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Voltage-clamp Experiments (voltage-clamp + experiment)
Selected AbstractsExcitatory actions of substance P in the rat lateral posterior nucleusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2010Kush Paul Abstract The lateral posterior nucleus (LP) receives inputs from both neocortex and superior colliculus (SC), and is involved with integration and processing of higher-level visual information. Relay neurons in LP contain tachykinin receptors and are innervated by substance P (SP)-containing SC neurons and by layer V neurons of the visual cortex. In this study, we investigated the actions of SP on LP relay neurons using whole-cell recording techniques. SP produced a graded depolarizing response in LP neurons along the rostro-caudal extent of the lateral subdivision of LP nuclei (LPl), with a significantly larger response in rostral LPl neurons compared with caudal LPl neurons. In rostral LPl, SP (5,2000 nm) depolarized nearly all relay neurons tested (> 98%) in a concentration-dependent manner. Voltage-clamp experiments revealed that SP produced an inward current associated with a decreased conductance. The inward current was mediated primarily by neurokinin receptor (NK)1 tachykinin receptors, although significantly smaller inward currents were produced by specific NK2 and NK3 receptor agonists. The selective NK1 receptor antagonist RP67580 attenuated the SP-mediated response by 71.5% and was significantly larger than the attenuation of the SP response obtained by NK2 and NK3 receptor antagonists, GR159897 and SB222200, respectively. The SP-mediated response showed voltage characteristics consistent with a K+ conductance, and was attenuated by Cs+, a K+ channel blocker. Our data suggest that SP may modulate visual information that is being processed and integrated in the LPl with inputs from collicular sources. [source] Apical SK potassium channels and Ca2+ -dependent anion secretion in endometrial epithelial cellsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Melissa L. Palmer Apical uridine triphosphate (UTP) stimulation was shown to increase short circuit current (Isc) in immortalized porcine endometrial gland epithelial monolayers. Pretreatment with the bee venom toxin apamin enhanced this response. Voltage-clamp experiments using amphotericin B-permeablized monolayers revealed that the apamin-sensitive current increased immediately after UTP stimulation and was K+ dependent. The current,voltage relationship was slightly inwardly rectifying with a reversal potential of ,52 ± 2 mV, and the PK/PNa ratio was 14, indicating high selectivity for K+. Concentration,response relationships for apamin and dequalinium had IC50 values of 0.5 nm and 1.8 ,m, respectively, consistent with data previously reported for SK3 channels in excitable cells and hepatocytes. Treatment of monolayers with 50 ,m BAPTA-AM completely blocked the effects of UTP on K+ channel activation, indicating that the apamin-sensitive current was also Ca2+ dependent. Moreover, channel activation was blocked by calmidazolium (IC50= 5 ,m), suggesting a role for calmodulin in Ca2+ -dependent regulation of channel activity. RT-PCR experiments demonstrated expression of mRNA for the SK1 and SK3 channels, but not SK2 channels. Treatment of monolayers with 20 nm oestradiol-17, produced a 2-fold increase in SK3 mRNA, a 2-fold decrease in SK1 mRNA, but no change in GAPDH mRNA expression. This result correlated with a 2.5-fold increase in apamin-sensitive K+ channel activity in the apical membrane. We speculate that SK channels provide a mechanism for rapidly sensing changes in intracellular Ca2+ near the apical membrane, evoking immediate hyperpolarization necessary for increasing the driving force for anion efflux following P2Y receptor activation. [source] Evidence for a Single Nucleotide Polymorphism in the KCNQ1 Potassium Channel that Underlies Susceptibility to Life-Threatening ArrhythmiasJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 11 2001TOMOYUKI KUBOTA M.D. Ion Channel Polymorphism and Cardiac Arrhythmia. Introduction: Congenital long QT syndrome (LQTS) is a genetically heterogeneous arrhythmogenic disorder caused by mutations in at least five different genes encoding cardiac ion channels. It was suggested recently that common polymorphisms of LQTS-associated genes might modify arrhythmia susceptibility in potential gene carriers. Methods and Results: We examined the known LQTS genes in 95 patients with definitive or suspected LQTS. Exon-specific polymerase chain reaction single-strand conformation polymorphism and direct sequence analyses identified six patients who carried only a single nucleotide polymorphism in KCNQ1 that is found in , 11% of the Japanese population. This 1727G> A substitution that changes the sense of its coding sequence from glycine to serine at position 643 (G643S) was mostly associated with a milder phenotype, often precipitated by hypokalemia and bradyarrhythmias. When heterologously examined by voltage-clamp experiments, the in vitro cellular phenotype caused by the single nucleotide polymorphism revealed that G643S- KCNQ1 forms functional homomultimeric channels, producing a significantly smaller current than that of the wild-type (WT) channels. Coexpression of WT- KCNQ1 and G643S- KCNQ1 with KCNE1 resulted in , 30% reduction in the slow delayed rectifier K+ current IKs without much alteration in the kinetic properties except its deactivation process, suggesting that the G643S substitution had a weaker dominant-negative effect on the heteromultimeric channel complexes. Conclusion: We demonstrate that a common polymorphism in the KCNQ1 potassium channel could be a molecular basis for mild IKs dysfunction that, in the presence of appropriate precipitating factors, might predispose potential gene carriers to life-threatening arrhythmias in a specific population. [source] Ethanol potentiates the function of the human dopamine transporter expressed in Xenopus oocytesJOURNAL OF NEUROCHEMISTRY, Issue 5 2001R. Dayne Mayfield Ethanol alters a variety of properties of brain dopaminergic neurons including firing rate, synthesis, release, and metabolism. Recent studies suggest that ethanol's action on central dopamine systems may also involve modulation of dopamine transporter (DAT) activity. The human DAT was expressed in Xenopus oocytes to examine directly the effects of ethanol on transporter function. [3H]Dopamine (100 nm) accumulation into DAT-expressing oocytes increased significantly in response to ethanol (10 min; 10,100 mm). In two-electrode voltage-clamp experiments, DAT-mediated currents were also enhanced significantly by ethanol (10,100 mm). The magnitude of the ethanol-induced potentiation of DAT function depended on ethanol exposure time and substrate concentration. Cell surface DAT binding ([3H]WIN 35,428; 4 nm) also increased as a function of ethanol exposure time. Thus, the increase in dopamine uptake was associated with a parallel increase in the number of DAT molecules expressed at the cell surface. These experiments demonstrate that DAT-mediated substrate translocation and substrate-associated ionic conductances are sensitive to intoxicating concentrations of ethanol and suggest that DAT may represent an important site of action for ethanol's effects on central dopaminergic transmission. A potential mechanism by which ethanol acts to enhance DAT function may involve regulation of DAT expression on the cell surface. [source] Phosphate uptake in Chara: membrane transport via Na/Pi cotransportPLANT CELL & ENVIRONMENT, Issue 2 2000R. J. Reid ABSTRACT Phosphate uptake in the freshwater charophyte plant Chara corallina was found to be strongly dependent on the presence of Na in the external medium. Based on the reciprocal stimulations of 32Pi uptake by Na and 22Na uptake by Pi, the logical mechanism for Pi uptake appears to be a nNa/Pi symport with a half-maximal stimulation (Km) for Na of approximately 300 ,M and a Km for Pi of approximately 10 ,M. Comparison of the stimulations of 32Pi and 22Na influxes at pH 6 gives a stoichiometry of Na : Pi of 5·68. The reduction in Pi influx with increasing pH is consistent with the transported species being the monovalent H2PO4,. In voltage-clamp experiments, currents elicited by Pi in the presence of Na were equivalent to an influx of positive charge which exceeded the measured influxes of 32P by a factor of 6·26. Intracellular perfusion was used to examine the dependence of Pi influx on ATP and Na. In perfused cells, Pi influx was low when ATP was absent from the internal medium or Na was absent from the external medium. Addition of ATP alone had little effect whereas addition of Na alone increased the 32Pi influx slightly. Addition of both ATP and Na together restored Pi influx to rates comparable to those of intact cells. It is suggested that the ATP is required for membrane hyperpolarization which in turn drives the highly electrogenic flux of Pi with up to 6 Na. However, consideration of the electrochemical potential differences for Na and Pi at pH less than 6 shows that nNa/Pi would not be feasible. It is suggested that at low pH, H+ may substitute for Na. [source] Aromatic residues at position 55 of rat ,7 nicotinic acetylcholine receptors are critical for maintaining rapid desensitizationTHE JOURNAL OF PHYSIOLOGY, Issue 4 2008Elaine A. Gay The rat ,7 nicotinic acetylcholine receptor (nAChR) can undergo rapid onset of desensitization; however, the mechanisms of desensitization are largely unknown. The contribution of a tryptophan (W) residue at position 55 of the rat ,7 nAChR subunit, which lies within the ,2 strand, was studied by mutating it to other hydrophobic and/or aromatic amino acids, followed by voltage-clamp experiments in Xenopus oocytes. When mutated to alanine, the ,7-W55A nAChR desensitized more slowly, and recovered from desensitization more rapidly, than wildtype ,7 nAChRs. The contribution of desensitization was validated by kinetic modelling. Mutating W55 to other aromatic residues (phenylalanine or tyrosine) had no significant effect on the kinetics of desensitization, whereas mutation to various hydrophobic residues (alanine, cysteine or valine) significantly decreased the rate of onset and increased the rate of recovery from desensitization. To gain insight into possible structural rearrangements during desensitization, we probed the accessibility of W55 by mutating W55 to cysteine (,7-W55C) and testing the ability of various sulfhydryl reagents to react with this cysteine. Several positively charged sulfhydryl reagents blocked ACh-induced responses for ,7-W55C nAChRs, whereas a neutral sulfhydryl reagent potentiated responses; residue C55 was not accessible for modification in the desensitized state. These data suggest that W55 plays an important role in both the onset and recovery from desensitization in the rat ,7 nAChR, and that aromatic residues at position 55 are critical for maintaining rapid desensitization. Furthermore, these data suggest that W55 may be a potential target for modulatory agents operating via hydrophobic interactions. [source] Functional role of cyclic nucleotide-gated channels in rat medial vestibular nucleus neuronsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Maria Vittoria Podda Although cyclic nucleotide-gated (CNG) channels are expressed in numerous brain areas, little information is available on their functions in CNS neurons. The aim of the present study was to define the distribution of CNG channels in the rat medial vestibular nucleus (MVN) and their possible involvement in regulating MVN neuron (MVNn) excitability. The majority of MVNn expressed both CNG1 and CNG2 A subunits. In whole-cell current-clamp experiments carried out on brainstem slices containing the MVNn, the membrane-permeant analogues of cyclic nucleotides, 8-Br-cGMP and 8-Br-cAMP (1 mm), induced membrane depolarizations (8.9 ± 0.8 and 9.2 ± 1.0 mV, respectively) that were protein kinase independent. The cGMP-induced depolarization was associated with a significant decrease in the membrane input resistance. The effects of cGMP on membrane potential were almost completely abolished by the CNG channel blockers, Cd2+ and l - cis -diltiazem, but they were unaffected by blockade of hyperpolarization-activated cyclic nucleotide-gated channels. In voltage-clamp experiments, 8-Br-cGMP induced non-inactivating inward currents (,22.2 ± 3.9 pA) with an estimated reversal potential near 0 mV, which were markedly inhibited by reduction of extracellular Na+ and Ca2+ concentrations. Membrane depolarization induced by CNG channel activation increased the firing rate of MVNn without changing the action potential shape. Collectively, these findings provide novel evidence that CNG channels affect membrane potential and excitability of MVNn. Such action should have a significant impact on the function of these neurons in sensory,motor integration processes. More generally, it might represent a broad mechanism for regulating the excitability of different CNS neurons. [source] | ||||||||||