neurons within the IC (see Chapter 5). In addition, the IC receives converging auditory, somatosensory, visual, and motor information. Thus, IC neurons are influenced by ascending, descending, and internal excitatory and inhibitory synaptic inputs while processing auditory and other signals. © 2005 Springer Science+Business Media, Inc. All rights reserved.
The voltage-, time-, and K § properties of a G protein-activated inwardly rectif~ng K + channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K + (Vx = 0 mV), voltage jumps from Vx to negative membrane potentials activated inward GIRK1 K + currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants q'f r~50 ms and x, ,-0400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from Vx. Voltage-dependent GIRK1 availability, reveaied by tail currents at-80 mV after long prepulses, was greatest at potentials negative to Vx and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with VK and was half maximal at VK-20 mV; the equivalent gating charge was ,'d.6 e-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein 131~/2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K + channels.
This chapter describes the properties of G-protein-gated inwardly rectifying potassium channel subunit (GIRKs), its potential roles in brain, and the pathophysiology of the GIRK2 mutation in weaver mice. The weaver mouse is one of the first animal models of neurodegenerative disease and is proving invaluable to neuroscientists. Weaver mice are a direct result of a mutation in ion channel, rather than an adhesion or migratory molecule as originally speculated. This mutation affects both striatal dopaminergic and cerebellar granule cells, ultimately leading to cell death. This, in turn, indirectly affects many other cell types and systems. The cellular defects in weaver mice correlate well with their motor and gross anatomical abnormalities. Weaver mice are also providing some insights into human neuropathophysiology. The anomalous and repetitive behavior of weaver mice is similar to human compulsive spectrum disorders, such as Parkinson's disease.
This chapter discusses the distribution of inwardly rectifying potassium (kir) channel subunits in the brain. Expression and localization of Kir subunit mRNAs in the mammalian brain has mainly been demonstrated using in situ hybridization. Because mRNAs are predominantly found in somata, this analysis defines brain nuclei and cell populations expressing a given channel subunit. Relative amounts of observed hybridization signals also reflect strong or weak expression in these brain nuclei. More recent immunocytochemical studies that evaluate the localization of Kir2.1, Kir3.1, Kir3.2, and Kir3.4 proteins using specific antibodies reach beyond the information provided by mRNA localization. Both light and electron microscopic studies have identified Kir subunits at high cellular resolution, allowing localization at pre- or postsynaptic sites, or defining neuronal compartments with several subunits coexpressed.
Das Wirkspektrum von Flupirtin umfaßt analgetische, muskelentspannende und neuroprotektive Eigenschaften. Der Wirkmechanismus der Substanz war bislang unzureichend bekannt. In den letzten Jahren verdichteten sich jedoch Hinweise auf eine Interaktion von Flupirtin mit dem glutamatergen N-Methyl-D-Aspartat (NMDA)-Rezeptor. Obwohl eine direkte Wirkung am NMDA-Rezeptor nicht nachweisbar war. sprachen alle Befunde für eine indirekte Beeinflussung des NMDA-Rezeptors im Sinne eines funktionellen NMDA-Antagonismus. Es wurde somit postuliert, daß ein Wirkort ,,up- oder downstream" vom NMDA-Rezeptor beeinflußt wird. Als solcher erwiesen sich die G-Protein gesteuerten einwärts gleichrichtenden K"-Kanale (GIRK), deren Öffnung zu einer Stabilisierung des Ruhemembranpotentials neuronaler Zellen führt und dadurch eine indirekte Hemmung des NMDA-Rezeptors bewirkt. Flupirtin ist in therapeutisch relevanten Konzentrationen ein neuronaler K'-Kanalöffner (neuronal potassium Channel opener). Dieser Mechanismus vermag das Wirkspektrum von Flupirtin zu erklären. Damit erweist sich die selektive neuronale K⁺-Kanaloffnung (SNEPCO) als ein neues Wirkprinzip und Flupirtin als Prototyp einer neuen Substanzklasse mit analgetischen, muskelrelaxierenden und neuroprotektiven Eigenschaften. Die experimentellen Grundlagen dieser Arbeitshypothese und der daraus resultierenden Modellvorstellungen werden in einer vierstufigen Betrachtungsebene vorgestellt.
The past three years have seen remarkable progress in research on the molecular basis of inward rectification, with significant implications for basic understanding and pharmacological manipulation of cellular excitability. Expression cloning of the first inward rectifier K channel (Kir) genes provided the necessary break-through that has led to isolation of a family of related clones encoding channels with the essential functional properties of classical inward rectifiers, ATP-sensitive K channels, and muscarinic receptor-activated K channels. High-level expression of cloned channels led to the discovery that classical inward so-called anomalous rectification is caused by voltage-dependent block of the channel by polyamines and Mg2+ ions, and it is now clear that a similar mechanism results in inward rectification of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-kainate receptor channels. Knowledge of the primary structures of Kir channels and the ability to mutate them also has led to the determination of many of the structural requirements of inward rectification.
Xenopus oocytes injected with GIRK1 mRNA express inwardly rectifying K+ channels resembling IKACh. Yet IKACh, the atrial G protein-regulated ion channel, is a heteromultimer of GIRK1 and CIR. Reasoning that an oocyte protein might be substituting for CIR, we cloned XIR, a CIR homolog endogenously expressed by Xenopus oocytes. Coinjecting XIR and GIRK1 mRNAs produced large, inwardly rectifying K+ currents responsive to m2-muscarinic receptor stimulation. The m2-stimulated currents of oocytes expressing GIRK1 alone decreased 80% after injecting antisense oligonucleotides specific to the 5' untranslated region of XIR, but GIRK1/CIR currents were unaffected. Thus, GIRK1 without XIR or CIR only ineffectively produces currents in oocytes. This result suggests that GIRK1 does not form native homomultimeric channels.
The cloning and characterisation of the Drosophila melanogaster Shaker potassium channel has led to the discovery of many diverse potassium channel types. It has only recently been appreciated that a number of disorders are the result of mutations in potassium channel sequences or due to potassium channel dysfunction. Compounds that modulate potassium channels have been tested for efficacy in a variety of peripheral and central diseases. Agents that block potassium channels have been advocated for the treatment of a number of CNS disorders such as multiple sclerosis, Alzheimer's disease and chronic CNS trauma. Although these compounds have some beneficial effects, especially in multiple sclerosis, at present their use appears to be limited. Potassium channel activators represent a relatively new class of drugs that has great potential for the treatment of a variety of CNS disorders, particularly ischaemia and acute CNS trauma. Some novel applications of these agents have recently been suggested, including their use in Alzheimer's disease and schizophrenia.
This chapter discusses GABAB receptor antagonists. The most abundant inhibitory neurotransmitter in the mammalian central nervous system is γ-aminobutyric acid (GABA), which interacts with two types of receptors designated GABAA and GABAB by Hill and Bowery in 1981. GABAA receptors are linked to chloride channels and transmit fast synaptic inhibition. The actions of GABAB receptors are mediated indirectly through heterotrimeric G-proteins of the subtypes Go and Gil. Agonists for GABAB receptors are GABA and the (R)-(-)- enantiomer of the antispastic agent, baclofen, synthesized in 1962. GABAB receptor subtypes are located on postsynaptic as well as on presynaptic sites. Postsynaptic GABAB receptors are coupled via pertussis toxin (PTX) sensitive G-proteins to potassium channels of the GIRK class. One of the physiological roles of the postsynaptic GABAB receptors is to mediate the late inhibitory postsynaptic potential. The chapter discusses GABAB receptor antagonists as tools to elucidate mechanisms of induction of long-term potentiation as well as their therapeutic potential. Extensive in vivo investigations suggest that GABAB receptor antagonists may have therapeutic potential for the treatment of cognition deficits, absence epilepsy, anxiety and depression.
In GH3/B6 cells at least two different inward K+ currents are observed that are regulated by thyrotropin-releasing hormone and somatostatin, respectively. Using a polymerase chain reaction based approach a cDNA was isolated and functionally expressed in human embryonic kidney cells that encodes an inward rectifier K+ channel, rIRK3, with a predicted molecular mass of 49.7 kDa. Corresponding transcripts of 2.6 kb have been detected in rat brain, pituitary and GH3/B6 cells. In situ hybridization revealed that rIRK3 mRNA is distributed throughout the brain and occurs predominantly in the piriform cortex, indusium griseum, supraoptic nucleus, facial nucleus and cerebellar Purkinje cells.
Potassium channels are highly selective for potassium ions over other cations. They have been broadly classified into two
main families (Hille 1992). So-called “voltage-gated” K channels are typically closed at negative membrane potentials and
open following depolarization beyond about —40mV. “Inward rectifier” K channels show an almost opposite dependence on membrane
potential. They are open at negative membrane potentials and close following depolarization. The change of conductance with
voltage is referred to as “rectification”, and the term is used to indicate both voltage-dependent channel “gating” and voltage-dependence
of the open channel current. Strong inward rectification (Fig.1A) was first described in skeletal muscle (Katz 1949), and
is very prominent in cardiac myocytes, and in glial cells and neurons in the central nervous system (Nakajima et al. 1988;
Newman 1993; Brismar and Collins 1989). Rectification of these channels is such that conductance declines to zero about 40mV
positive to the potassium reversal potential (Noble 1965; Vandenberg 1994). The high conductance at negative voltages allows
cells to maintain a stable resting potential, but the reduced conductance at positive potentials avoids short-circuiting the
action potential. “Weak” (Fig.1A) inward rectifier ATP-sensitive K+ (KA
T
P) channels allow substantial outward current to flow at positive potentials (Noma 1983).
Upon stimulation of vagal nerves, “Vagusstoff,” which was afterwards identified as acetylcholine (ACh), is released from the
axonal terminals of the vagal nerve and decelerates the heart beat. This historical discovery by OTTOLOEWI in the 1920s, established the concept of synaptic chemical transmission (LOEWI 1921; LOEwI and NAVARATIL 1926). Since then, many physiologists have been trying to elucidate the mechanisms underlying ACh-induced bradycardia. DEL-CASTILLO and KATZ (1955) described hyperpolarization of the membrane induced by ACh in frog heart. HUTTER and TRAUTWEIN (1955) measured an increase of the K+ efflux across the cardiac cell membrane under vagal stimulation. TRAUTWEIN and DUDEL (1958) showed an increase of K+ conductance under the voltage clamp condition. TRAUTWEIN and his colleagues (NOMA and TRAUTWEIN 1978; OSTERRIEDER et al. 1981) further analyzed the relaxation kinetics of the ACh-induced K+ current in the rabbit sinoatrial node and proposed
that ACh induces activation of a specific population of K+ channels, named muscarinic K+ (KACN) channels, to decelerate the pacemaker activity. The single channel currents of the KACN channels were recorded for the first time by Sakmann et al. (1983), who showed that the channel exhibits an inwardly rectifying
property but gating kinetics different from that of the background inwardly rectifying K+ (IK1) channel in cardiac myocytes. In 1985-6, it was discovered that pertussis toxin (PTX)-sensitive heterotrimeric G proteins
are involved in the activation of the KACN channel by m2-muscarinic and A1-,purinergic receptors (PFAFFINGER et al. 1985; BREITWIESER and SZABO 1985; KURACHI et al. 1986a,b,c). Because the KACh channel could be activated by intracellular GTP (in the presence of agonists) and GTPγS (even in the absence of agonists)
in cell-free inside-out patches (KURACHI et al. 1986a,b,c), the system is delimited to the cell membrane, leading to the proposal that the channel is directly activated
by G protein subunits. The G protein responsible for activation of KACh channels was designated GK according to its function(BREITWIESER and SZABO 1985).
Southern blot analysis of RT-PCR products from brain and heart revealed multiple products for a C-terminal region of Kir3.1. Sequencing yielded clones for wild-type Kir3.1 and three Kir3.1 C-terminal alternative splice variants, including a unique alternative exon. Two of these variants encoded truncated Kir3.1 molecules. Tissue distribution and electrophysiological characterization of a single truncated variant, Kir3.100 were then examined. Kir3.1 channels are gated by G-protein βγ-subunits binding to the C-terminal domain, thus, the truncation of Kir3.100 removes a major functional domain. When incorporated into heteromeric channels with other family members (Kir3.1, 3.2 or 3.4) several functional changes were observed: (1) Kir3.100 changes G-protein activation of Kir3 channels; (2) Kir3.100 is restricted in its ability to assemble with other channel subunits as heteromers; and (3) incorporation of Kir3.100 into heteromeric channel complexes alters the kinetics of channel re-activation.
We have raised affinity-purified polyclonal antibodies specific for the inward rectifying K+ channel (IRK1/Kir2.1) and the G protein-activated inward rectifying K+ channel (GIRK1/Kir3.1) examined their distributions in the rat brain immunohistochemically. The regional expression pattern of the IRK1 and GIRK1 proteins were similar to those of mRNA of the previous in situ hybridization study. The subcellular distribution was studied in the cerebellum, cerebral cortex and hippocampus. In the cerebellum, the IRK1 protein was clearly detected in the somata and proximal dendrites of Purkinje cells, while the GIRK1 protein was present in the somata and clustered dendrites of granule cells. In the cerebral cortex and hippocampus, both IRK1- and GIRK1-immunoreactivities were detected in the somata and apical dendrites of the pyramidal cells. The presence of IRK1 or GIRK1 proteins in the axons could not proved by the present study. The developmental changes of the expression pattern of the GIRK1 protein were also investigated in the hippocampus and in the cerebellum of postnatal day (P) 7 to P17 rats. The GIRK1 protein was detected neither in the subgranular zone of the dentate gyrus nor in the proliferative zone of the external granule cell layer of the cerebellum, in which granule cell precursors are reported to proliferate, while it was clearly detected in the adjacent layer in which postmitotic but immature cells exist. These results imply that the expression of the GIRK1 protein starts just after the neuronal precursors finished the last mitotic cell division. ©1997 Elsevier Science B.V. All rights reserved.
We report the detailed gene expression of all subunits within the Kir2 and Kir3 inwardly rectifying K+ channel subfamilies in the developing rat. Using in situ hybridization, onset of expression and cellular distribution of transcripts in embryonic and postnatal rat brains as well as in peripheral tissues is evaluated. Beginning at embryonic day 13 (E13), except "forebrain" Kir2.3 subunits which are absent from the body and brain until E21, all subunits appear with distinct and mainly nonoverlapping expression patterns. During ontogenic development, expression in the CNS becomes more widespread, leading to widely overlapping mRNA patterns as observed in the adult rat. Subunits are mainly found in regions of the developing brain that are also positive in the adult. Most subunits, in particular Kir3.2 and Kir3.4, are expressed transiently in distinct brain nuclei during ontogeny. Appearance of Kir transcripts is not generally related to the progressive and recessive phases during neurogenesis, but rather regulated differentially for each subunit and any specific group of neurons. It is demonstrated for the first time that several subunits, and most abundantly Kir2.2, are present early in the peripheral nervous system, i.e., in dorsal root-, sensory cranial-, and sympathetic ganglia. Also, of all subunits Kir3.3 is ubiquitously expressed in the entire embryonic nervous system and throughout the body. In summary, analysis of ontogenic Kir channel expression helps deciphering the importance of Kir channels (as exemplified for the defective weaver Kir3.2 gene) during proliferation, differentiation, and synaptogenesis in the CNS.
The levels and cellular localization of the mRNA encoding the inwardly rectifying potassium ion channel Kir4.1 were investigated in the embryonic rat brain by Northern blots and in situ hybridization. This transcript was absent at embryonic day 13 (E13), whereas it was clearly present in E14-15 preparations, principally in the neuroepithelium of the cerebral cortex, thalamus, and hypothalamus. At later embryonic stages (E17-20), Kir4.1 mRNA levels increased and expanded to the mantle zone, such as the cortical plate, hippocampus, thalamus, and hypothalamus. The early appearance of Kir4.1 mRNA in various brain regions suggests an involvement of the channel in cell proliferation, migration and differentiation in the rat CNS.
This paper extended the evaluation of the depressant and antinociceptive activities of hesperidin in order to determine its effectiveness by the intraperitoneal and oral routes, its pharmacological interaction with diverse pathways of neurotransmission and the role of its aglycone, hesperetin. The capacity of hesperidin and hesperetin to bind to μ-opioid receptor and their actions on μ-opioid receptor co-expressed with GIRK1/GIRK2 channels (G protein-activated inwardly rectifying K+ channels) in Xenopus laevis oocytes were also determined. Hesperidin exhibited a depressant activity in the hole board and locomotor activity tests, antinociceptive activities in the abdominal writhing and hot plate tests and no motor incoordination in the inverted screen and rotarod assays, only by the intraperitoneal route. Hesperetin did not show any effects in vivo in mice in these models, but in vitro it displaced the [³H]DAMGO binding with low-affinity and inhibited inward currents through the expressed GIRK1/2 channels. Although hesperidin actions in vivo demonstrated to be mediated by an opioid mechanism of action, it failed to directly bind to and activate the μ-opioid receptor or produce any change on inward GIRK1/2 currents in vitro. However, it should be considered that hesperidin may be metabolized, possibly resulting in crucial changes in its biological activity.
Die Heterodimerisierung von G- Protein gekoppelten Rezeptoren (GPCR) stellt ein aktuelles Forschungsgebiet dar, das molekulare Erklärungsmöglichkeiten für die Vielfalt der Signalwege über solche Rezeptoren aufzeigt. Die genauen Funktionen diese Konstrukte in vivo sind bisher erst in Ansätzen erforscht, ebenso wenig die molekularbiologischen Mechanismen. Für die beiden Serotoninrezeptoren 5-HT1A und 5-HT7 konnte Heterodimerisierung molekular nachgewiesen werden, in ihren physiologischen Mechanismen und Effekten sollte daher eine Charakterisierung vorgenommen werden. Mittels elektrophysiologischer Messverfahren wurden Ströme an dem heterologen Expressionsmodell der Oozyten des Krallenfrosches Xenopus laevis mittels Voltage-Clamp Technik an Kaliumionenkanälen (Kir3 und TASK-1) gemessen. Hierbei konnte gezeigt werden, dass die heterodimere Koexpression beider Rezeptoren eine signifikante Reduktion des Rezeptor-aktivierten Kanalstroms im Vergleich zur homomeren Expression zur Folge hatte. Weitere Experimente konnten dann zeigen, dass diese Effekte spezifisch für dieses Rezeptorheterodimer sind, und dass die Effekte von der Dosis bzw. dem Verhältnis der exprimierten cRNA abhängen. In Fluoreszenzmessung konnte zudem gezeigt werden, dass die Reduktion der Stromamplitude in der heterodimeren Expression nicht auf eine Reduktion von Kanalproteinen in der Zellmembran zurückzuführen ist. Zur weiteren Charakterisierung des bisher erst in Ansätzen erforschten 5-HT7 Rezeptors wurde dieser abschließend mit einem ß- adrenergen Rezeptor verglichen, der über den gleichen Signalweg bzw. Ionenkanal funktioniert. Auch hier zeigte sich eine signifikante Reduktion des Kanalstroms beim 5-HT7 Rezeptor. Die physiologische Relevanz dieser Ergebnisse liegt darin begründet, dass ein weiterführendes Verständnis von 5-HT Rezeptor vermittelten Signalwegen, insbesondere von der Bedeutung und den Mechanismen ihrer Heterodimersierung, neue pathophysiologische Zusammenhänge verdeutlicht. Speziell im Hinblick auf Erkrankungen, die mit den 5-HT Rezeptoren assoziiert sind, wie etwa Depressionen und Angststörungen, soll sich hieraus die Möglichkeit spezifischerer Therapien ergeben. Heteromerisation of G-protein coupled receptors (GPCR)is a current object of research to find out diversity of signaling pathways. The functional details of those constructs in vivo are not yet understood, also molecular mechanisms. For the Serotonin receptors 1A and 7 heteromerisation recently could be shown, therefore intention now is to make a physiological characterization. By electrophysiological methods, i.e. voltage clamp, currents of potassium channels (Kir3 and TASK-1) could be detected using the heterologous expression system of oocytes of xenopus leavis. So we could demonstrate, that heterologous expression of both receptors leads to a reduced current amplitude in comparison to homologous expression of one receptor. Those effects where shown to be specific and dependend of cRNA dose. By using fluorescense tagged Kir-channel we could demonstrate that the effect doesn't base on less channel protein in cell surface of the oocytes. Another point of interest was the characterization of Serotonin-7 channel. Therefore we analyzed a dose-response-relationship, afterwards we compared data with a ß-adrenergic receptore in heteromeric expression with Serotonin-1A. The physiological relevance of those experiments is to understand serotonin pathways an metabolism that is very important in development of mental disorders of fear or major depression.
Previous studies have shown that flupirtine, a centrally acting, non‐opioid analgesic agent, also exhibits neuroprotective activity in focal cerebral ischaemia in mice and reduces apoptosis induced by NMDA, gp 120 of HIV, prior protein fragment or lead acetate as well as necrosis induced by glutamate or NMDA in cell culture. To study the potential mechanism of the neuroprotective action of flupirtine, we investigated whether flupirtine is able to modulate potassium or NMDA‐induced currents in rat cultured hippocampal neurones by use of the whole‐cell configuration of the patch‐clamp technique.
We demonstrated that 1 μ M flupirtine activated an inwardly rectifying potassium current (K ir ) in hippocampal neurones (Δ I =−39±18 pA at −130 mV; n =10). This effect was dose‐dependent (EC 50 =0.6 μ M ). The reversal potential for K ir was in agreement with the potassium equilibrium potential predicted from the Nernst equation showing that K ir was predominantly carried by K ⁺ . Furthermore, the induced current was blocked completely by Ba ²⁺ (1 m M ), an effect typical for K ir .
The activation of K ir by flupirtine was largely prevented by pretreatment of the cells with pertussis toxin (PTX) indicating the involvement of a PTX‐sensitive G‐protein in the transduction mechanism (Δ I =−3±6 pA at −130 mV; n =8). Inclusion of cyclic AMP in the intracellular solution completely abolished the activation of K ir ( n =7).
The selective α 2 ‐adrenoceptor antagonist SKF‐86466 (10 μ M ), the selective 5‐HT 1A antagonist NAN 190 as well as the selective GABA B antagonist 2‐hydroxysaclofen (10 μ M ) failed to block the flupirtine effect on the inward rectifier.
Flupirtine (1 μ M ) could not change the current induced by 50 μ M NMDA.
These results show that in cultured hippocampal neurones flupirtine activates an inwardly rectifying potassium current and that a PTX‐sensitive G‐protein is involved in the transduction mechanism.
The voltage-, time-, and K(+)-dependent properties of a G protein-activated inwardly rectifying K+ channel (GIRK1/KGA/Kir3.1) cloned from rat atrium were studied in Xenopus oocytes under two-electrode voltage clamp. During maintained G protein activation and in the presence of high external K+ (VK = 0 mV), voltage jumps from VK to negative membrane potentials activated inward GIRK1 K+ currents with three distinct time-resolved current components. GIRK1 current activation consisted of an instantaneous component that was followed by two components with time constants tau f approximately 50 ms and tau s approximately 400 ms. These activation time constants were weakly voltage dependent, increasing approximately twofold with maximal hyperpolarization from VK. Voltage-dependent GIRK1 availability, revealed by tail currents at -80 mV after long prepulses, was greatest at potentials negative to VK and declined to a plateau of approximately half the maximal level at positive voltages. Voltage-dependent GIRK1 availability shifted with VK and was half maximal at VK -20 mV; the equivalent gating charge was approximately 1.6 e-. The voltage-dependent gating parameters of GIRK1 did not significantly differ for G protein activation by three heterologously expressed signaling pathways: m2 muscarinic receptors, serotonin 1A receptors, or G protein beta 1 gamma 2 subunits. Voltage dependence was also unaffected by agonist concentration. These results indicate that the voltage-dependent gating properties of GIRK1 are not due to extrinsic factors such as agonist-receptor interactions and G protein-channel coupling, but instead are analogous to the intrinsic gating behaviors of other inwardly rectifying K+ channels.
Four cDNA-encoding G-activated inwardly rectifying K+ channels have been cloned recently (Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y.(1993) Nature 364, 802-806; Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T., Lazdunski, M., and Hugnot, J. P. (1994) FEBS Lett. 353, 37-42; Krapivinsky, G., Gordon, E. A., Wickman, K., Velimirovic, B., Krapivinsky, L., and Clapham, D. E. (1995) Nature 374, 135-141). We report the cloning of a mouse GIRK2 splice variant, noted mGIRK2A. Both channel proteins are functionally
expressed in Xenopus oocytes upon injection of their cRNA, alone or in combination with the GIRK1 cRNA. Three GIRK channels, mGIRK1-3, are shown
to be present in the brain. Colocalization in the same neurons of mGIRK1 and mGIRK2 supports the hypothesis that native channels
are made by an heteromeric subunit assembly. GIRK3 channels have not been expressed successfully, even in the presence of
the other types of subunits. However, GIRK3 chimeras with the amino- and carboxyl-terminal of GIRK2 are functionally expressed
in the presence of GIRK1. The expressed mGIRK2 and mGIRK1, −2 currents are blocked by Ba2+ and Cs+ ions. They are not regulated by protein kinase A and protein kinase C. Channel activity runs down in inside-out excised patches,
and ATP is required to prevent this rundown. Since the nonhydrolyzable ATP analog AMP-PCP is also active and since addition
of kinases A and C as well as alkaline phosphatase does not modify the ATP effect, it is concluded that ATP hydrolysis is
not required. An ATP binding process appears to be essential for maintaining a functional state of the neuronal inward rectifier
K+ channel. A Na+ binding site on the cytoplasmic face of the membrane acts in synergy with the ATP binding site to stabilize channel activity.
We have isolated a novel inward rectifier K+ channel predominantly expressed in glial cells of the central nervous system. Its amino acid sequence exhibited 53% identity with ROMK1 and approximately 40% identity with other inward rectifier K+ channels. Xenopus oocytes injected with cRNA derived from this clone expressed a K+ current, which showed classical inward rectifier K+ channel characteristics. Intracellular Mg.ATP was required to sustain channel activity in excised membrane patches, which is consistent with a Walker type-A ATP-binding domain on this clone. We designate this new clone as KAB-2 (the second type of inward rectifying K+ channel with an ATP-binding domain). In situ hybridization showed KAB-2 mRNA to be expressed predominantly in glial cells of the cerebellum and forebrain. This is the first description of the cloning of a glial cell inward rectifier potassium channel, which may be responsible for K+ buffering action of glial cells in the brain.
The family of G-protein-activated inward-rectifiers K+ channels presently comprise at least 3 cloned members called GIRK1, GIRK2 and GIRK3. A close structural parent of GIRK channels has recently been described as being an ATP-sensitive K+ channel. This paper shows that Xenopus expression of this new channel that we call GIRK4 does not produce an ATP-inhibitable activity with a pharmacological activation by pinacidil as previously described but instead a G-protein activated inward-rectifier. While oocyte expression of single subunits is infrequent and relatively modest in intensity, expression of GIRK1,2, GIRK1,4 and GIRK2,4 mixtures leads to routine and robust expression of K+ channels indicating that heterologous subunit assembly is necessary for activity. Activity of GIRK1,2, GIRK1,4 and GIRK2,4 channels required the presence of ATP acting as an activator at the cytoplasmic face and is further activated by the beta gamma subunits.
We cloned the mouse brain G-protein-activated K+ channel 1 (mGIRK1) cDNA and determined the complete nucleotide and amino acid sequences of the coding region. In in situ hybridization using specific oligonucleotide probes, the signals for the three mGIRK (mGIRK1, mGIRK2 and mGIRK3) mRNAs were shown to be distributed widely as well as differently in most brain regions except for the caudate-putamen. Further, at least one, usually several, mGIRK mRNA with variable combinations was observed in most brain regions. These findings suggested that mGIRK channels may be essential in most brain regions in a signal transduction mediated by various G-protein-coupled receptors and that different subunit organizations of the mGIRK channel might occur in different neurons, resulting in diversity of their channel function in vivo.
In hippocampal neurons, 5-hydroxytryptamine (5-HT) activates an inwardly rectifying K+ current via G protein. We identified the K+ channel activated by 5-HT (K5-HT channel) and studied the effects of G protein subunits and nucleotides on the K+ channel kinetics in adult rat hippocampal neurons. In inside-out patches with 10 microM 5-HT in the pipette, application of GTP (100 microM) to the cytoplasmic side of the membrane activated an inwardly rectifying K+ channel with a slope conductance of 36 +/- 1 pS (symmetrical 140 mM K+) at -60 mV and a mean open time of 1.1 +/- 0.1 msec (n = 5). Transducin beta gamma activated the K5-HT channels and this was reversed by alpha-GDP. Whether the K5-HT channel was activated endogenously (GTP, GTP gamma S) or exogenously (beta gamma), the presence of 1 mM ATP resulted in a approximately 4-fold increase in channel activity due in large part to the prolongation of the open time duration. These effects of ATP were irreversible and not mimicked by AMPPMP, suggesting that phosphorylation might be involved. However, inhibitors of protein kinases A and C (H-7, staurosporine) and tyrosine kinase (tyrphostin 25) failed to block the effect of ATP. These results show that G beta gamma activates the G protein-gated K+ channel in hippocampal neurons, and that ATP modifies the gating kinetics of the channel, resulting in increased open probability via as yet unknown pathways.
Serotonin (5-HT) produced 3 different types of responses in neurons of mouse hippocampal and striatal cell cultures. These 3 responses have been characterized in terms of their pharmacological specificity, physiological mechanism, and dependence on cytoplasmic components. The most frequently observed response was inhibitory and was the result of a receptor-mediated activation of an inwardly rectifying potassium conductance. Typically, the response peaked within 1–3 sec of agonist application and did not exhibit desensitization. 5-Methoxy-N,N- dimethyltryptamine also produced this response in both striatal and hippocampal cultures and had no effect on the other 5-HT currents observed in this study. The selective 5-HT agonists--8-hydroxy-2-(di-n- propylamino)-tetralin, 1-(m-chlorophenyl) piperazine, and 1-(2- methoxyphenyl) piperazine--did not activate this outward current response. Methysergide did not block the 5-HT-activated outward current and often acted as an agonist. The response was lost in low-series- resistance recordings which facilitate solution exchange between the patch electrode and the cell. The loss of this response was prevented by using high-resistance patch electrodes, which retard this exchange. The 2 other responses described in this study were excitatory. They were seen less often than the inhibitory response. One of the excitatory responses was fast, with a time to peak of approximately 200 msec and a duration of 2–4 sec. The other was slow, with a time to peak of 7–10 sec and a duration of approximately 30–40 sec. Both of these responses were accompanied by a conductance increase. The fast excitatory response reversed at depolarized potentials and desensitized with a rate that varied with voltage. Metoclopramide and d-tubocurarine completely and reversibly blocked this fast excitatory response, while methysergide had no effect. The fast excitatory response was not lost during intracellular dialysis of cells in cultures from either striatum or hippocampus. In cultures from both brain regions, the slow excitatory response was blocked by methysergide. The slow excitatory response was lost even in patch-clamp recordings with high-resistance electrodes. This response was similar to responses to dopamine, norepinephrine, and forskolin, all of which are known to activate adenylate cyclase in the CNS.
Serotonin (5-HT) produced 3 different types of responses in neurons of mouse hippocampal and striatal cell cultures. These 3 responses have been characterized in terms of their pharmacological specificity, physiological mechanism, and dependence on cytoplasmic components. The most frequently observed response was inhibitory and was the result of a receptor-mediated activation of an inwardly rectifying potassium conductance. Typically, the response peaked within 1-3 sec of agonist application and did not exhibit desensitization. 5-Methoxy-N,N-dimethyltryptamine also produced this response in both striatal and hippocampal cultures and had no effect on the other 5-HT currents observed in this study. The selective 5-HT agonists--8-hydroxy-2-(di-n-propylamino)-tetralin, 1-(m-chlorophenyl) piperazine, and 1-(2-methoxyphenyl) piperazine--did not activate this outward current response. Methysergide did not block the 5-HT-activated outward current and often acted as an agonist. The response was lost in low-series-resistance recordings which facilitate solution exchange between the patch electrode and the cell. The loss of this response was prevented by using high-resistance patch electrodes, which retard this exchange. The 2 other responses described in this study were excitatory. They were seen less often than the inhibitory response. One of the excitatory responses was fast, with a time to peak of approximately 200 msec and a duration of 2-4 sec. The other was slow, with a time to peak of 7-10 sec and a duration of approximately 30-40 sec. Both of these responses were accompanied by a conductance increase. The fast excitatory response reversed at depolarized potentials and desensitized with a rate that varied with voltage. Metoclopramide and d-tubocurarine completely and reversibly blocked this fast excitatory response, while methysergide had no effect. The fast excitatory response was not lost during intracellular dialysis of cells in cultures from either striatum or hippocampus. In cultures from both brain regions, the slow excitatory response was blocked by methysergide. The slow excitatory response was lost even in patch-clamp recordings with high-resistance electrodes. This response was similar to responses to dopamine, norepinephrine, and forskolin, all of which are known to activate adenylate cyclase in the CNS.
Acetylcholine activates inwardly rectifying potassium channels (IK.ACh) in the heart through muscarinic receptor binding and activation of pertussis-toxin-sensitive G proteins. Experiments showing that only the beta gamma-subunit (G beta gamma) activates IK.ACh (ref. 4) were challenged by reports that only the activated alpha-subunit (G alpha) was effective. Here we examine IK.ACh regulation using purified brain and recombinant G-protein subunits. Six recombinant G beta gamma-subunits activated IK.ACh with apparent half-maximal activation concentrations of 3-30 nM. Activation of IK.ACh by recombinant G alpha-GTP gamma S was observed, but this was probably due to release of GTP gamma S from the protein. Importantly, IK.ACh activity elicited by GTP gamma S was inhibited by purified brain and recombinant G alpha-GDP, suggesting that native G beta gamma plays a major role in this pathway. We conclude that G beta gamma is a primary regulator of IK.ACh activity.
Activity of several ion channels is controlled by heterotrimeric GTP-binding proteins (G proteins) via a membrane-delimited pathway that does not involve cytoplasmic intermediates. The best studied example is the K+ channel activated by muscarinic agonists in the atrium, which plays a crucial role in regulating the heartbeat. To enable studies of the molecular mechanisms of activation, this channel, denoted KGA, was cloned from a rat atrium cDNA library by functional coupling to coexpressed serotonin type 1A receptors in Xenopus oocytes. KGA displays regions of sequence homology to other inwardly rectifying channels as well as unique regions that may govern G-protein interaction. The expressed KGA channel is activated by serotonin 1A, muscarinic m2, and delta-opioid receptors via G proteins. KGA is activated by guanosine 5'-[gamma-thio]triphosphate in excised patches, confirming activation by a membrane-delimited pathway, and displays a conductance equal to that of the endogenous channel in atrial cells. The hypothesis that similar channels play a role in neuronal inhibition is supported by the cloning of a nearly identical channel (KGB1) from a rat brain cDNA library.
Guanine nucleotide binding proteins, interchangeably called N or G proteins, seem to be the primary signal-transducing components of various agonist-induced cell membrane functions. In the heart, G proteins have been implicated in beta-adrenergic modulation of the slow inward Ca2+ current. We have investigated the role of G proteins in muscarinic activation of an inwardly rectifying, acetylcholine (ACh)-induced K+ current (IACh), and beta-adrenergic activation of an (isoprenaline)-induced Ca2+ current (Isi). Here we report that intracellular application of the non-hydrolysable GTP analogue 5'-guanylylimidodiphosphate (GppNHp) brought about an agonist-induced, antagonist-resistant, persistent activation of IACh and Isi. This functional uncoupling of channel from receptor suggests that the muscarinic receptor and the IACh channel are separate molecular structures. Membrane conductance responses to sequential activation of muscarinic and beta-adrenergic receptors demonstrate that in contrast to the muscarinic inhibition of Isi, muscarinic stimulation of IACh is mediated by a G protein via a pathway that does not involve adenylate cyclase. Taken together, the results support the notion that agonist is required to induce GppNHp binding and/or activation of the G proteins. Once triggered by agonist, the control system remains maximally activated, thereby transforming the cell so that it no longer responds to subsequent homologous receptor-mediated signals.
Binding of acetylcholine (ACh) to cardiac muscarinic ACh receptors (mAChR) activates a potassium channel that slows pacemaker activity. Although the time course of this activation suggests a multi-step process with intrinsic delays of 30-100 ms, no second-messenger system has been demonstrated to link the mAChR to the channel. Changes in cyclic nucleotide levels (cyclic AMP and cyclic GMP) do not affect this K channel or its response to muscarinic agonists. Indeed, electrophysiological experiments argue against the involvement of any second messenger that diffuses through the cytoplasm. We report here that coupling of the mAChR in embryonic chick atrial cells to this inward rectifying K channel requires intracellular GTP. Furthermore, pretreatment of cells with IAP (islet-activating protein from the bacterium Bordetella pertussis) eliminates the ACh-induced inward rectification. As IAP specifically ADP-ribosylates two GTP-binding proteins, Ni and No, that can interact with mAChRs, we conclude that a guanyl nucleotide-binding protein couples ACh binding to channel activation. This represents the first demonstration that a GTP-binding protein can regulate the function of an ionic channel without acting through cyclic nucleotide second messengers.
Both serotonin and the selective gamma-aminobutyric acidB (GABAB) agonist, baclofen, increase potassium (K+) conductance in
hippocampal pyramidal cells. Although these agonists act on separate receptors, the potassium currents evoked by the agonists
are not additive, indicating that the two receptors share the same potassium channels. Experiments with hydrolysis-resistant
guanosine triphosphate (GTP) and guanosine diphosphate analogs and pertussis toxin indicate that the opening of the potassium
channels by serotonin and GABAB receptors involves a pertussis toxin-sensitive GTP-binding (G) protein, which may directly
couple the two receptors to the potassium channel.
The mammalian heart rate is regulated by the vagus nerve, which acts via muscarinic acetylcholine receptors to cause hyperpolarization of atrial pacemaker cells. The hyperpolarization is produced by the opening of potassium channels and involves an intermediary guanosine triphosphate-binding regulatory (G) protein. Potassium channels in isolated, inside-out patches of membranes from atrial cells now are shown to be activated by a purified pertussis toxin-sensitive G protein of subunit composition alpha beta gamma, with an alpha subunit of 40,000 daltons. Thus, mammalian atrial muscarinic potassium channels are activated directly by a G protein, not indirectly through a cascade of intermediary events. The G protein regulating these channels is identified as a potent Gk; it is active at 0.2 to 1 pM. Thus, proteins other than enzymes can be under control of receptor coupling G proteins.
Guanine nucleotide binding (G) proteins (subunit composition alpha beta gamma) dissociate on activation with guanosine triphosphate
(GTP) analogs and magnesium to give alpha-guanine nucleotide complexes and free beta gamma subunits. Whether the opening of
potassium channels by the recently described Gk in isolated membrane patches from mammalian atrial myocytes was mediated by
the alpha k subunit or beta gamma dimer was tested. The alpha k subunit was found to be active, while the beta gamma dimer
was inactive in stimulating potassium channel activity. Thus, Gk resembles Gs, the stimulatory regulatory component of adenylyl
cyclase, and transducin, the regulatory component of the visual system, in that it regulates its effector function--the activity
of the ligand-gated potassium channel--through its guanine nucleotide binding subunit.
Somatostatin (SS) inhibits secretion from many cells, including clonal GH3 pituitary cells, by a complex mechanism that involves a pertussis toxin (PTX)-sensitive step and is not limited to its cAMP lowering effect, since secretion induced by cAMP analogs and K+ depolarization are also inhibited. SS also causes membrane hyperpolarization which may lead to decreases in intracellular Ca2+ need for secretion. Using patch clamp techniques we now demonstrate: 1) that both (SS) and acetylcholine applied through the patch pipette to the extracellular face of a patch activate a 55-picosiemens K+ channel without using a soluble second messenger; 2) that, after patch excision, the active state of the ligand-stimulated channel is dependent on GTP in the bath, is abolished by treatment of the cytoplasmic face of the patch with activated PTX and NAD+, and after inactivation by PTX, is restored in a GTP-dependent manner by addition of a nonactivated human erythrocyte PTX-sensitive G protein, and 3) that the 55-picosiemens K+ channel can also be activated in a ligand-independent manner with guanosine [gamma-thio] triphosphate (GTP gamma S) or with Mg2+/GTP gamma S-activated erythrocyte G protein. We call this protein GK. It is an alpha-beta-gamma trimer of which we have previously shown that the alpha-subunit is the substrate for PTX and that it dissociates on activation with Mg2+/GTP gamma S into alpha-GTP gamma S plus beta-gamma. A similarly activated and dissociated preparation of GS, the stimulatory regulatory component of adenylyl cyclase, having a different alpha-subunit but the same beta-gamma-dimer, was unable to cause K+ opening.(ABSTRACT TRUNCATED AT 250 WORDS)
Potassium channels in neurons are linked by guanine nucleotide binding (G) proteins to numerous neurotransmitter receptors.
The ability of Go, the predominant G protein in the brain, to stimulate potassium channels was tested in cell-free membrane
patches of hippocampal pyramidal neurons. Four distinct types of potassium channels, which were otherwise quiescent, were
activated by both isolated brain G0 and recombinant Go alpha. Hence brain Go can couple diverse brain potassium channels to
neurotransmitter receptors.
A complementary DNA encoding an ATP-regulated potassium channel has been isolated by expression cloning from rat kidney. The predicted 45K protein, which features two potential membrane-spanning helices and a proposed ATP-binding domain, represents a major departure from the basic structural design characteristic of voltage-gated and second messenger-gated ion channels. But the presence of an H5 region, which is likely to form the ion conduction pathway, indicates that the protein may share a common origin with voltage-gated potassium channel proteins.
Ion channels are signaling molecules and by themselves perform no work. In this regard they are unlike the usual membrane enzyme effectors for G proteins. The pathways of G protein receptor, G protein and ion channels are, therefore, purely informational in function. Because a single G protein may have several ion channels as effectors, the effects should be coordinated and this seems to be the case. Inhibition of Ca2+ current and stimulation of K+ currents would have a greater impact than either alone. Additional flexibility is provided by spontaneous noise in the complexes of G protein receptor, G protein, and ion channel. By having a non-zero setpoint, the range of control is extended and the responses become bi-directional.
A complementary DNA encoding an inward rectifier K+ channel (IRK1) was isolated from a mouse macrophage cell line by expression cloning. This channel conducts inward K+ current below the K+ equilibrium potential but passes little outward K+ current. The IRK1 channel contains only two putative transmembrane segments per subunit and corresponds to the inner core structure of voltage-gated K+ channels. The IRK1 channel and an ATP-regulated K+ channel show extensive sequence similarity and constitute a new superfamily.
1. Single inwardly rectifying K+ channel currents were recorded from acutely isolated adult serotonergic dorsal raphe (DR) neurones using the cell-attached and outside-out patch clamp configuration. 2. Four equally spaced conductance levels were observed in both outside-out and cell-attached patch recordings with conductance levels averaging 11, 21, 30 and 40 pS. Larger conductance openings (50-120 pS) were seen less frequently. 3. When using 136 [K+]0 the single channel I-V relation was linear in the range 0 mV to -100 mV in all cases. 4. Transitions between the various conductance levels were observed, as were apparent direct opening and closing to each individual conductance level. Furthermore openings of 11, 21 and 30 pS were observed in almost all the patches. These results suggest that the different-sized events result from substrates of a single channel rather than several different channels with different conductances. 5. Unitary K+ channel current probability of opening, recorded in cell-attached patch, was unchanged after 5-hydroxytryptamine (5-HT) was added to the bath outside the patch pipette which suggests that no easily diffusible second messenger was involved. 6. The single K+ channel activity, however, was increased on average by 670% following the addition of 5-HT to the bath when recording channel activity in the outside-out configuration. Usually all K+ channel subconductance levels increased in activity but the largest increases occurred in the events with 30 and 40 pS conductance. 7. These results suggest that 5-HT enhances the probability of opening of the resting K+ channel activity, which can open to several levels of conductance, and that no new channel or freely diffusible second messenger is involved in the response.
We have cloned an inward-rectifier potassium channel from a mouse brain cDNA library, studied its distribution in the brain by in situ hybridization and determined the chromosomal localization of the gene. A mouse brain cDNA library was screened using a fragment of the mouse macrophage IRK1 cDNA as a probe. Two duplicate clones of approximately 5.5 kb were obtained. Xenopus ococytes injected with cRNA derived from the clone expressed a potassium channel with inwardly rectifying channel characteristics. The amino acid sequence of the clone was identical to that of IRK1 recently cloned from a mouse macrophage cell line. In situ hybridization study showed the mouse brain IRK1 to be generally distributed throughout the brain, but in particular subsets of neurons at high levels. The gene was placed in the distal region of mouse chromosome 11, which contains several uncloned neurological mutations. These results provide the first demonstration of the cloning and distribution of an inward rectifier potassium channel from the nervous system.
Parasympathetic nerve stimulation causes slowing of the heart rate by activation of muscarinic receptors and the subsequent opening of muscarinic K+ channels in the sinoatrial node and atrium. This inwardly rectifying K+ channel is coupled directly with G protein. Based on sequence homology with cloned inwardly rectifying K+ channels, ROMK1 (ref. 11) and IRK1 (ref. 12), we have isolated a complementary DNA for a G-protein-coupled inwardly rectifying K+ channel (GIRK1) from rat heart. The GIRK1 channel probably corresponds to the muscarinic K+ channel because (1) its functional properties resemble those of the atrial muscarinic K+ channel and (2) its messenger RNA is much more abundant in the atrium than in the ventricle. In addition, GIRK1 mRNA is expressed not only in the heart but also in the brain.
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