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– Effects of ApC on the action potential of DRG cells. Action potentials elicited by 100 pA, 2.5 ms stimuli were recorded under control conditions and after 3 μ M ApC application. ApC increased the duration of action potential 1670% (n = 7). The dotted line indicates the zero voltage level.
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We have characterized the actions of ApC, a sea anemone polypeptide toxin isolated from Anthopleura elegantissima, on neuronal sodium currents (I(Na)) using current and voltage-clamp techniques. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study. These cells express tetrodotoxin-sensitive (TTX-S) an...
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... a unimodal histogram that was well fitted (r 2 = 0.97) by a Gaussian function with the mean = 51 ± 13 pF, corresponding to medium size DRG cells with a diameter of about 40 μm. The average duration of the action potentials measured at 50% of its amplitude (D 50 ) was 2.1 ± 0.5 ms (n = 7). Application of 3 μM ApC increased D 50 to 34.7 ± 7.8 ms (Fig. 1). The maximum effect was always achieved within the first minute after perfusion with ApC. Washout (5 min) removed the toxin effects in 95 ± 2%. The mean membrane potential in these experiments was −59 ± 0.4 mV. ApC 3 μM produced a sig- nificant (P < 0.05, Student's t test) depolarization in the cell membrane potential of 4.8 ± 0.4 mV. ...
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... slowing of the inactivation kinetics and the reduced voltage depen- dence of the steady-state availability curve indicate that ApC modifies the transition rate of both the open state to the inactivated state and the inactivated state to the open state of the Na + channel. Anemone toxins were first completely purified from A. sulcata (Beress et al., 1975a,b). Since then, successful efforts leading to the isolation and identification of several peptide toxins with high activity on excitable mem- branes have been made with various sea anemone species. ...
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... sodium channels (Na channels) are integral membrane proteins responsible for the generation and propagation of action potentials in most excitable tissues. These channels are specific targets for a variety of neurotoxins that bind to various sites on the channel protein (Caterall, 2000) and therefore have been used as pharmacological tools to investigate the structure and function of Na + channels (Gordon et al., 1996; Caterall, 2000; Chen et al., 2002). To date, six neurotoxin-receptor sites have been identified by direct radiolabeled toxin studies (Strichartz et al., 1987; Fainzilber et al., 1994; Gilles et al., 2002). Typically, sea anemone toxins and scorpion α -toxins bind to the so-called neurotoxin receptor site-3 which has been located in the short extracellular loop IVS3-S4 of the Na + channel α -subunit with an important participation of the glutamic acid in position 1613 (rat brain Na + channel) (Rogers et al., 1996) or the aspartic acid in position 1612 (cardiac Na + channel) (Benzinger et al., 1998). Mutation of these acidic residues from the transmembrane segment IVS3 reduces the binding of sea anemone toxins and scorpion α -toxins. Both the IVS3-S4 loop and the amino acid residues noted could also be involved in the coupling of channel activation to fast inactivation. Beside, several other regions of the α -subunit may contribute to the receptor site, particularly the IS5-S6 and IVS5-S6 extracellular loops. Site-3 neurotoxins comprise a structurally diverse group of peptide toxins isolated from scorpions (Meves et al., 1986; Possani et al., 1999), sea anemones (Norton, 1991), spiders (Fletcher et al., 1997; Little et al., 1998), and wasps (Sahara et al., 2000; Kinoshita et al., 2001). These compounds increase the action potential duration and often produce spontaneous firing. The site-3 neurotoxins modify the inactivation process, although they act from the extracellular side, making them interesting for use as probes to study the inactivation mechanism of Na + channels. Also, the analysis of peptide sequences of the toxins may allow the identification of the amino acid residues of the channel that are essential for binding of the toxin. Moreover, the electrophysiological effects of site-3 toxins are analogous to those displayed by a number of naturally occurring mutant channel forms observed in periodic pathologies, such as paramyotonia congenita and hyperkalemic periodic paralysis (Cannon and Corey, 1993; Lehmann-Horn and Jurkat-Rott, 1999; Jurkat-Rott et al., 2000; Blumenthal and Seibert, 2003), constituting for this reason a potential tool for the development of animal models of these pathologies. Finally, because some site-3 toxins are highly efficacious and potent insect-selective toxins (Bosmans et al., 2002), these compounds could constitute a prototype for the design of new insecticides. In this work, we have characterized the electrophysiological effects of APC, a peptide toxin obtained from the sea anemone Anthopleura elegantissima , on mammalian neurons. A total of 92 neurons were successfully current- or voltage- clamped for a sufficient time to allow the study of ApC actions. The capacitances of the DRG cells formed a unimodal histogram that was well fitted ( r 2 = 0.97) by a Gaussian function with the mean = 51 ± 13 pF, corresponding to medium size DRG cells with a diameter of about 40 μ m. The average duration of the action potentials measured at 50% of its amplitude ( D 50 ) was 2.1 ± 0.5 ms ( n = 7). Application of 3 μ M ApC increased D 50 to 34.7 ± 7.8 ms (Fig. 1). The maximum effect was always achieved within the first minute after perfusion with ApC. Washout (5 min) removed the toxin effects in 95 ± 2%. The mean membrane potential in these experiments was − 59 ± 0.4 mV. ApC 3 μ M produced a significant ( P < 0.05, Student's t test) depolarization in the cell membrane potential of 4.8 ± 0.4 mV. Washout (5 min) repolar- ized the cell membrane to − 58 ± 0.5 mV. In voltage-clamp experiments, the percentage of change in peak amplitude and activation and inactivation time constants were calculated for ionic currents from both TTX-S and TTX-R Na + currents before and about 2 min after toxin perfusion. Concentration – response curves for these variables were built using 0.1, 0.3, 1, 3, and 10 μ M ApC. The main effect of ApC was a concentration-dependent increase in the TTX-S I Na inactivation time course (Fig. 2A) with no significant effects ( P > 0.05, Student's t test) on the activation time course or peak current amplitude. In the presence of 1 μ M ApC, the inactivation time constant ( τ h ) increased 35.7 ± 4.6% ( n = 4), whereas perfusion with 3 μ M ApC produced 94 ± 20% ( n = 12) increase in τ h . The maximum effect of ApC on τ h occurred within the first minute after perfusion with toxin. Washout (5 min) removed 91 ± 9% of the effect of 3 μ M ApC. The IC 50 value derived from concentration – response curve (Fig. 2B) was 1.25 ± 0.9 μ M. The ratio I 5ms / I peak in control conditions at − 30 mV had a value of 0.1 ± 0.02. Use of 3 μ M ApC ( n = 12) significantly increased I 5ms / I peak to 0.34 ± 0.02, whereas 10 μ M ApC increased the ratio to 0.39 ± 0.04. A concentration of 3 μ M was chosen for the remaining experiments in this study because its effect was close to the maximum, and because supplies of ApC were limited. Current density versus voltage curves were obtained from current – voltage relationships by dividing peak ionic-current amplitudes by the corresponding cell-membrane capacity. Under control conditions, TTX-S I Na activated at about − 60 mV, reached its maximum at − 30 mV and reversed at approximately + 20 mV. After perfusion with 3 μ M ApC ( n = 8), there was a slight hyperpolarizing shift in the voltage dependence of Na + channel activation (Fig. 3). ApC also reduced the threshold of activation to about − 65 mV. Despite this, there were no significant changes ( P > 0.05, Student's t test) in the voltage at which the maximum current density was achieved nor in the reversal potential of TTX-S I Na . Likewise, no significant difference ( P > 0.05, Student's t test) in the maximum current density was produced when 3 μ M ApC was used; − 185 ± 9 pA/pF (control) versus − 190 ± 9 pA/pF (ApC). The maximum sodium conductance ( gNa max ) at several potential values was calculated as a chord conductance from the corresponding peak current. A Boltzmann function fit to normalized conductance curves yields the half-maximum voltage of activation, V 1/2 act , and the corresponding slope factor, k . A slight difference in the voltage dependence of gNa / gNa max after perfusion with 3 μ M ApC ( n = 8) was observed (Fig. 4): from V 1/2 act = − 39.4 ± 0.6 mV (control) to V 1/2 act = − 42.4 ± 0.9 mV (ApC). However, taking into account the time-dependent spontaneous shift for V 1/2 act occurring between the control measurements and the time at which toxin was applied, as follows from control experiments (about 0.9 mV), this difference was not significant. Also, no significant change was produced in the slope factors (5.3 ± 0.2 mV versus 5.6 ± 0.3 mV). ApC (3 μ M, n = 8) produced a significant ( P < 0.05, Student's t test) hyperpolarizing shift in the voltage at which 50% of the channels are inactivated ( V 1/2 inact . ) from a control value of − 74.5 ± 1.6 mV to − 82.4 ± 1.5 mV after ApC (Fig. 4). These changes in the presence of ApC could not be explained by spontaneous time-dependent shift in the inactivation curve (about 1.1 mV as derived from control experiments). A significant change ( P < 0.05, Student's t test) was also observed in the slope factor for these curves; 8.0 ± 0.6 mV (control) and 9.8 ± 0.5 mV. To explore the state of the Na + channel on which ApC (3 μ M) exerts its action, a pair of voltage pulse trains (60 squared pulses named p 1 – p 60 ) separated by a rest period were applied ( n = 3). A significant change in τ h ( P < 0.05, Student's t test) was observed from control train p 60 to test train p 1 (Fig. 5); no significant difference was observed between test train p 1 and p 60 indicating that the action of ApC was not use-dependent. TTX-R I Na recorded for this study was compatible with the two subtypes previously described in literature for DRG neurons (Elliot and Elliot, 1993; Baker and Wood, 2001): (1) a slowly inactivating current ( τ h = 4.5 ± 1.8 ms, n = 3), and (2) a current that fails to inactivate, giving rise to a large late component ( n = 3). These currents are mainly due to Na v 1.8 and Na v 1.9 sodium channels (Novakovik et ...
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We have characterized the effects of BgII and BgIII, two sea anemone peptides with almost identical sequences (they only differ by a single amino acid), on neuronal sodium currents using the whole-cell patch-clamp technique. Neurons of dorsal root ganglia of Wistar rats (P5-9) in primary culture (Leibovitz's L15 medium; 37 degrees C, 95% air/5% CO2...
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... Some-such as AFT-II from Anthopleura fuscoviridis, ApC from Anthopleura elegantissima, Bc-III from Bunodosoma caissarum, CGTX-II from Bunodosoma cangicum, CgNa from Condylactis gigantea, or RTX-III from Heteractis crispa-exhibit selectivity for specific Na V isoforms. 64,96,[107][108][109]120,142 In addition, phospholipase-A 2 is found in venoms across all cnidarian classes, as are pore-forming toxins. 92 However, the contribution of these toxins to pain and nociception has not been explored systematically, in part because many jellyfish venoms in particular suffer from poor stability and venom extraction is, compared with other venomous animals, somewhat more difficult. ...
... Methodology Generally, the study of biocompatible materials in electrophysiology use gold, titanium, parylene or platinum-black microelectrodes [21]. However, these materials have electrical limitations such as very high impedance and oxidation in electrolyte environment. ...
... However, these materials have electrical limitations such as very high impedance and oxidation in electrolyte environment. Recently, some alternative materials have been studied to improve the electrical characteristics between the electrolytic environment and microelectrodes [21]. The a-Si 1-x Ge x :H films were fabricated on a Corning 2947 glass substrate deposited by a LF-PECVD plasma system (planar reactor Reinberg, model AMP3300) with the following conditions: Glow discharge was maintained for one hour at 110 kHz with a substrate temperature of 300 °C, a low pressure (8.10 -4 ), and a power of 600 Watts. ...
... These procedures were made to minimize animal suffering and to reduce the number of animals used, as outlined in the ''Guide for the Care and Use of Laboratory Animals" that is issued by the National Academy of Sciences. For the cell culture, we used Wistar rats of postanatal days 7-10 (P7-P10) that were euthanized by decapitation [21]. The dorsal root ganglia were isolated and were incubated in culture medium L-15 (Gibco) added with 0.125% collagenase and 0.125% trypsin for 30 min at 37 °C. ...
We studied the surface morphology and biocompatibility of hydrogenated amorphous silicon-germanium (a-Si1-xGex:H) thin films prepared by Low Frequency Plasma Enhanced Chemical Vapor Deposition (LF-PECVD). These films were deposited on a Corning 2947 glass substrate having a thickness of 3 μm, the electrical performance showed a decreased electrical resistance for low regime voltage. The root mean square (RMS) surface roughness of the films was measured by atomic force microscopy (AFM) in a non-contact mode. A biocompatibility tests was carried out using primary cultures of dorsal root ganglion (DRG) of Wistar rats. The DRG neurons were incubated for 18 hours on a-Si1-xGex:H thin films, and subsequent electrophysiological recording was performed. These neurons displayed typical ionic currents, including a fast-inward current at the beginning of voltage clamp pulse (Na+ current) and ensuing outward currents (K+ current). In current clamp experiments, depolarizing current pulse injection caused typical action potential discharge of the neurons. These results confirmed the feasibility of using a-Si1-xGex:H thin films as a biocompatible material
(PDF) Biocompatibility and surface properties of hydrogenated amorphous silicon-germanium thin films prepared by LF-PECVD. Available from: https://www.researchgate.net/publication/336347777_Biocompatibility_and_surface_properties_of_hydrogenated_amorphous_silicon-germanium_thin_films_prepared_by_LF-PECVD [accessed Oct 09 2019].
... Then, the use of snail neurons as a model for the characterization of toxins provides an adequate system, since their ion channels could be targeted by these toxins under natural conditions. The use of DRG neurons, despite they are very different taxonomically, represents an important step for comparison with previous results obtained from other sea anemone toxins [52][53][54]. Furthermore, depending on the route of administration, when tested in mice, some anemone toxins may have LD 50 levels that are several orders of magnitude below those obtained when tested on crabs. ...
Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 µM), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new β-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.
... As observed, both CGTX-II and -AITX-Bcg1a induce different effects on Nav1.1 and 1.2. On Nav1.1 and 1.6, the peptides indeed shifted the Boltzmann inactivation curves to more depolarized potentials and maintain a pedestal (seeFig. 2 ), by the induction of a persistent current (steadystate current – A ss ), in contrary to that observed for the other clones investigated and also reports by other authors [27,28] . This characterizes a population of bound channels that do not inactivate. ...
... This clearly suggests that the binding site of type 1 toxins is not restricted only to the supposed site 3, between segments S3 and S4 of domain IV, in agreement with previous results [23] . Also, a similar discrete shift of activation toward more hyperpolarized potentials was only observed in the toxin ApC when tested in rat DRG neurons [27], suggesting that these sea anemone type 1 toxins might act in some way as a -scorpion fashion, facilitating depolarization of affected cells. Thus, further site-directed mutagenesis studies in other regions of Navs should be performed in order to determine the other contact regions between channel and sea anemone toxins, as obviously other topological areas of such channels are involved in these interactions. ...
During their evolution, animals have developed a set of cysteine-rich peptides capable of binding various extracellular sites of voltage-gated sodium channels (VGSC). Sea anemone toxins that target VGSCs delay their inactivation process, but little is known about their selectivities. Here we report the investigation of three native type 1 toxins (CGTX-II, δ-AITX-Bcg1a and δ-AITX-Bcg1b) purified from the venom of Bunodosoma cangicum. Both δ-AITX-Bcg1a and δ-AITX-Bcg1b toxins were fully sequenced. The three peptides were evaluated by patch-clamp technique among Nav1.1-1.7 isoforms expressed in mammalian cell lines, and their preferential targets are Na(v)1.5>1.6>1.1. We also evaluated the role of some supposedly critical residues in the toxins which would interact with the channels, and observed that some substitutions are not critical as expected. In addition, CGTX-II and δ-AITX-Bcg1a evoke different shifts in activation/inactivation Boltzmann curves in Nav1.1 and 1.6. Moreover, our results suggest that the interaction region between toxins and VGSCs is not restricted to the supposed site 3 (S3-S4 linker of domain IV), and this may be a consequence of distinct surface of contact of each peptide vs. targeted channel. Our data suggest that the contact surfaces of each peptide may be related to their surface charges, as CGTX-II is more positive than δ-AITX-Bcg1a and δ-AITX-Bcg1b.
... Based on structural differences and activity profile, these potassium channel toxins can be divided into 4 structural classes [11,[13][14][15]. Up to date 6 toxins from A. elegantissima have been isolated and characterized: APE1-1, APE1-2, APE2-2 and ApC which are type 1 sodium channel toxins; APETx1 a selective modifier of the human ether a go-go related gene K + channel (hERG) and APETx2 which specifically inhibits the Acid Sensing Ion Channel (ASIC3) [15][16][17][18]. In this work we present the purification, biochemical analysis and electrophysiological characterization of a very potent and selective K V 1.1 blocker which represents the newest member of the sea anemone type 2 potassium channel toxins. ...
Sea anemone venom is a known source of interesting bioactive compounds, including peptide toxins which are invaluable tools for studying structure and function of voltage-gated potassium channels. APEKTx1 is a novel peptide isolated from the sea anemone Anthopleura elegantissima, containing 63 amino acids cross-linked by 3 disulfide bridges. Sequence alignment reveals that APEKTx1 is a new member of the type 2 sea anemone peptides targeting voltage-gated potassium channels (K(V)s), which also include the kalicludines from Anemonia sulcata. Similar to the kalicludines, APEKTx1 shares structural homology with both the basic pancreatic trypsin inhibitor (BPTI), a very potent Kunitz-type protease inhibitor, and dendrotoxins which are powerful blockers of voltage-gated potassium channels. In this study, APEKTx1 has been subjected to a screening on a wide range of 23 ion channels expressed in Xenopus laevis oocytes: 13 cloned voltage-gated potassium channels (K(V)1.1-K(V)1.6, K(V)1.1 triple mutant, K(V)2.1, K(V)3.1, K(V)4.2, K(V)4.3, hERG, the insect channel Shaker IR), 2 cloned hyperpolarization-activated cyclic nucleotide-sensitive cation non-selective channels (HCN1 and HCN2) and 8 cloned voltage-gated sodium channels (Na(V)1.2-Na(V)1.8 and the insect channel DmNa(V)1). Our data show that APEKTx1 selectively blocks K(V)1.1 channels in a very potent manner with an IC(50) value of 0.9nM. Furthermore, we compared the trypsin inhibitory activity of this toxin with BPTI. APEKTx1 inhibits trypsin with a dissociation constant of 124nM. In conclusion, this study demonstrates that APEKTx1 has the unique feature to combine the dual functionality of a potent and selective blocker of K(V)1.1 channels with that of a competitive inhibitor of trypsin.
... In the light of the observed preference of CgNa for insect Na V channels, the substitution of the outermost C-terminally aromatic Phe residue in the IVS3–S4 loop into a Leu residue definitely deserves further attention in these mutagenesis studies. Another intriguing observation is that the TTX-R peripheral nervous system subtypes rNa V 1.8 (and rNa V 1.9) are in general quite resistant to site 3 toxins from sea anemones (Bosmans et al., 2002; Salceda et al., 2002 Salceda et al., , 2006), scorpions (Saab et al., 2002; Maertens et al., 2006), and spiders (Nicholson et al., 2004; Yamaji et al., 2009 ). This could, at least in part, be due to low conservation of important amino acid residues and longer size of the IVS3–S4 loop (seeFigure 4). ...
Because of their prominent role in electro-excitability, voltage-gated sodium (NaV) channels have become the foremost important target of animal toxins. These toxins have developed the ability to discriminate between closely related NaV subtypes, making them powerful tools to study NaV channel function and structure. CgNa is a 47-amino acid residue type I toxin isolated from the venom of the Giant Caribbean Sea Anemone Condylactis gigantea. Previous studies showed that this toxin slows the fast inactivation of tetrodotoxin-sensitive NaV currents in rat dorsal root ganglion neurons. To illuminate the underlying NaV subtype-selectivity pattern, we have assayed the effects of CgNa on a broad range of mammalian isoforms (NaV1.2–NaV1.8) expressed in Xenopus oocytes. This study demonstrates that CgNa selectively slows the fast inactivation of rNaV1.3/β1, mNaV1.6/β1 and, to a lesser extent, hNaV1.5/β1, while the other mammalian isoforms remain unaffected. Importantly, CgNa was also examined on the insect sodium channel DmNaV1/tipE, revealing a clear phyla-selectivity in the efficacious actions of the toxin. CgNa strongly inhibits the inactivation of the insect NaV channel, resulting in a dramatic increase in peak current amplitude and complete removal of fast and steady-state inactivation. Together with the previously determined solution structure, the subtype-selective effects revealed in this study make of CgNa an interesting pharmacological probe to investigate the functional role of specific NaV channel subtypes. Moreover, further structural studies could provide important information on the molecular mechanism of NaV channel inactivation.
... The same has been observed for ATX-II and LqTx (from the scorpion L. quinquestriatus) when applied on Na v 1.2 channels [35]. Similarly, the sea anemone toxins BgII, BgIII (both from Bunodosoma granulifera), ApC (from Anthopleura elegantissima) and CgNa (from C. gigantea) did not affect the current amplitude when applied to DRG neurons [37][38][39]. Therefore it is possible that some site-3 toxins may also produce some degree of channel blockade. ...
... In previous works [37][38][39] we have shown that other anemone toxins, when applied to DRG neurons, shifted the steady-state inactivation curve to hyperpolarizing values and caused a decrease in the voltage dependence of sodium channel inactivation by increasing the slope factor of the h 1 curve. This latter effect has been also observed with site-3 toxins from scorpions [14], and wasps [36]. ...
Sea anemone toxins bind to site 3 of the sodium channels, which is partially formed by the extracellular linker connecting S3 and S4 segments of domain IV, slowing down the inactivation process. In this work we have characterized the actions of BcIII, a sea anemone polypeptide toxin isolated from Bunodosoma caissarum, on neuronal sodium currents using the patch clamp technique. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study (n=65). The main effects of BcIII were a concentration-dependent increase in the sodium current inactivation time course (IC(50)=2.8 microM) as well as an increase in the current peak amplitude. BcIII did not modify the voltage at which 50% of the channels are activated or inactivated, nor the reversal potential of sodium current. BcIII shows a voltage-dependent action. A progressive acceleration of sodium current fast inactivation with longer conditioning pulses was observed, which was steeper as more depolarizing were the prepulses. The same was observed for other two anemone toxins (CgNa, from Condylactis gigantea and ATX-II, from Anemonia viridis). These results suggest that the binding affinity of sea anemone toxins may be reduced in a voltage-dependent manner, as has been described for alpha-scorpion toxins.
... Single dorsal root ganglion (DRG) neurons were isolated from 7-to 9-d-old BALB/c mice as described previously (Salceda et al., 2006) with slight modifications. In short, a dorsal laminectomy was performed and DRGs with the corresponding spinal roots were dissected out. ...
Presynaptic gamma-aminobutyric acid type B receptors (GABA(B)Rs) regulate transmitter release at many central synapses by inhibiting Ca(2+) channels. However, the mechanisms by which GABA(B)Rs modulate neurotransmission at descending terminals synapsing on motoneurons in the spinal cord remain unexplored. To address this issue, we characterized the effects of baclofen, an agonist of GABA(B)Rs, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of the dorsolateral funiculus (DLF) terminals in a slice preparation from the turtle spinal cord. We found that baclofen depressed neurotransmission in a dose-dependent manner (IC(50) of approximately 2 microM). The membrane time constant of the motoneurons did not change, whereas the amplitude ratio of the evoked EPSPs in response to a paired pulse was altered in the presence of the drug, suggesting a presynaptic mechanism. Likewise, the use of N- and P/Q-type Ca(2+) channel antagonists (omega-conotoxin GVIA and omega-agatoxin IVA, respectively) also depressed EPSPs significantly. Therefore, these channels are likely involved in the Ca(2+) influx that triggers transmitter release from DLF terminals. To determine whether the N and P/Q channels were regulated by GABA(B)R activation, we analyzed the action of the toxins in the presence of baclofen. Interestingly, baclofen occluded omega-conotoxin GVIA action by approximately 50% without affecting omega-agatoxin IVA inhibition, indicating that the N-type channels are the target of GABA(B)Rs. Lastly, the mechanism underlying this effect was further assessed by inhibiting G-proteins with N-ethylmaleimide (NEM). Our data show that EPSP depression caused by baclofen was prevented by NEM, suggesting that GABA(B)Rs inhibit N-type channels via G-protein activation.
... For electrophysiological recordings, cells were lifted off plates, reseeded on poly-L-lysine (0.05%)-precoated glass coverslips and used 2– 6 h after plating. Single dorsal root ganglion (DRG) neurons were isolated from 7-to 9-d-old BALB/c mice as described previously (Salceda et al., 2006) with slight modifications. In short, a dorsal laminectomy was performed and DRGs with the corresponding spinal roots were dissected out. ...
Auxiliary gamma subunits are an important component of high-voltage-activated calcium (Ca(V)) channels, but their precise regulatory role remains to be determined. In the current report, we have used complementary approaches including molecular biology and electrophysiology to investigate the influence of the gamma subunits on neuronal Ca(V) channel activity and expression. We found that coexpression of gamma2 or gamma3 subunits drastically inhibited macroscopic currents through recombinant N-type channels (Ca(V)2.2/beta3/alpha2delta) in HEK-293 cells. Using inhibitors of internalization, we found that removal of functional channels from the plasma membrane is an improbable mechanism of current regulation by gamma. Instead, changes in current amplitude could be attributed to two distinct mechanisms. First, gamma subunit expression altered the voltage dependence of channel activity. Second, gamma subunit expression reduced N-type channel synthesis via activation of the endoplasmic reticulum unfolded protein response. Together, our findings (1) corroborate that neuronal gamma subunits significantly downregulate Ca(V)2.2 channel activity, (2) uncover a role for the gamma2 subunit in Ca(V)2.2 channel expression through early components of the biosynthetic pathway, and (3) suggest that, under certain conditions, channel protein misfolding could be induced by interactions with the gamma subunits, supporting the notion that Ca(V) channels constitute a class of difficult-to-fold proteins.
... 41,42 Las neuronas provenientes de estos ganglios se cultivaron como se ha descrito previamente. 41,[43][44][45] Brevemente, los ganglios disecados se colocaron en medio de cultivo L-15 al 100% en presencia de tripsina y colagenasa IA (1.25 mg/ml) por 30 min a 37º C, tras lo cual las células fueron lavadas y disociadas mecánicamente. Una vez disociadas, se colocaron las células en cajas de Petri recubiertas con poli-L-lisina y se mantuvieron en cultivo de 8 a 20 horas en medio L-15 modificado con 10% de SBF a 37º C y con una atmósfera de CO 2 al 5%. ...
SUMMARY Background. Acid sensing ion channels (ASIC) are ionic channels activated by transient pH reductions in the extracellular environment. Although the activation mechanism is not fully elucidated, it is clear that the channel is activated by proton binding to its extracellular domain, a process that is modulated by calcium and zinc. Objective. The fact that divalent cations are able to modify ASIC operation, lead us to consider if lead, another divalent cation and widely distributed neurotoxicant, is also capable to affect ASIC function. Methods. For this purpose, we recorded ASIC currents in rat dorsal root ganglion neurons using the whole cell patch-clamp technique. Results. The results indicated that lead inhibits ASIC currents in a concentration-dependent fashion. Conclusions. These results contribute to the understanding of the activation mechanism of ASIC and to explain some of the toxic mechanisms of lead in the organism.
