Most biological scientists conduct experiments to look for effects, and test the results statistically. We have already described Student's t test which is very commonly used. However, this test concentrates on a very limited question. We assume that there is no effect in the experiment, and then estimate the possibility that we could have obtained these results. The question concerns what we may deduce, if the samples measured came from a single theoretical population of known characteristics. We calculate the probability that we might obtain the results we did, or more extreme results, on the basis of this premise. The statistical procedure is called frequentist because the results are expressed in terms of ‘how frequently would a result like this be observed, given the exact definition of the theoretical population that we proposed?’ (Fig. 1).
1. Parameters derived from frequency-domain analysis of heart period and blood pressure variability are gaining increasing importance in clinical practice. However, the underlying physiological mechanisms in human subjects are not fully understood. Here we address the question as to whether the low frequency variability (approximately 0.1 Hz) of the heart period may depend on a baroreflex-mediated response to blood pressure oscillations, induced by the alpha-sympathetic drive on the peripheral resistance. 2. Heart period (ECG), finger arterial pressure (Finapres) and respiratory airflow were recorded in eight healthy volunteers in the supine position with metronome respiration at 0.25 Hz. We inhibited the vascular response to the sympathetic vasomotor activity with a peripheral alpha-blocker (urapidil) and maintained mean blood pressure at control levels with angiotensin II. 3. We performed spectral and cross-spectral analysis of heart period (RR) and systolic pressure to quantify the power of low- and high-frequency oscillations, phase shift, coherence and transfer function gain. 4. In control conditions, spectral analysis yielded typical results. In the low-frequency range, cross-spectral analysis showed high coherence (> 0.5) and a negative phase shift (-65.1 +/- 18 deg) between RR and systolic pressure, which indicates a 1-2 s lag in heart period changes in relation to pressure. In the high-frequency region, the phase shift was close to zero, indicating simultaneous fluctuations of RR and systolic pressure. During urapidil + angiotensin II infusion the low-frequency oscillations of both blood pressure and heart period were abolished in five cases. In the remaining three cases they were substantially reduced and lost their typical cross-spectral characteristics. 5. We conclude that in supine rest conditions, the oscillation of RR at low frequency is almost entirely accounted for by a baroreflex mechanism, since it is not produced in the absence of a 0.1 Hz pressure oscillation. 6. The results provide physiological support for the use of non-invasive estimates of the closed-loop baroreflex gain from cross-spectral analysis of blood pressure and heart period variability in the 0.1 Hz range.
1. Charge movement was studied in the cut twitch fibres of Rana temporaria with a double Vaseline-gap voltage-clamp technique. 2. In fibres containing 0.1 mM EGTA, the I gamma hump component of charge movement was either unresolvable or had a brief duration at all potentials. In the steady-state charge-voltage (Q-V) plots that were separable into the less voltage dependent (Q beta) and more voltage dependent (Q gamma) components, the amount of Q gamma was 0.9 +/- 0.1 nC microF-1. (mean +/- S.E.M., n = 18). 3. Fibres containing 20 mM EGTA showed broad I gamma humps at about -45 mV. The addition of 1.8 mM total Ca2+ to the end-pools accelerated the rising phase and abbreviated the duration of the hump. 4. I gamma in fibres containing 0.1 mM EGTA resembled those in intact fibres when recorded at similar low temperatures. 5. These results explain the controversy concerning the variable appearance of I gamma humps in cut fibres.
Previous studies in rodents and sheep show that maternal nutrient restriction during pregnancy alters fetal renal development. To date, no studies using fetal baboon RNA with human Affymetrix gene chips have been published. In the present study we have (1) evaluated the specificity of the Affymetrix human gene array 'Laboratory on a Chip' system for use with fetal baboon mRNA and (2) investigated the effects of moderate maternal global nutrient restriction (NR; 70% of ad libitum animals) from early (30 days gestation (dG)) to mid-gestation (90 dG; term = 184 dG) on the fetal baboon kidney. Morphometric and blood measurements were made on 12 non-pregnant baboons before they were bred. All baboons were fed ad libitum until 30 days pregnant, at which time six control baboons continued to feed ad libitum (control - C) while six received 70% of the C diet on a weight adjusted basis. Fetal kidneys were collected following caesarean section at 90 dG, with samples flash frozen and fixed for histological assessment. Fetal hip circumference was decreased in the NR group (68 +/- 2 versus 75 +/- 2 mm), while fetal body weight and all other measurements of fetal size were not different between C and NR at 90 dG. Maternal body weight was decreased in the NR group (12.16 +/- 0.34 versus 13.73 +/- 0.55 kg). Having established the specificity of the Affymetrix system for fetal baboon mRNA, gene expression profiling of fetal kidneys in the context of our maternal nutrient restriction protocol shows that NR resulted in a down-regulation of genes in pathways related to RNA, DNA and protein biosynthesis, metabolism and catabolism. In contrast, genes in cell signal transduction, communication and transport pathways were up-regulated in the NR group. These changes indicate that even a moderate level of maternal global NR impacts fetal renal gene pathways. Our histological assessment of renal structure indicates decreased tubule density within the cortex of NR kidneys compared with controls. The number of glomerular cross-sections per unit area were unaffected by NR, suggesting that tubule tortuosity and/or tubule length was decreased in the NR kidney. Taken together the changes indicate that NR results in accelerated fetal renal differentiation. The negative impact of poor maternal nutrition on the fetal kidney may therefore be in part due to shortening of critical phases of renal growth resulting in decreased functional capacity in later life. These findings may have important implications for postnatal renal function, thereby contributing to the observed increased predisposition to hypertension and renal disease in the offspring of nutrient restricted mothers.
1. Electrophysiologically identified thalamocortical neurones have been intra- and extracellularly recorded in acutely prepared cats, under different anaesthetic conditions. 2. A slow (0.5-4 Hz) membrane potential oscillation was observed in thalamocortical cells recorded in motor, sensory, associational and intralaminar thalamic nuclei. The oscillation consisted of rhythmic low-threshold spikes alternating with after-hyperpolarizations. 3. About 80% of the neurones with intact cortical connections were set into the slow oscillatory mode by bringing their membrane potential to between -68 and -90 mV. The oscillation did not depend upon the occurrence of fast action potentials and did not outlast the imposed hyperpolarization. 4. Anatomical or functional disconnection from related cortical areas resulted in a membrane potential hyperpolarization of about 9 mV and in the occurrence of spontaneous slow oscillations in virtually all recorded neurones. The intrinsic nature of the phenomenon was supported by the lack of rhythmic postsynaptic potentials as the cells were prevented from oscillating by outward current injection. 5. In contrast with other thalamic nuclei, the slow oscillation has not been observed in anterior thalamic neurones despite their having similar basic electrophysiological properties. 6. Barbiturate administration suppressed the slow oscillatory mode, an effect accompanied by a decrease in the membrane input resistance. 7. Multiunit recordings of spontaneously oscillating cells showed epochs characterized by phase-related firing. This synchronous discharge was paralleled by a clear-cut build-up of field potentials in the frequency range of electroencephalogram slow or delta waves. 8. These results demonstrate that the majority of thalamocortical neurones are endowed with electrophysiological properties allowing them to oscillate at 0.5-4 Hz, if they have a membrane potential more negative than -65 mV and a high input resistance. Such a condition is physiologically achieved in the deepest stages of electroencephalogram-synchronized sleep, as a result of brain stem-thalamic as well as cortico-thalamic deafferentation. We postulate a thalamic contribution in the genesis of electroencephalogram delta waves during slow wave sleep, once independently oscillating thalamocortical cells become in phase.
Amino acid infusions increase renal blood flow (RBF) and glomerular filtration rate (GFR) and stimulate tubular reabsorption in adults. To characterize the effects of amino acids on fetal renal haemodynamics, tubular sodium reabsorption, acid-base homeostasis and plasma renin levels, 11 chronically catheterized fetal sheep aged 121 +/- 1 days (term ~150 days) were infused I.V. for 4 h with alanine, glycine, proline and serine (0.1, 0.1, 0.06 and 0.06 mmol min(-1), respectively) in 0.15 M saline at 0.165 ml min(-1). Eight control fetuses were given saline. During amino acid infusion, plasma amino acid levels increased up to 20-fold (P < 0.005). GFR increased by 50 +/- 8 % (P < 0.001); there was only a small transient increase in RBF. Proximal fractional sodium reabsorption fell from 74.6 +/- 2.9 to 55.5 +/- 5.4 % (P < 0.005). Distal sodium delivery increased, but a smaller percentage of this distal sodium load was reabsorbed (P < 0.005). Thus fractional sodium reabsorption fell from 95.5 +/- 0.9 to 81.4 +/- 2.0 % (P < 0.005). There was a large diuresis, natriuresis, kaliuresis and increase in osmolar excretion (P < 0.005). Plasma sodium and chloride concentrations fell (P < 0.005). Plasma osmolality did not change. Plasma renin levels fell (P < 0.05), cortisol levels increased (P < 0.05), and there was a compensated metabolic acidosis. Thus the fetal sheep kidney demonstrated a remarkable functional capacity to respond to amino acid infusion. The increase in filtration fraction and the lack of an increase in RBF suggest that efferent arteriolar vasoconstriction occurred, a very different response from the renal vasodilatation seen in adult animals.
1. The transduction mechanisms involved in the activation and modulation of the noradrenaline-activated cation current (Icat) were investigated with whole-cell patch clamp techniques in rabbit portal vein smooth muscle cells. 2. Intracellular application of guanosine 5-O-(3-thiotriphosphate) (GTP gamma S, 500 microM) evoked a 'noisy' inward current at -50 mV with a similar current-voltage relationship and reversal potential to the current evoked by bath application of noradrenaline (100 microM). Guanosine 5-O-(2-thiodiphosphate) (GDP beta S, 1 mM) markedly inhibited noradrenaline-activated Icat. 3. The phospholipase C (PLC) inhibitor U73122 inhibited the amplitude of the noradrenaline-activated Icat in a concentration- and time-dependent manner and the IC50 was about 180 nM. U73122 had similar effects on the cation current evoked by GTP gamma S. 4. Intracellular application of myo-inositol 1,4,5-trisphosphate (IP3, 100 microM) from the patch pipette did not activate any membrane current in cells where intracellular calcium concentration ([Ca2+]i) was buffered to 14 nM, but subsequent addition of noradrenaline evoked Icat. 5. Bath application of the 1,2-diacyl-sn-glycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG, 10 microM) activated Icat, whereas the phorbol ester phorbol 12,13-dibutyrate (PDBu, 0.1-5 microM) failed to activate Icat, in every cell examined. Icat activated by OAG after bath application of PDBu was not significantly different from OAG-activated Icat in the absence of PDBu. The DAG lipase inhibitor RHC80267 (10 microM) activated Icat in some cells, whereas the DAG kinase inhibitor R59949 (10 microM) never activated Icat. 6. Bath application of the protein kinase C inhibitor chelerythrine (1-10 microM) had no effect on either OAG-or noradrenaline-activated Icat. 7. It is concluded that noradrenaline activates Icat via a G-protein coupled to PLC and that the resulting DAG product plays a central role in the activation of cation channels via a protein kinase C-independent mechanism.
1. The validity of estimations of plasma fatty acid oxidation using tracers has often been questioned. The appearance of isotopic markers in breath CO2 is delayed and incomplete. Recently suggestions have been made that substantial amounts of tracer are incorporated into products of the tricarboxylic acid cycle (e.g. glucose, glutamine and glutamate) and that an acetate correction factor can be used to correct for tracer fixation. In the present study we investigated whether the appearance of 13CO2 during a separate infusion of [1,2-13C]acetate could be used for correction of [U-13C]palmitate oxidation rates in studies lasting <2 h and we quantified the appearance of tracer in the glutamine, glutamate and glucose pools of the body. 2. An infusion of either [1,2-13C]acetate (0.104 micromol min-1 kg-1) or [U-13C]palmitate (0.013 micromol min-1 kg-1) was given to eight male subjects and continued for 2 h at rest. In six subjects the infusion of [1,2-13C]acetate was repeated to determine reproducibility of the acetate recovery. 3. Fractional recovery in breath from [1,2-13C]acetate gradually increased during the infusion period at rest from 14.1 +/- 0.6% at 60 min to 26.5 +/- 0.5% at 120 min after the start of the infusion. Intersubject coefficient of variance was 8.3 +/- 0.6% and intrasubject coefficient of variance of the acetate recovery tests was 4.0 +/- 1.5%. After 2 h of [1,2-13C]acetate infusion, 12.4 +/- 0.8 and 10.3 +/- 0.9% of infused 13C was incorporated in the glutamine and glutamate pools, respectively. 4. In conclusion, the [1,2-13C]acetate recovery factor can be used for correcting the rate of [U-13C]palmitate oxidation in infusing studies of 2 h in resting conditions. Failure to use this recovery factor leads to a substantial underestimation of the rate of plasma free fatty acid oxidation. The extent of label fixation could largely be explained by accumulation of tracer carbon in glutamine and glutamate, and the accumulation in glucose is negligible.
1. The effect of inositol 1,3,4,5-tetrakisphosphate (InsP4) on long-term potentiation (LTP) was investigated in the CA1 region of rat hippocampal slices. Intracellular application of InsP4 and EPSP recordings were carried out using the whole-cell configuration. 2. Induction of LTP in the presence of InsP4 (100 microM) resulted in a substantial enhancement of the LTP magnitude compared with control potentiation. Using an intrapipette perfusion system, it was established that application of InsP4 was required during induction of potentiation for this enhancement to occur. An enhancement of LTP was not observed if a non-metabolizable inositol 1,4,5-trisphosphate (InsP3) analogue (2,3-dideoxy-1,4,5-trisphosphate, 100 microM) was applied intracellularly. 3. Current-voltage relations of NMDA receptor-mediated EPSCs were not altered by InsP4 application. The presence of InsP4 was slightly effective in relieving a D-(-)-2-amino-5-phosphonopentanoic acid (D-APV)-induced block of LTP. 4. The peak current amplitude of voltage-gated calcium channels (VGCCs) was increased by InsP4. omega-Conotoxin GVIA inhibited the InsP4-induced LTP facilitation. 5. These data indicate that InsP4 can modify the extracellular Ca2+ entry through upregulation of VGCCs, which may in turn contribute to the observed enhancement of LTP induced by InsP4. 6. To investigate the possible involvement of intracellular Ca2+ release in the facilitatory effect of InsP4 on LTP, different inhibitors of the endoplasmic reticulum-dependent Ca2+ release were applied (heparin, ryanodine, cyclopiazonic acid). The results suggest that InsP4 activates postsynaptic InsP3-dependent Ca2+ release which normally does not contribute to the calcium-induced calcium release-dependent LTP.
1. To examine the role of the phosphoinositide cascade triggered by disturbed Ca2+ homeostasis in ischaemic neurones, inositol 1,3,4,5-tetrakisphosphate (InsP4) was applied to the cytoplasmic face of membrane patches isolated from CA1 pyramidal neurones in the gerbil hippocampus. 2. In outside-out recordings, InsP4 induced an inward current which was increased by raising the extracellular [Ca2+]. In contrast, no clear channel openings could be observed in patches from neurones of sham-operated gerbils. 3. Open probabilities of InsP4-activated channels were significantly decreased upon application of omega-conotoxin but were not affected by omega-agatoxin or nifedipine. 4. In inside-out patches using high concentrations of Ca2+, Ba2+ or Sr2+ in the pipette solution, InsP4 enhanced inward currents. 5. Application of the isomers of InsP4 slightly enhanced the currents, but inositol 1,4,5-trisphosphate (InsP3) had no effect. 6. In the absence of InsP4 there was a single main Ba2+ current peak of 4.0 pA in amplitude, whereas upon its application two main peaks of 3.0 and 7.2 pA were present. 7. The open probabilities of these channels were apparently increased by InsP4. 8. These findings support the view that a disturbed phosphoinositide cascade occurs in the hippocampal pyramidal neurones after ischaemia and the InsP4 thus formed plays an important role in promoting the Ca2+ accumulation which results in neuronal death.
1. Intracellular calcium concentration ([Ca2+]i) and force were measured from isolated single mouse skeletal muscle fibres at rest and during tetani. The actions of 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump, were examined at a range of concentrations (100-1000 nM). 2. TBQ increased resting [Ca2+]i, increased tetanic [Ca2+]i and slowed the rate of decline of [Ca2+]i after a tetanus. TBQ produced a small increase in tetanic force and a large slowing of the rate of relaxation after a tetanus. All these effects were reversible. 3. TBQ had no important effects on the Ca2+ sensitivity or the maximum force produced by the myofibrillar proteins. 4. Analysis of the SR Ca2+ pump function confirmed that under control conditions and at very low levels of [Ca2+]i, the relationship between [Ca2+]i and SR pump rate was a 4th power function. TBQ caused a pronounced inhibition of the pump rate and reduced the power function to < 3. 5. Muscle fibres were fatigued by repeated tetani until tetanic [Ca2+]i and force were reduced and the rate of decline of [Ca2+]i after a tetanus was slowed. Under these conditions application of TBQ caused a further slowing of the rate of decline of [Ca2+]i but still increased tetanic [Ca2+]i and force. This result suggests that slowing of the SR pump rate is not the cause of the decline in tetanic [Ca2+]i and force at the late stage of fatigue. 6. A simple model of the interactions of Ca2+, TBQ and pump proteins is described, which predicts the 4th power function of the normal pump, inhibition by TBQ, and the reduced power function in the presence of TBQ. 7. A model of Ca2+ movements and force development in muscle is described, which closely matches the experimental results under control conditions. Inhibition of the SR pump by TBQ using the model of the pump described above simulates qualitatively all the observed effects of TBQ on [Ca2+]i and force. 8. In conclusion, TBQ is a potent, specific and reversible inhibitor of the SR Ca2+ pump in intact mouse skeletal muscle. Inhibition of the pump directly affects intracellular Ca2+ handling and force production.
Cardiac pacemaking initiated by the sinus node is attributable to the interplay of several membrane currents. These include the depolarizing "funny current" (If ) and the sodium-calcium exchanger current (INCX ). The latter is activated by ryanodine receptor (RyR)-mediated calcium (Ca(2+) ) release from the sarcoplasmic reticulum (SR). Another SR Ca(2+) release channel, the inositol-1,4,5-triphosphate receptor (IP3 R), has been implicated in the generation of spontaneous Ca(2+) release in atrial and ventricular cardiomyocytes. Whether IP3 R-mediated Ca(2+) release also influences SAN automaticity is controversial, in part due to the confounding influence of periodic Ca(2+) flux through the sarcolemma accompanying each beat. We took advantage of atrial-specific NCX knockout (KO) SAN cells to study the influence of IP3 signalling on cardiac pacemaking in a system where periodic intracellular Ca(2+) cycling persists despite the absence of depolarization or Ca(2+) flux across the sarcolemma. We recorded confocal line scans of spontaneous Ca(2+) release in WT and NCX KO SAN cells in the presence or absence of an IP3 R blocker (2-APB), or during block of IP3 production by the phospholipase C inhibitor U73122. 2-APB and U73122 decreased the frequency of spontaneous Ca(2+) transients and waves in WT and NCX KO cells respectively. Alternatively, increased IP3 production induced by phenylephrine increased Ca(2+) transient and wave frequency. We conclude that IP3 R-mediated SR Ca(2+) flux is crucial for initiating and modulating the RyR-mediated Ca(2+) cycling that regulates SAN pacemaking. Our results in NCX KO SAN cells also demonstrate that RyRs, but not NCX, are required for IP3 to modulate Ca(2+) clock frequency. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Various cardio-active stimuli, including endothelin-1 (ET-1), exhibit potent arrhythmogenicity, but the underlying cellular mechanisms of their actions are largely unclear. We used isolated rat atrial myocytes and related changes in their subcellular Ca(2+) signalling to the ability of various stimuli to induce diastolic, premature extra Ca(2+) transients (ECTs). For this, we recorded global and spatially resolved Ca(2+) signals in indo-1- and fluo-4-loaded atrial myocytes during electrical pacing. ET-1 exhibited a higher arrhythmogenicity (arrhythmogenic index; ratio of number of ECTs over fold-increase in Ca(2+) response, 8.60; n = 8 cells) when compared with concentrations of cardiac glycosides (arrhythmogenic index, 4.10; n = 8 cells) or the beta-adrenergic agonist isoproterenol (arrhythmogenic index, 0.11; n = 6 cells) that gave similar increases in the global Ca(2+) responses. Seventy-five percent of the ET-1-induced arrhythmogenic Ca(2+) transients were accompanied by premature action potentials, while for digoxin this proportion was 25 %. The beta-adrenergic agonist failed to elicit a significant number of ECTs. Direct activation of inositol 1,4,5-trisphosphate (InsP(3)) receptors with a membrane-permeable InsP(3) ester (InsP(3) BM) mimicked the effect of ET-1 (arrhythmogenic index, 14.70; n = 6 cells). Inhibition of InsP(3) receptors using 2 microM 2-aminoethoxydiphenyl borate, which did not display any effects on Ca(2+) signalling under control conditions, specifically suppressed the arrhythmogenic action of ET-1 and InsP(3) BM. Immunocytochemistry indicated a co-localisation of peripheral, junctional ryanodine receptors with InsP(3)Rs. Thus, the pronounced arrhythmogenic potency of ET-1 is due to the spatially specific recruitment of Ca(2+) sparks by subsarcolemmal InsP(3)Rs. Summation of such sparks efficiently generates delayed after depolarisations that trigger premature action potentials. We conclude that the particular spatial profile of cellular Ca(2+) signals is a major, previously unrecognised, determinant for arrhythmogenic potency and that the InsP(3) signalling cassette might therefore be a promising new target for understanding and managing atrial arrhythmia.
1. The cellular mechanism by which acetylcholine (ACh) potentiates neuronal responses to N-methyl-D-aspartate (NMDA) was investigated in CA1 neurones of hippocampal slices using current- and voltage-clamp techniques. 2. Loading cells with 5'-guanylylimidodiphosphate (GppNHp) caused a gradual increase in response to NMDA. Pulses of ACh accelerated this increase. Guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) blocked the potentiating effect of ACh on responses to NMDA. 3. Acute LiCl caused a gradual decrease in the potentiating effect of ACh, while the potentiation was completely prevented by 3 day chronic 6 mequiv/kg (I.P.) LiCl treatment and restored by acute treatment with 10 mM-inositol. 4. Loading cells with a general protein kinase inhibitor, H-7, enhanced the potentiating effect of ACh on responses to NMDA and blocked the effect of ACh on the after-hyperpolarization (AHP). 5. Ultraviolet irradiation of cells loaded with a photolabile inositol 1,4,5-trisphosphate (InsP3) caused a transient increase in responses to NMDA, while penetrating cells with active InsP3-containing pipettes caused a gradual BAPTA-sensitive increase in responses to NMDA. 6. Reducing the rate of InsP3 metabolism, with 2,3-diphosphoglyceric acid (DPG), caused an increase and prolongation of the potentiating effect of ACh, while blocking the InsP3 receptor with heparin prevented the cholinergic potentiation. 7. NMDA, by itself, potentiated subsequent responses to NMDA, an effect that was blocked when [Ca2+]i was chelated with BAPTA. NMDA and ACh were also found to compete in potentiating responses to NMDA. Finally, the cholinergic potentiation was blocked when cells were loaded with BAPTA. 8. We propose that activation of the InsP3 branch of the phosphoinositide pathway potentiated responses to NMDA and that InsP3 exerted this effect by elevating [Ca2+]i.
1. The effects of activation of guanine nucleotide-binding protein (G protein) by guanine nucleotides or sodium fluoride on the release of intracellular Ca2+ and on tension development were determined in chemically skinned strips of rabbit main pulmonary arteries (MPA). Ca2+ movements were monitored with Fura-2, as the change in free Ca2+ concentration in the bath medium surrounding the skinned MPA. 2. Sodium fluoride or non-hydrolysable analogues of GTP, guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) and guanosine 5'-[beta,gamma-imido]triphosphate (GMP-PNP), induced sustained and dose-dependent contraction of skinned MPA. GTP (100 microM) induced transient contraction of skinned MPA. GTP gamma S did not contract intact MPA. We also confirmed that inositol 1,4,5-trisphosphate (InsP3) released sufficient Ca2+ to induce contraction of skinned, but not intact, MPA. 3. Guanosine 5'-[beta-thio]diphosphate (GDP beta S), a non-hydrolysable analogue of GDP that competitively inhibits the binding of guanine nucleotides to G proteins, inhibited the contractions induced by GTP gamma S. Neomycin (1 mM) inhibited the GTP gamma S-induced contractions, but also, to a lesser extent, contractions induced by caffeine. 4. Depletion of Ca2+ from the sarcoplasmic reticulum (SR) or treatment with Triton X-100 inhibited the GTP gamma S-induced contractions. The effects of Ca2+ depletion was reversible, while that of Triton X-100 was irreversible. GTP gamma S (up to 100 microM) had no apparent effect on the pCa-tension curve of freeze-glycerinated MPA. 5. GTP gamma S- or InsP3-induced contractions occurred in the presence of 20 mM-procaine, while this agent completely blocked the contraction induced by caffeine. 6. Both GTP gamma S and InsP3 induced an increase in the Fura-2 fluorescence signal of the bath medium surrounding the skinned MPA, indicating that GTP gamma S releases intracellular Ca2+. The release of Ca2+ induced by GTP gamma S was inhibited by GDP beta S. 7. During the initial phasic contraction induced by GTP gamma S, added InsP3 had little or no additive effect, in contrast to its additive effect during the latter sustained contraction induced by GTP gamma S.(ABSTRACT TRUNCATED AT 400 WORDS)
1. The action of bradykinin (BK), inositol 1,4,5-trisphosphate (InsP3), and phorbol dibutyrate (PDBu) on the release of acetylcholine (ACh) was studied electrophysiologically on short-distance (less than 20 micron) synapses formed between cultured NG108-15 mouse neuroblastoma x rat glioma hybrid cells and rat muscle cells. Action potentials in NG108-15 cells did not usually evoke an excitatory junction potential (EJP) in the muscle cell in this system. 2. Ionophoretic application of BK onto the somatic surface of an NG108-15 cell produced an increase in frequency of miniature end-plate potentials (MEPPs) for 40-50s in the paired myotube. Some MEPPs were evoked during BK-induced hyperpolarization (10-20 s) of the hybrid cell soma. A few MEPPs were also elicited during BK-induced depolarization. 3. Ionophoretic injection of Ca2+ into an NG108-15 cell soma generated MEPPs for a very brief period (less than 3 s), coincident with somatic hyperpolarization. No increase was observed during a subsequent somatic depolarization induced by a larger current of Ca2+. 4. Ionophoretic injection of InsP3 into the cytoplasm of an NG108-15 cell soma transiently evoked MEPPs during the InsP3-induced hyperpolarizing phase. A large InsP3 injection caused sustained generation of MEPPs for 2-4 min, associated with InsP3-evoked depolarization. 5. Within 3-5 min after exposure of NG108-15-myotube pairs to 1 microM-PDBu, the MEPP frequency increased by 2-5 times and reached a plateau after 8 min. The increase continued after wash-out of the drug. The PDBu-induced increase of MEPPs was still observed when the membrane potential of the NG108-15 cell was clamped at -30 mV. 6. The data suggest that the BK-induced facilitation results from the action of two intracellular second messengers: an InsP3-dependent release of Ca2+ from the intracellular storage sites and protein phosphorylation by diacyclglycerol (DAG)-activated protein kinase C.
Phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP(3)R) by PKA represents an important, common route for regulation of Ca(2+) release. Following phosphorylation of the S2 splice variant of InsP(3)R-1 (S2-InsP-1), Ca(2+) release is markedly potentiated. In this study we utilize the plasma membrane (PM) expression of InsP(3)R-1 and phosphorylation state mutant InsP(3)R-1 to study how this regulation occurs at the single InsP(3)R-1 channel level. DT40-3KO cells stably expressing rat S2- InsP(3)R-1 were generated and studied in the whole-cell mode of the patch clamp technique. At hyperpolarized holding potentials, small numbers of unitary currents (average approximately 1.7 per cell) were observed which were dependent on InsP(3) and the presence of functional InsP(3)R-1, and regulated by both cytoplasmic Ca(2+) and ATP. Raising cAMP markedly enhanced the open probability (P(o)) of the InsP(3)R-1 and induced bursting activity, characterized by extended periods of rapid channel openings and subsequent prolonged refractory periods. The activity, as measured by the P(o) of the channel, of a non-phosphorylatable InsP(3)R-1 construct (Ser1589Ala/Ser1755Ala InsP(3)R-1) was markedly less than wild-type (WT) InsP(3)R-1 and right shifted some approximately 15-fold when the concentration dependency was compared to a phosphomimetic construct (Ser1589Glu/Ser1755Glu InsP(3)R-1). No change in conductance of the channel was observed. This shift in apparent InsP(3) sensitivity occurred without a change in InsP(3) binding or Ca(2+) dependency of activation or inactivation. Biophysical analysis indicated that channel activity can be described by three states: an open state, a long lived closed state which manifests itself as long interburst intervals, and a short-lived closed state. Bursting activity occurs as the channel shuttles rapidly between the open and short-lived closed state. The predominant effect of InsP(3)R-1 phosphorylation is to increase the likelihood of extended bursting activity and thus markedly augment Ca(2+) release. These analyses provide insight into the mechanism responsible for augmenting InsP(3)R-1 channel activity following phosphorylation and moreover should be generally useful for further detailed investigation of the biophysical properties of InsP(3)R.
We investigated synergism between inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) and diacylglycerol (DAG) on TRPC6-like channel activity in rabbit portal vein myocytes using single channel recording and immunoprecipitation techniques. Ins(1,4,5)P(3) at 10 microm increased 3-fold TRPC6-like activity induced by 10 microm 1-oleoyl-2-acetyl-sn-glycerol (OAG), a DAG analogue. Ins(1,4,5)P(3) had no effect on OAG-induced TRPC6 activity in mesenteric artery myocytes. Anti-TRPC6 and anti-TRPC7 antibodies blocked channel activity in portal vein but only anti-TRPC6 inhibited activity in mesenteric artery. TRPC6 and TRPC7 proteins strongly associated in portal vein but only weakly associated in mesenteric artery tissue lysates. Therefore in portal vein the conductance consists of TRPC6/C7 subunits, while OAG activates a homomeric TRPC6 channel in mesenteric artery myocytes. Wortmannin at 20 microm reduced phosphatidylinositol 4,5-bisphosphate (PIP(2)) association with TRPC6 and TRPC7, and produced a 40-fold increase in OAG-induced TRPC6/C7 activity. Anti-PIP(2) antibodies evoked TRPC6/C7 activity, which was blocked by U73122, a phospholipase C inhibitor. DiC8-PIP(2), a water-soluble PIP(2) analogue, inhibited OAG-induced TRPC6/C7 activity with an IC(50) of 0.74 microm. Ins(1,4,5)P(3) rescued OAG-induced TRPC6/C7 activity from inhibition by diC8-PIP(2) in portal vein myocytes, and this was not prevented by the Ins(1,4,5)P(3) receptor antagonist heparin. In contrast, Ins(1,4,5)P(3) did not overcome diC8-PIP(2)-induced inhibition of TRPC6 activity in mesenteric artery myocytes. 2,3,6-Tri-O-butyryl-Ins(1,4,5)P(3)/AM (6-Ins(1,4,5)P(3)), a cell-permeant analogue of Ins(1,4,5)P(3), at 10 microm increased TRPC6/C7 activity in portal vein and reduced association between TRPC7 and PIP(2), but not TRPC6 and PIP(2). In contrast, 10 microm OAG reduced association between TRPC6 and PIP(2), but not between TRPC7 and PIP(2). The present work provides the first evidence that Ins(1,4,5)P(3) modulates native TRPC channel activity through removal of the inhibitory action of PIP(2) from TRPC7 subunits.
We tested the hypothesis that vasomotor control is differentially regulated between feed arteries and downstream arterioles from the cremaster muscle of C57BL/6 mice. In isolated pressurized arteries, confocal Ca(2+) imaging of smooth muscle cells (SMCs) revealed Ca(2+) sparks and Ca(2+) waves. Ryanodine receptor (RyR) antagonists (ryanodine and tetracaine) inhibited both sparks and waves but increased global Ca(2+) and myogenic tone. In arterioles, SMCs exhibited only Ca(2+) waves that were insensitive to ryanodine or tetracaine. Pharmacological interventions indicated that RyRs are functionally coupled to large-conductance, Ca(2+)-activated K(+) channels (BK(Ca)) in SMCs of arteries, whereas BK(Ca) appear functionally coupled to voltage-gated Ca2+ channels in SMCs of arterioles. Inositol 1,4,5-trisphosphate receptor (IP3R) antagonists (xestospongin D or 2-aminoethoxydiphenyl borate) or a phospholipase C inhibitor (U73122) attenuated Ca(2+) waves, global Ca(2+) and myogenic tone in arteries and arterioles but had no effect on arterial sparks. Real-time PCR of isolated SMCs revealed RyR2 as the most abundant isoform transcript; arteries expressed twice the RyR2 but only 65% the RyR3 of arterioles and neither vessel expressed RyR1. Immunofluorescent localisation of RyR protein indicated bright, clustered staining of arterial SMCs in contrast to diffuse staining in arteriolar SMCs. Expression of IP(3)R transcripts and protein immunofluorescence were similar in SMCs of both vessels with IP(3)R1>IP(3)R2>IP(3)R3. Despite similar expression of IP(3)Rs and dependence of Ca(2+) waves on IP(3)Rs, these data illustrate pronounced regional heterogeneity in function and expression of RyRs between SMCs of the same vascular resistance network. We conclude that vasomotor control is differentially regulated in feed arteries vs. downstream arterioles.
Spontaneous, rhythmical contractions, or vasomotion, can be recorded from cerebral vessels under both normal physiological and pathophysiological conditions. Using electrophysiology to study changes in membrane potential, the ratiometric calcium indicator Fura-2 AM to study changes in [Ca(2+)](i) in both the arterial wall and in individual smooth muscle cells (SMCs), and video microscopy to study changes in vessel diameter, we have investigated the cellular mechanisms underlying vasomotion in the juvenile rat basilar artery. During vasomotion, rhythmical oscillations in both membrane potential and [Ca(2+)](i) were found to precede rhythmical contractions. Nifedipine depolarized SMCs and abolished rhythmical contractions and depolarizations. [Ca(2+)](i) oscillations in the arterial wall became reduced and irregular, while [Ca(2+)](i) oscillations in adjacent SMCs were no longer synchronized. BAPTA-AM, thapsigargin and U73122 hyperpolarized SMCs, relaxed the vessel, decreased basal calcium levels and abolished vasomotion. Chloride substitution abolished rhythmical activity, depolarized SMCs, increased basal calcium levels and constricted the vessel, while niflumic acid and DIDS abolished vasomotion. Ryanodine, charybdotoxin and TRAM-34, but not iberiotoxin, 4-aminopyridine or apamin, each depolarized SMCs and increased the frequency of rhythmical depolarizations and [Ca(2+)](i) oscillations. We conclude that vasomotion in the basilar artery depends on the release of intracellular calcium from IP(3) (inositol 1,4,5,-trisphosphate)-sensitive stores which activates calcium-dependent chloride channels to depolarize SMCs. Depolarization in turn activates voltage-dependent calcium channels, synchronizing contractions of adjacent cells through influx of extracellular calcium. Subsequent calcium-induced calcium release from ryanodine-sensitive stores activates an intermediate conductance potassium channel, hyperpolarizing the SMCs and providing a negative feedback pathway for regeneration of the contractile cycle.
1. Inositol 1,4,5-trisphosphate (InsP3) injected into unfertilized golden hamster eggs elicits a hyperpolarizing response (HR) that is due to stimulation of calcium-activated potassium channels in the egg plasma membrane. 2. A single injection of InsP3 gave a single HR above a threshold value of 0.3 nM. At 5 nM and above, InsP3 induced HRs with no detectable latency. At concentrations between these two values a latency was observed. The amplitude of the HR was independent of InsP3 concentration. 3. A second HR could be elicited by injection of InsP3, but five times more InsP3 was required to trigger a second HR, and 10-100 times more to give an HR of similar magnitude to the first, and there was no latency. 4. The increase in [Ca2+]i in response to an initial injection of 1 nM InsP3 could be resolved into two distinct components: a slow, early rise immediately after InsP3 injection (phase I) followed by a larger and more rapid increase (phase II). The initiation of an HR coincided with the second component of the [Ca2+]i increase. 5. Further injection of InsP3 resulted only in slow, smaller increases in [Ca2+]i that resembled phase I and often did not cause an HR. Phase II appeared to be absent. However, 100-fold greater InsP3 concentrations gave slow, larger Ca2+ transients (and HRs) with no detectable latency. 6. If large amounts of InsP3 were allowed to leak into the eggs constantly from a pipette, repetitive calcium transients were seen. Unlike the sustained repetitive responses seen at fertilization, they were often smaller than the initial transient and less well sustained. However, a subsequent transient could still be elicited on injection of very large concentrations of InsP3. 7. InsP3 can induce regenerative, all-or-none [Ca2+]i increase (CICR) in hamster eggs, often with a long latency, as well as non-regenerative increases. InsP3 injections desensitize CICR and cannot mimic all the features of Ca2+ signalling at fertilization in the hamster egg, in particular, the sensitization of CICR caused by the sperm.
1. Vascular smooth muscle cells were isolated from the coronary artery of the guinea-pig. At 2.5 mM [Ca2+]o and 36 degrees C, whole cell membrane currents were recorded under voltage-clamp and the concentration of ionized calcium in the cytoplasm ([Ca2+]i) was monitored by indo-1 fluorescence. 2. At -60 mV, [Ca2+]i was 143 +/- 36 mM (mean +/- S.D.) and was insensitive to clamp steps to +100 mV. During 1 min application of acetylcholine (ACh, 10 microM) [Ca2+]i increased within approximately 2 s to 1480 +/- 250 nM. During the subsequent slow decay, [Ca2+]i was transiently increased by depolarizing clamp steps and decreased during hyperpolarizing steps. [Ca2+]i transients in response to caffeine (10 mM) could not be modulated by voltage steps. The results suggest that modulation of [Ca2+]i by membrane potential involves inositol 1,4,5-trisphosphate (Ins(1,4,5)P3)-induced Ca2+ release (IICR). 3. Modulation of IICR by membrane potential did not depend on sarcolemmal Ca2+ fluxes; it persisted after block of sarcolemmal Ca2+ fluxes with 3 mM lanthanum or after a change to nominally Ca(2+)-free bathing solutions. 4. Modulation of [Ca2+]i by membrane potential was recorded during cell dialysis of 50 microM GTP-gamma-S in the absence of ACh. Cell dialysis of exogenous Ins(1,4,5)P3 (50 or 100 microM) did not mimic the effects. The sensitivity of [Ca2+]i to depolarizing clamp steps was also induced by cell dialysis of lithium ions which, presumably, inhibited the breakdown of Ins(1,4,5)P3. The results are compatible with the idea that the membrane potential modulates the liberation of Ins(1,4,5)P3. 5. Modulation of IICR by membrane potential is discussed as a new mechanism that contributes to the regulation of activator calcium and to the modulation of contraction in vascular smooth muscle cells.
1. Effects of membrane hyperpolarization induced by pinacidil on Ca2+ mobilization induced by noradrenaline (NA) were investigated by measuring intracellular Ca2+ concentration ([Ca2+]i), isometric tension, membrane potential and production of inositol 1,4,5-trisphosphate (IP3) in smooth muscle cells of the rabbit mesenteric artery. 2. Pinacidil (0.1-10 microM) concentration dependently hyperpolarized the smooth muscle membrane with a reduction in membrane resistance. Glibenclamide (1 microM) blocked the membrane hyperpolarization induced by 1 microM-pinacidil. NA (10 microM) depolarized the smooth muscle membrane with associated oscillations. Pinacidil (1 microM) inhibited this response and glibenclamide (1 microM) prevented the action of pinacidil on both the NA-induced events. 3. In thin smooth muscle strips, 10 microM-NA produced a large phasic and a subsequent small tonic increase in [Ca2+]i with associated oscillations. These changes in [Ca2+]i seemed to be coincident with phasic, tonic and oscillatory contractions, respectively. Pinacidil (0.1-1 microM) inhibited the increases in [Ca2+]i and in tension induced by NA, but not by 128 mM-K+. Glibenclamide inhibited these actions of pinacidil. Pinacidil (1 microM) also inhibited the contraction induced by 10 microM-NA in strips treated with A23187 (which functionally removes cellular Ca2+ storage sites), suggesting that membrane hyperpolarization inhibits Ca2+ influxes activated by NA. 4. In Ca2(+)-free solution containing 2 mM-EGTA, NA (10 microM) transiently increased [Ca2+]i, tension and synthesis of IP3. Pinacidil (over 0.1 microM) inhibited the increases in [Ca2+]i, tension and synthesis of IP3 induced by 10 microM-NA in Ca2(+)-free solution containing 5.9 mM-K+, but not in a similar solution containing 40 or 128 mM-K+. Glibenclamide (1 microM) inhibited these actions of pinacidil. These inhibitory actions of pinacidil were still observed in solutions containing low Na+ or low Cl-. These results suggest that pinacidil inhibits NA-induced Ca2+ release from storage sites through an inhibition of IP3 synthesis resulting from its membrane hyperpolarizing action. 5. In beta-escin-treated skinned strips, NA (10 microM) or IP3 (20 microM) increased Ca2+ in Ca2(+)-free solution containing 50 microM-EGTA and 3 microM-guanosine triphosphate (GTP) after brief application of 0.3 microM-Ca2+, suggesting Ca2+ is released from intracellular storage sites. Heparin (500 micrograms/ml, an inhibitor of the IP3 receptor), but not pinacidil (1 microM) or glibenclamide (1 microM), inhibited the Ca2+ release from storage sites induced by NA or IP3. These results suggest that membrane hyperpolarization is essential for the inhibitory action of pinacidil on the NA-induced Ca2(+)-releasing mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)
In rabbit portal vein smooth muscle cells, store-operated Ca2+-permeable cation channels (SOCs) display multi-modal gating mechanisms. SOCs are activated by depletion of intracellular Ca2+ stores but also may be stimulated in a store-independent manner by noradrenaline acting on alpha-adrenoceptors and by diacylglycerol (DAG) via protein kinase C (PKC). In the present study we have investigated whether inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) modulates SOC activity in freshly dispersed rabbit portal vein myocytes with patch pipette recording techniques. Inclusion of 1 mum Ins(1,4,5)P3 in the patch pipette solution increased whole-cell currents evoked by the Ca2+-ATPase inhibitor cyclopiazonic acid (CPA) by about 3-fold at -80 mV. In the cell-attached configuration the cell-permeable Ca2+ chelator BAPTA-AM stimulated SOC activity and after excision of an isolated inside-out patch bath application of 1 mum Ins(1,4,5)P3 increased open channel probability (NP(o)) by approximately 3-fold. Ins(1,4,5)P3 also produced a similar increase in NP(o) of SOCs stimulated by the phorbol ester, phorbol 12,13-dibutyrate (PDBu) in inside-out patches and these channel currents had a unitary conductance of about 2 pS. The equilibrium constant of Ins(1,4,5)P3 on increasing PDBu-evoked SOC activity was about 0.4 mum. The facilitatory effect of Ins(1,4,5)P3 was also manifest as markedly increasing the rate of activation of SOCs. The synergistic effect of Ins(1,4,5)P3 was mimicked by the metabolically stable analogue 3-fluoro-Ins(1,4,5)P3 and Ins(1,4)P2, a metabolite of Ins(1,4,5)P3, but was not inhibited by the classical Ins(1,4,5)P3 receptor antagonist heparin. Finally Ins(1,4,5)P3 also increased NP(o) of SOCs activated by a PKC catalytic subunit. It is concluded that Ins(1,4,5)P3 facilitates SOC opening via a heparin-insensitive mechanism at, or close to, the channel protein.
1. The role of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (DAG) as possible mediators of the membrane current responses of NG108-15 neuroblastoma x glioma hybrid cells to bradykinin (BK, Brown & Higashida, 1988b) has been tested using intracellular ionophoresis of InsP3 and external application of phorbol dibutyrate (PDBu) and 1-oleoyl-2-acetylglycerol (OAG). 2. Intracellular ionophoresis of InsP3 into cells clamped at -30 to -50 mV produced (i) a transient outward current, (ii) a transient outward current followed by an inward current, or (iii) an inward current. All currents were accompanied by an increased input conductance. 3. The transient outward current reversed at between -80 and -90 mV. The reversal potential was shifted to more positive potentials on raising extracellular [K+], suggesting that it resulted from an increased K+ conductance. 4. The outward current was inhibited by apamin (0.4 microM) or d-tubocurarine (0.2-0.5 mM); these drugs also inhibit the outward current produced by BK or by intracellular Ca2+ injections (Brown & Higashida, 1988 a, b). The outward current was also slowly reduced in 0 mM [Ca2+] or 0.5 mM [Cd2+] plus 2 mM [Co2+] solution. 5. Ionophoretic injection of inositol 1,3,4-trisphosphate and inositol 1,3,4,5-tetrakisphosphate, guanosine trisphosphate or inorganic phosphate did not evoke an outward current but produced only an inward current with an increased conductance, reversing at between -10 and -20 mV. 6. Bath application of PDBu (10 nM-1 microM) or OAG (1-10 microM) produced an inward current with a fall in input conductance. The inward current was voltage dependent and was accompanied by an inhibition of the time-dependent current relaxations associated with activation or deactivation of the voltage-dependent K+ current, IM. 7. PDBu did not clearly reduce the Ca2+ current or the Ca2+-dependent K+ current recorded in these cells. During superfusion with PDBu, the outward current produced by intracellular ionophoresis of InsP3 was greatly enhanced. 8. The results support the view that the two membrane current responses to BK might both result from accelerated membrane phosphatidylinositide hydrolysis. One product, InsP3, releases Ca2+ and activates an apamin-curare-sensitive outward K+ current; this effect is imitated by intracellular InsP3 ionophoresis. The second product, DAG; activates protein kinase C to inhibit the voltage-dependent K+ current IM and generate an inward current; this effect is imitated by external application of PDBu or OAG.
1. The membrane response to acetylcholine (ACh), inositol 1,4,5-trisphosphate (IP3) and intracellular Ca2+ was studied in Xenopus laevis oocytes under voltage-clamp conditions. 2. Shallow, submembranal injections of IP3 in the animal hemisphere of the oocyte evoked a two-component response comprised of a rapid, transient component followed by a slow, sustained component. 3. When the injection pipette was inserted further into the cell (to 300 microns below the cell membrane), the fast component diminished and the slow component remained unchanged or even increased. 4. The rapid component exhibited an apparent higher sensitivity to IP3 compared to the slow component. 5. The two components of the IP3 response were retained in a Ca2+-free environment. 6. Injection of a single large dose (20-50 pmol) of CaCl2 into the oocyte evoked a typical two-component response, whereas repetitive threshold doses (0.1 pmol CaCl2) elicited large current fluctuations which developed into a small depolarization current. 7. The delay in the peak of the slow component of the response to either IP3 or to CaCl2 injections appeared too long to be accounted for by diffusion alone. 8. Depletion of oocyte Ca2+ by the divalent cation ionophore A23187 (greater than 1 microM) inhibited the response to ACh and IP3. Low concentrations of A23187 selectively inhibited the rapid component of the ACh response, though not the rapid component of the IP3 response. 9. Our data suggest that the two-component membrane response to ACh in Xenopus oocytes can be accounted for by ACh-induced elevation of IP3 and subsequent IP3-induced release of intracellular Ca2+.
We studied inositol-1,4,5-trisphosphate (IP(3)) receptor-dependent intracellular Ca(2+) waves in CA1 hippocampal and layer V medial prefrontal cortical pyramidal neurons using whole-cell patch-clamp recordings and Ca(2+) fluorescence imaging. We observed that Ca(2+) waves propagate in a saltatory manner through dendritic regions where increases in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) were large and fast ('hot spots') separated by regions where increases in [Ca(2+)](i) were comparatively small and slow ('cold spots'). We also observed that Ca(2+) waves typically initiate in hot spots and terminate in cold spots, and that most hot spots, but few cold spots, are located at dendritic branch points. Using immunohistochemistry, we found that IP(3) receptors (IP(3)Rs) are distributed in clusters along pyramidal neuron dendrites and that the distribution of inter-cluster distances is nearly identical to the distribution of inter-hot spot distances. These findings support the hypothesis that the dendritic locations of Ca(2+) wave hot spots in general, and branch points in particular, are specially equipped for regenerative IP(3)R-dependent internal Ca(2+) release. Functionally, the observation that IP(3)R-dependent [Ca(2+)](i) rises are greater at branch points raises the possibility that this novel Ca(2+) signal may be important for the regulation of Ca(2+)-dependent processes in these locations. Futhermore, the observation that Ca(2+) waves tend to fail between hot spots raises the possibility that influences on Ca(2+) wave propagation may determine the degree of functional association between distinct Ca(2+)-sensitive dendritic domains.
1. The Ca2+ release in response to inositol 1,4,5-trisphosphate (InsP3) was studied in single patch-clamped smooth muscle cells of rat portal vein. InsP3 was photochemically produced from a caged InsP3 precursor included in the pipette solution. Changes in internal Ca2+ concentration ([Ca2+]i) were monitored by measuring Ca(2+)-activated K+ current. 2. Photoreleased InsP3 evoked a transient K+ current which was abolished when 10 mM EGTA or 5 mg ml-1 heparin was included in the pipette. The amplitude and time course of the K+ current responses depended on the light-flash intensity. The amplitude increased, and the latency and the time to peak decreased, with increasing flash intensity, suggesting that the amount of released Ca2+ varied as a function of the amount of InsP3 photoreleased. 3. The K+ current response to photolysis of caged InsP3 was abolished in the presence of 10 mM caffeine; conversely, caffeine was inefficient at inducing at K+ current when applied immediately after a light flash of maximal intensity. 4. The time course of the recovery of the K+ response evoked by a light flash of supramaximal intensity was similar to that obtained for the 10 mM caffeine-induced K+ current. The response recovered to 50% of control with an interval (t1/2) of about 10 s between pulses. The time course of the recovery of submaximal response to photoreleased InsP3 was considerably slower (t1/2 = 1 min), and did not correspond to that obtained for a response of similar amplitude evoked by 2 mM caffeine. 5. Responses to photoreleased InsP3 obtained after the cells were bathed for 3 min in Ca(2+)-free solution were compared with those obtained in 2 mM Ca2+ solution. Responses to light flashes of submaximal intensity were proportionally more inhibited than those evoked by supramaximal stimulations. 6. In portal vein smooth muscle cells, the InsP3-sensitive Ca2+ store seems also to be sensitive to caffeine. Our results suggest that the InsP3-induced Ca2+ release was modulated by regulatory mechanisms.
Embryonic stem cell-derived cardiomyocytes (ESdCs) have been proposed as a source for cardiac cell-replacement therapy. The aim of this study was to determine the Ca2+-handling mechanisms that determine the frequency and duration of spontaneous Ca2+ transients in single ESdCs. With laser scanning confocal microscopy using the Ca2+-sensitive dye Fluo-4/AM, we determined that spontaneous Ca2+ transients in ESdCs at the onset of beating (day 9) depend on Ca2+ entry across the plasma membrane (50%) whereas Ca2+-induced Ca2+ release is the major contributor to Ca2+ transients in ESdCs after 16 days (72%). Likewise, Ca2+ extrusion in 9-day-old ESdCs depends on Na+-Ca2+ exchange (50.0+/-8%) whereas Ca2+ reuptake by the sarco(endo)plasmic Ca2+ ATPase (72+/-5%) dominates in further differentiated cells. Spontaneous Ca2+ transients were suppressed by the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) blocker 2-aminoethoxydiphenyl borate (2-APB) and the phospholipase C blocker U73122 but continued in the presence of caffeine. Stimulation of IP3 production by phenylephrine or endothelin-1 had a positive chronotropic effect that could be reversed by U73122 and 2-APB. The presence of Ca2+-free solution and block of L-type Ca2+ channels by nifedipine also resulted in a cessation of spontaneous activity. Overall, IP3R-mediated Ca2+ release in ESdCs is translated into a depolarization of the plasma membrane and a whole-cell Ca2+ transient is subsequently induced by voltage-dependent Ca2+ influx. Although ryanodine receptor-mediated Ca2+ release amplifies the IP3R-induced trigger for the Ca2+ transients and modulates its frequencies, it is not a prerequisite for spontaneous activity. The results of this study offer important insight into the role of IP3R-mediated Ca2+ release for pacemaker activity in differentiating cardiomyocytes.
1. The dependence on pH of inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release was studied in saponin-skinned smooth muscle cells from guinea-pig portal vein, using the indicator fura-2 to monitor Ca2+ release. 2. Increasing pH between 6.7 and 7.3 enhanced the rate of IP3-induced Ca2+ release at all the Ca2+ concentrations above 30 nM without changing the bell-shaped dependence of the Ca2+ release rate on Ca2+ concentration with a peak near 300 nM. 3. The ascending limb of the biphasic Ca2+ dependence was shifted slightly toward the lower Ca2+ concentration at pH 7.3, suggesting an increase in the Ca2+ sensitivity of IP3-induced Ca2+ release at the higher pH. 4. With the elevation in pH from 6.7 to 7.3 at 100 nM Ca2+, about 7-fold higher IP3 concentration was required to release half of the Ca2+ in the store within 15 s. This pH-dependent change in the IP3 sensitivity was smaller at 1 microM Ca2+ and was indiscernible in the absence of Ca2+. 5. These results suggest that H+ may inhibit binding of IP3 and Ca2+ to the modulator sites of the Ca2+ release mechanism. However, these effects on the binding sites may not fully explain the complex effect of pH, and there may be pH-dependent step(s) involved in the gating mechanism of IP3 channels. The present study demonstrates the importance of pH as a modulator of IP3-induced Ca2+ release.
1. Single smooth muscle cells were isolated by enzymic digestion from the longitudinal muscle layer of rabbit jejunum, and the response of the cells to calcium (Ca2+) release by InsP3 (D-myo-inositol 1,4,5-trisphosphate) was studied. Changes in internal Ca2+ concentration were monitored by measuring Ca(2+)-activated K+ currents (outward currents) using the whole-cell voltage-clamp technique. 2. At break-through from cell-attached patch to whole-cell recording mode using a 100 microM-InsP3-filled pipette, cells exhibited a brief outward current which reached its peak in 1.1 s and terminated within 10 s. Following this the generation of spontaneous transient outward currents (STOCs) was inhibited. (STOCs are considered to represent bursts of openings of Ca(2+)-activated K+ channels in response to spontaneous discharges of Ca2+ from the stores.) When a pipette filled with 20 microM-InsP3 was used, similar current responses were also evoked, but some cells failed to respond. 3. The InsP3-induced outward current at membrane break-through was similar in size and time course to the outward current response of normal cells to bath-applied carbachol (CCh, 100 microM) or caffeine (20 mM). 4. Dialysis with InsP3-containing solution inhibited the caffeine-induced outward current, depending on the pipette InsP3 concentration. Inclusion of heparin (5 mg/ml) in the pipette completely prevented inhibition by InsP3 of the caffeine response and of STOC discharge. However, the InsP3-induced current at break-through remained unchanged, probably because of the slower rate of diffusion of heparin. 5. In cells dialysed with pipette solution containing 30 or 100 microM-caged InsP3, flash photolysis (producing up to 1.5 microM-InsP3) induced an outward current response after a latency of 31.0 +/- 1.8 ms (n = 15), which was followed by inhibition of STOCs. The reversal potential of the current to flash-release of InsP3 followed closely the Nernst potential for K+ ions (EK), suggesting negligible contributions from channels other than Ca(2+)-activated K+ channels. 6. Photolysis of caged InsP3 (30 or 100 microM) still produced a current response after 3-6 min in Ca(2+)-free (3 mM-EGTA added) bathing solution, but no response occurred if the cell was exposed to either caffeine (20 mM) or CCh (100 microM) to deplete Ca stores.(ABSTRACT TRUNCATED AT 400 WORDS)
To clarify the nature of the noradrenaline (NA)-induced contraction, the effects of NA on inositol phospholipid metabolism and the actions of inositol 1,4,5-trisphosphate (InsP3) on skinned muscle of the rabbit mesenteric artery were investigated. NA, in concentrations over 1 nM, reduced the amount of phosphatidylinositol 4,5-bisphosphate (PI-P2) and increased the amount of phosphatidic acid (PA). The maximum reduction in the amount of PI-P2 and the maximum increase in the amount of PA were observed in the presence of 1 microM-NA. With prolonged application of NA, the PI-P2 was gradually restored to near the control level, but with repeated applications of NA separated by rinses with Krebs solution, there was a consistent reduction of PI-P2. The NA-induced PI-P2 breakdown was inhibited by the alpha 1-adrenoceptor blocking agent, prazosin. After incubation of the tissue with radioactive inositol-containing solution, NA transiently increased the amount of radioactive InsP3 which was followed by increases in the amount of inositol 1,4-bisphosphate (InsP2) and inositol monophosphate (InsP). After accumulation of Ca by saponin-treated muscle cells of the dog mesenteric artery dispersed by collagenase, InsP3 released Ca stored in cells but InsP2 did not. A23187 (5 microM) but not InsP3 (up to 10 microM), depleted Ca accumulated in the presence of ATP. In saponin-treated skinned muscle tissues, InsP3 in concentrations over 0.3 microM, produced contraction following accumulation of Ca into the store site. InsP3 released Ca from the same source as caffeine. The release of Ca by InsP3 appeared in a concentration-dependent manner and this release also depended on the amount of Ca stored in cells (the median effective dose (ED50) was 3.0 microM in 0.1 microM-Ca and 1.0 microM in 0.3 microM-Ca). We concluded that NA activates alpha 1-adrenoceptors, thus hydrolysing PI-P2 and synthesizing InsP3. This product can release Ca stored in cells as estimated from the contraction in skinned muscle tissues, and also reduces the residual amount of Ca stored in skinned dispersed muscle cells. Contraction evoked by NA through pharmacomechanical coupling can be explained as a function of InsP3.
Ca2+ influx via Ca(V)1/Ca(V)2 channels drives processes ranging from neurotransmission to muscle contraction. Association of a pore-forming alpha(1) and cytosolic beta is necessary for trafficking Ca(V)1/Ca(V)2 channels to the cell surface through poorly understood mechanisms. A prevalent idea suggests beta binds the alpha(1) intracellular I-II loop, masking an endoplasmic reticulum (ER) retention signal as the dominant mechanism for Ca(V)1/Ca(V)2 channel membrane trafficking. There are hints that other alpha(1) subunit cytoplasmic domains may play a significant role, but the nature of their potential contribution is unclear. We assessed the roles of all intracellular domains of Ca(V)1.2-alpha(1C) by generating chimeras featuring substitutions of all possible permutations of intracellular loops/termini of alpha(1C) into the beta-independent Ca(V)3.1-alpha(1G) channel. Surprisingly, functional analyses demonstrated alpha(1C) I-II loop strongly increases channel surface density while other cytoplasmic domains had a competing opposing effect. Alanine-scanning mutagenesis identified an acidic-residue putative ER export motif responsible for the I-II loop-mediated increase in channel surface density. beta-dependent increase in current arose as an emergent property requiring four alpha(1C) intracellular domains, with the I-II loop and C-terminus being essential. The results suggest beta binding to the alpha(1C) I-II loop causes a C-terminus-dependent rearrangement of intracellular domains, shifting a balance of power between export signals on the I-II loop and retention signals elsewhere.
1. The blocking action of 4-aminopyridine (4-AP) on a delayed rectifier Kv1.2 K+ channel expressed in oocytes was investigated at room temperature (22 degrees C) and physiological temperature (34 degrees C) using the double-electrode voltage clamp and patch clamp techniques. 2. At room temperature, 4-AP (100 microM) inhibition occurred only after activation of current. The rate of onset of block was dependent upon the length of time current was activated by a depolarizing step. Similarly, removal of block required current activation. The degree of steady-state block by 4-AP was not reduced by increasingly more depolarized step potentials. The degree of steady-state block also did not change over the duration of a 1 s step. 3. When channels were nearly fully inactivated, 4-AP produced no additional block of a subsequent depolarizing step, suggesting that 4-AP did not bind when channels were in the inactivated state. In single channel experiments, 4-AP decreased the mean open time in a dose-dependent manner but did not alter the single-channel current amplitude. 4. At 34 degrees C the I-V relationship and inactivation curve shifted to more negative potentials. Increasing the temperature to 34 degrees C did not alter the degree of block by 4-AP, although the rate of onset of block was greatly enhanced. 5. Results suggest that 4-AP binds to the open state of the Kv1.2 channel and is trapped when the channel closes. 4-AP cannot bind when the channel is closed or inactivated prior to the addition of the drug. C-type inactivation and 4-AP binding to the channel are mutually exclusive. A model for the proposed mechanism of action of 4-AP on the Kv1.2 channel is proposed based on experimental data.
We examined changes in ionic and gating currents in Ca(V)1.2 channels when extracellular Ca(2+) was reduced from 10 mm to 0.1 microm. Saturating gating currents decreased by two-thirds (K(D) approximately 40 microm) and ionic currents increased 5-fold (K(D) approximately 0.5 microm) due to increasing Na(+) conductance. A biphasic time dependence for the activation of ionic currents was observed at low [Ca(2+)], which appeared to reflect the rapid activation of channels that were not blocked by Ca(2+) and a slower reversal of Ca(2+) blockade of the remaining channels. Removal of Ca(2+) following inactivation of Ca(2+) currents showed that Na(+) currents were not affected by Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation also induced a negative shift of the reversal potential for ionic currents suggesting that inactivation alters channel selectivity. Our findings suggest that activation of Ca(2+) conductance and Ca(2+)-dependent inactivation depend on extracellular Ca(2+) and are linked to changes in selectivity.
Cav1.3 channels mediate Ca2+ influx that triggers exocytosis of glutamate at cochlear inner hair cell (IHC) synapses. Harmonin is a PDZ-domain containing protein that interacts with the C-terminus of the Cav1.3 α1 subunit (α1 1.3) and controls cell-surface Cav1.3 levels by promoting ubiquitin-dependent proteosomal degradation. However, PDZ-domain containing proteins have diverse functions and regulate other Cav1.3 properties, which could collectively influence presynaptic transmitter release. Here, we report that harmonin binding to the α1 1.3 distal C-terminus (dCT) enhances voltage-dependent facilitation (VDF) of Cav1.3 currents both in transfected HEK293T cells and in mouse inner hair cells. In HEK293T cells, this effect of harmonin was greater for Cav1.3 channels containing the auxiliary Cav β1 than with the β2 auxiliary subunit. Cav1.3 channels lacking the α11.3 dCT were insensitive to harmonin modulation. Moreover, the "deaf-circler" dfcr mutant form of harmonin, which does not interact with the α11.3 dCT, did not promote VDF. In mature IHCs from mice expressing the dfcr harmonin mutant, Cav1.3 VDF was less than in control IHCs. This difference was not observed between control and dfcr IHCs prior to hearing onset. Membrane capacitance recordings from dfcr IHCs revealed a role for harmonin in synchronous exocytosis and in increasing the efficiency of Ca2+ influx for triggering exocytosis. Collectively, our results indicate a multifaceted presynaptic role of harmonin in IHCs in regulating Cav1.3 Ca2+ channels and exocytosis.
1. Using the two-microelectrode, 'cut open' oocyte, and 'torn off' macropatch voltage clamp techniques, we studied the blocking effects of 4-aminopyridine (4-AP) on two cloned K+ channels expressed in Xenopus oocytes, an inactivating K+ channel isolated from ferret ventricle (FK1), and its NH2-terminal deletion mutant (delta NCO) which lacks fast N-type inactivation. 2. Experiments with a permanently charged, impermeant 4-AP derivative, 4-aminopyridine-methyliodide, indicated that the cationic form of 4-AP blocks at an intracellular site. 3. Block accumulated from pulse to pulse and was sensitive to the applied potential during hyperpolarizing deactivating pulses, indicating trapping of 4-AP in deactivated channels. For long trains of depolarizing pulses (-90 to +50 mV, 0.1 Hz), 4-AP block increased with decreasing pulse duration. Block of FK1 was much more sensitive to pulse duration than was block of delta NCO, consistent with competition between N-type inactivation and 4-AP binding. 4. To elucidate these mechanisms further, in the absence of fast N-type inactivation the following results were obtained on delta NCO channels: (1) application of 4-AP caused the appearance of apparent inactivation; (2) 4-AP, however, did not cause cross-over of deactivating tail currents; (3) 4-AP block developed with time for potentials positive to -40 mV; and (4) trapping of 4-AP by delta NCO was insensitive to the degree of C-type inactivation. 5. We conclude that the kinetics of 4-AP block of FK1 and delta NCO channels cannot be accounted for by either a pure open channel or closed channel blocking scheme.
The inhibition by cocaine of the human heart Na+ channel (Na(v)1.5) heterologously expressed in Xenopus oocytes was investigated. Cocaine produced little tonic block of the resting channels but induced a characteristic, use-dependent inhibition during rapid, repetitive stimulation, suggesting that the drug preferentially binds to the open or inactivated states of the channel. To investigate further the state dependence, depolarizing pulses were used to inactivate the channels and promote cocaine binding. Cocaine produced a slow, concentration-dependent inhibition of inactivated channels, which had an apparent K(D) of 3.4 microM. Mutations of the interdomain III-IV linker that remove fast inactivation selectively abolished this high-affinity component of cocaine inhibition, which appeared to be linked to the fast inactivation of the channels. A rapid component of cocaine inhibition persisted in the inactivation-deficient mutant that was enhanced by depolarization and was sensitive to changes in the concentration of external Na+, properties that are consistent with a pore-blocking mechanism. Cocaine induced a use-dependent inhibition of the non-inactivating mutant and delayed the repriming at hyperpolarized voltages, indicating that the drug slowly dissociated when the channels were closed. Mutation of a conserved aromatic residue (Y1767) of the D4S6 segment weakened both the inactivation-dependent and the pore-blocking components of the cocaine inhibition. The data indicate that cocaine binds to a common site located within the internal vestibule and inhibits cardiac Na+ channels by blocking the pore and by stabilizing the channels in an inactivated state.
Mutations in the TTX-sensitive voltage-gated sodium channel subtype Nav1.7 have been implicated in the painful inherited neuropathy, hereditary erythromelalgia. Hereditary erythromelalgia can be difficult to treat and, although sodium channels are targeted by local anaesthetics such as lidocaine (lignocaine), some patients do not respond to treatment with local anaesthetics. This study examined electrophysiological differences in Nav1.7 caused by a hereditary erythromelalgia mutation (N395K) that lies within the local anaesthetic binding site of the channel. The N395K mutation produced a hyperpolarized voltage dependence of activation, slower kinetics of deactivation, and impaired steady-state slow inactivation. Computer simulations indicate that the shift in activation is the major determinant of the hyperexcitability induced by erythromelalgia mutations in sensory neurons, but that changes in slow inactivation can modulate the overall impact on excitability. This study also investigated lidocaine inhibition of the Nav1.7-N395K channel. We show that the N395K mutation attenuates the inhibitory effects of lidocaine on both resting and inactivated Nav1.7. The IC50 for lidocaine was estimated at 500 microM for inactivated wild-type Nav1.7 and 2.8 mM for inactivated Nav1.7-N395K. The N395K mutation also significantly reduced use-dependent inhibition of lidocaine on Nav1.7 current. In contrast, a different hereditary erythromelalgia mutation (F216S), not located in the local anaesthetic binding site, had no effect on lidocaine inhibition of Nav1.7 current. Our observation of reduced lidocaine inhibition on Nav1.7-N395K shows that the residue N395 is critical for lidocaine binding to Nav1.7 and suggests that the response of individuals with hereditary erythromelalgia to lidocaine treatment may be determined, at least in part, by their specific genotype.
The TTX-sensitive Na(v)1.7 (PN1) Na(+) channel alpha subunit protein is expressed mainly in small dorsal root ganglion (DRG) neurones. This study examines immunocytochemically whether it is expressed exclusively or preferentially in nociceptive primary afferent DRG neurones, and determines the electrophysiological properties of neurones that express it. Intracellular somatic action potentials (APs) evoked by dorsal root stimulation were recorded in L6/S1 DRG neurones at 30 +/- 2 degrees C in vivo in deeply anaesthetised young guinea-pigs. Each neurone was classified, from its dorsal root conduction velocity (CV) as a C-, Adelta- or Aalpha/beta-fibre unit and from its response to mechanical and thermal stimuli, as a nociceptive, low threshold mechanoreceptive (LTM) or unresponsive unit. Fluorescent dye was injected into the soma and Na(v)1.7-like immunoreactivity (Na(v)1.7-LI) was examined on sections of dye-injected neurones. All C-, 90 % of Adelta- and 40 % of Aalpha/beta-fibre units, including both nociceptive and LTM units, showed Na(v)1.7-LI. Positive units included 1/1 C-LTM, 6/6 C-nociceptive, 4/4 C-unresponsive (possible silent nociceptive) units, 5/6 Adelta-LTM (D hair), 13/14 Adelta-nociceptive, 2/9 Aalpha/beta-nociceptive, 10/18 Aalpha/beta-LTM cutaneous and 0/9 Aalpha/beta-muscle spindle afferent units. Overall, a higher proportion of nociceptive than of LTM neurones was positive, and the median relative staining intensity was greater in nociceptive than LTM units. Na(v)1.7-LI intensity was clearly positively correlated with AP duration and (less strongly) negatively correlated with CV and soma size. Since nociceptive units tend overall to have longer duration APs, slower CVs and smaller somata, these correlations may be related to the generally greater expression of Na(v)1.7 in nociceptive units.
We have examined the distribution of the sensory neuron-specific Na+ channel Nav1.8 (SNS/PN3) in nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons and whether its distribution is related to neuronal membrane properties. Nav1.8-like immunoreactivity (Nav1.8-LI) was examined with an affinity purified polyclonal antiserum (SNS11) in rat DRG neurons that were classified according to sensory receptive properties and by conduction velocity (CV) as C-, Adelta- or Aalpha/beta. A significantly higher proportion of nociceptive than low threshold mechanoreceptive (LTM) neurons showed Nav1.8-LI, and nociceptive neurons had significantly more intense immunoreactivity in their somata than LTM neurons. Results showed that 89, 93 and 60% of C-, Adelta- and Aalpha/beta-fibre nociceptive units respectively and 88% of C-unresponsive units were positive. C-unresponsive units had electrical membrane properties similar to C-nociceptors and were considered to be nociceptive-type neurons. Weak positive Nav1.8-LI was also present in some LTM units including a C LTM, all Adelta LTM units (D hair), about 10% of cutaneous LTM Aalpha/beta-units, but no muscle spindle afferent units. Nav1.8-LI intensity was negatively correlated with soma size (all neurons) and with dorsal root CVs in A- but not C-fibre neurons. Nav1.8-LI intensity was positively correlated with action potential (AP) duration (both rise and fall time) in A-fibre neurons and with AP rise time only in positive C-fibre neurons. It was also positively correlated with AP overshoot in positive neurons. Thus high levels of Nav1.8 protein may contribute to the longer AP durations (especially in A-fibre neurons) and larger AP overshoots that are typical of nociceptors.
Tetrodotoxin-resistant (TTX-R) sodium currents have been proposed to underlie sensory neuronal hyperexcitability in acute inflammatory models, but their role in chronic models is unknown. Since no pharmacological tools to separate TTX-R currents are available, this study employs Na(v)1.8 and Na(v)1.9 null mice to evaluate these currents roles in a chronic hyperexcitability model after the resolution of an inflammatory insult. Transient jejunitis was induced by infection with Nippostrongylus brasiliensis (Nb) in Na(v)1.9 and Na(v)1.8 null, wild-type and naïve mice. Retrogradely labelled dorsal root ganglia (DRG) neurons were harvested on day 20-24 post-infection for patch clamp recording. Rheobase and action potential (AP) parameters were recorded as measures of excitability, and Na(v)1.9 and Na(v)1.8 currents were recorded. DRG neuronal excitability was significantly increased in post-infected mice compared to sham animals, despite the absence of ongoing inflammation (sham = 1.9 +/- 0.3, infected = 3.6 +/- 0.7 APs at 2x rheobase, P = 0.02). Hyperexcitability was associated with a significantly increased amplitude of TTX-R currents. Hyperexcitability was maintained in Na(v)1.9(-/-) mice, but hyperexcitability was absent and APs were blunted in Na(v)1.8(-/-) mice. This study identifies a critical role for Na(v)1.8 in chronic post-infectious visceral hyperexcitability, with no contribution from Na(v)1.9. Nb infection-induced hyperexcitability is not observed in Na(v)1.8(-/-) mice, but is still present in Na(v)1.9(-/-) mice. It is not clear whether hyperexcitability is due to a change in the function of Na(v)1.8 channels or a change in the number of Na(v)1.8 channels.
Persistent tetrodotoxin-resistant (TTX-r) sodium currents up-regulated by intracellular GTP have been invoked as the site of action of peripheral inflammatory mediators that lower pain thresholds, and ascribed to the Na(V)1.9 sodium channel. Here we describe the properties of a global knock-out of Na(V)1.9 produced by replacing exons 4 and 5 in SCN11A with a neomycin resistance cassette, deleting the domain 1 voltage sensor and introducing a frameshift mutation. Recordings from small (< 25 microm apparent diameter) sensory neurones indicated that channel loss eliminates a TTX-r persistent current. Intracellular dialysis of GTP-gamma-S did not cause an up-regulation of persistent Na(+) current in Na(V)1.9-null neurones and the concomitant negative shift in voltage-threshold seen in wild-type and heterozygous neurones. Heterologous hNa(V)1.9 expression in Na(V)1.9 knock-out sensory neurones confirms that the human clone can restore the persistent Na(+) current. Taken together, these findings demonstrate that Na(V)1.9 underlies the G-protein pathway-regulated TTX-r persistent Na(+) current in small diameter sensory neurones that may drive spontaneous discharge in nociceptive nerve fibres during inflammation.
Potassium-selective ion channels regulate cardiac and neuronal excitability by stabilizing the resting membrane potential and by modulating shape and frequency of action potentials. The delicate control of membrane voltage requires structural and functional diversity of K+ channel subunits expressed in a given cell. Here we reveal a previously unrecognized biological mechanism. Tissue-specific mRNA splicing regulates alternative translation initiation (ATI) of human K(2P)10.1 K+ background channels via recombination of 5' nucleotide motifs. ATI-dependent expression of full-length protein or truncated subunits initiated from two downstream start codons determines macroscopic current amplitudes and biophysical properties of hK(2P)10.1 channels. The interaction between hK(2P)10.1 mRNA splicing, translation and function increases K+ channel complexity and is expected to contribute to electrophysiological plasticity of excitable cells.