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1.
We developed a multicompartmental Hodgkin-Huxley model of the Hermissenda type-B photoreceptor and used it to address the relative contributions of reductions of two K+ currents, I a and I C, to changes in cellular excitability and synaptic strength that occur in these cells after associative learning. We found that reductions of gC, the peak conductance of I C, substantially increased the firing frequency of the type-B cell during the plateau phase of a simulated light response, whereas reductions of gA had only a modest contribution to the plateau frequency. This can be understood at least in part by the contributions of these currents to the light-induced (nonspiking) generator potential, the plateau of which was enhanced by gC reductions, but not by gA reductions. In contrast, however, reductions of gA broadened the type-B cell action potential, increased Ca2+ influx, and increased the size of the postsynaptic potential produced in a type-A cell, whereas similar reductions of gC had only negligible contributions to these measures. These results suggest that reductions of I A and I C play important but different roles in type-B cell plasticity.  相似文献   

2.
Summary The presence of a Ca2+ channel in the plasmalemma of tonoplast-freeNitellopsis obtusa cells was demonstrated and its characteristics were studied using current- and voltage-clamp techniques. A long-lasting inward membrane current (I m ), recorded using a step voltage clamp, consisted of a single component without time-dependent inactivation. Increasing either [Ca2+] o or [Cl] o 1) enhanced the maximum amplitude of inwardI m ((I m ) p ) and 2) shifted the peak voltage ((V m ) p ) at(I m ) p to more positive values under ramp-shaped voltage clamping and 3) depolarized the peak value of action potentials. This behavior is consistent with predictions based on the Nernst equation for Ca2+ but not for Cl. DIDS (4,4-diisothiocyano-2,2-disulfonic acid stilbene) did not suppress(I m ) p in tonoplast-free cells, in contrast with its effect on normal cells. La3+ and nifedipine blocked(I m ) p irreversibly. On the other hand, Ca2+ channel agonist, BAY K 8644 irreversibly enhanced(I m ) p . Both Sr2+ influx and K+ efflux increased upon excitation. The charge carried by Sr2+ influx was compensated for by K+ efflux. It is concluded that only the Ca2+ channel is activated during plasmalemma excitation in tonoplast-free cells. In terms of the magnitude of(I m ) p , Sr2+ could replace Ca2+, but Mn2+, Mg2+ and Ba2+ could not. External pH affected(I m ) p and the membrane conductance (g m ) at(I m ) p ((g m ) p ). Increasing the external ionic strength caused increases in both(I m ) p and(g m ) p , and shifted(V m ) p to positive values. At the same time, Sr2+ influx increased. Thus Ca2+ channel activation seems to be enhanced by increasing external ionic strength. The possible involvement of surface potential is discussed.  相似文献   

3.
The pig is commonly used as an experimental model of human heart disease, including for the study of mechanisms of arrhythmia. However, there exist differences between human and porcine cellular electrophysiology: The pig action potential (AP) has a deeper phase-1 notch, a longer duration at 50% repolarization, and higher plateau potentials than human. Ionic differences underlying the AP include larger rapid delayed-rectifier and smaller inward-rectifier K+-currents (IKr and IK1 respectively) in humans. AP steady-state rate-dependence and restitution is steeper in pigs. Porcine Ca2+ transients can have two components, unlike human. Although a reliable computational model for human ventricular myocytes exists, one for pigs is lacking. This hampers translation from results obtained in pigs to human myocardium. Here, we developed a computational model of the pig ventricular cardiomyocyte AP using experimental datasets of the relevant ionic currents, Ca2+-handling, AP shape, AP duration restitution, and inducibility of triggered activity and alternans. To properly capture porcine Ca2+ transients, we introduced a two-step process with a faster release in the t-tubular region, followed by a slower diffusion-induced release from a non t-tubular subcellular region. The pig model behavior was compared with that of a human ventricular cardiomyocyte (O’Hara-Rudy) model. The pig, but not the human model, developed early afterdepolarizations (EADs) under block of IK1, while IKr block led to EADs in the human but not in the pig model. At fast rates (pacing cycle length = 400 ms), the human cell model was more susceptible to spontaneous Ca2+ release-mediated delayed afterdepolarizations (DADs) and triggered activity than pig. Fast pacing led to alternans in human but not pig. Developing species-specific models incorporating electrophysiology and Ca2+-handling provides a tool to aid translating antiarrhythmic and arrhythmogenic assessment from the bench to the clinic.  相似文献   

4.
Serotonin (5-HT) applied to the exposed but otherwise intact nervous system results in enhanced excitability of Hermissenda type-B photoreceptors. Several ion currents in the type-B photoreceptors are modulated by 5-HT, including the A-type K+ current (IK,A), sustained Ca2+ current (ICa,S), Ca-dependent K+ current (IK,Ca), and a hyperpolarization-activated inward rectifier current (Ih). In this study, we developed a computational model that reproduces physiological characteristics of type B photoreceptors, e.g. resting membrane potential, dark-adapted spike activity, spike width, and the amplitude difference between somatic and axonal spikes. We then used the model to investigate the contribution of different ion currents modulated by 5-HT to the magnitudes of enhanced excitability produced by 5-HT. Ion currents were systematically varied within limits observed experimentally, both individually and in combinations. A reduction of IK,A or IK,Ca, or an increase in Ih enhanced excitability by 20–50%. Decreasing ICa,S produced a dramatic decrease in excitability. Reductions of IK,V produced only minimal increases in excitability, suggesting that IK,V probably plays a minor role in 5-HT induced enhanced excitability. Combinations of changes in IK,A, IK,Ca, Ih and ICa,S produced increases in excitability comparable to experimental observations. After 5-HT application, the cell's depolarization force is shifted from the Ih–ICa,S combination to predominantly Ih.  相似文献   

5.
We have previously shown that the membrane conductance of mIMCD-3 cells at a holding potential of 0 mV is dominated by a Ca2+-dependent Cl current (ICLCA). Here we report that ICLCA activity is also voltage dependent and that this dependence on voltage is linked to the opening of a novel Al3+-sensitive, voltage-dependent, Ca2+ influx pathway. Using whole-cell patch-clamp recordings at a physiological holding potential (−60 mV), ICLCA was found to be inactive and resting currents were predominantly K+ selective. However, membrane depolarization to 0 mV resulted in a slow, sigmoidal, activation of ICLCA (T 0.5 ~ 500 s), while repolarization in turn resulted in a monoexponential decay in ICLCA (T 0.5 ~ 100 s). The activation of ICLCA by depolarization was reduced by lowering extracellular Ca2+ and completely inhibited by buffering cytosolic Ca2+ with EGTA, suggesting a role for Ca2+ influx in the activation of ICLCA. However, raising bulk cytosolic Ca2+ at −60 mV did not produce sustained ICLCA activity. Therefore ICLCA is dependent on both an increase in intracellular Ca2+ and depolarization to be active. We further show that membrane depolarization is coupled to opening of a Ca2+ influx pathway that displays equal permeability to Ca2+ and Ba2+ ions and that is blocked by extracellular Al3+ and La3+. Furthermore, Al3+ completely and reversibly inhibited depolarization-induced activation of ICLCA, thereby directly linking Ca2+ influx to activation of ICLCA. We speculate that during sustained membrane depolarization, calcium influx activates ICLCA which functions to modulate NaCl transport across the apical membrane of IMCD cells.  相似文献   

6.
Sarcolemmal Na+–Ca2+ exchange plays a central role in ion transport of the myocardium and the current carried with it contributes to the late phase of the action potential (AP) besides the contribution of outward K+-currents. In this study, the mathematical model for AP of the diabetic rat ventricular myocytes [34] was modified and used for the diabetic rat papillary muscle. We used our experimentally measured values of two K+-currents; transient outward current, Ito and steady-state outward current, Iss, as well as L-type Ca2+-current, ICaL, then compared with the simulated values. We have demonstrated that the prolongation in the AP of the papillary muscle of the diabetic rats are not due to the alteration of ICaL but mainly due to the inhibition of the K+-currents and also the Na+–Ca2+ exchanger current, INa–Ca. In combination with our experimental data on sodium-selenite-treated diabetic rats, our simulation results provide new information concerning plausible ionic mechanisms, and second a possible positive effect of selenium treatment on the altered INa–Ca for the observed changes in the AP duration of streptozotocin-induced diabetic rat heart. (Mol Cell Biochem 269: 121–129, 2005)  相似文献   

7.
Thein vivo effect of the mu agonist morphine and antagonist naloxone on [3H]nimodipine receptor binding in rat brain regions has been investigated. Morphine administration (15 mg/s.c.) for thirty minutes produced a 19% decrease in [3H]nimodipine receptor binding (B max 158.2 fmol to 128.9 fmol) in cortex and 29% decrease in cerebellum (65.3 fmol to 46.0 fmol). Lesser changes were observed in hippocampal and striatal regions with no changes in hypothalamus and brain stem. All effects were completely antagonized by naloxone pretreatment (1 mg/kg). The studies suggest that opiates in vivo can alter [3H]nimodipine binding to the Ca2+ channel receptor protein. These findings agree with the previously observed decreases in Ca2+ influx in nerve ending preparations and inhibition of ICa 2+ following opiate treatment and suggest opiates reduce Ca2+-dependent neurotransmitter release by altering the Ca2+ channel receptor protein in an allosteric fashion.  相似文献   

8.
In Hermissenda type-B photoreceptors, the spike is generated in the axon and back-propagated to the soma, resulting in smaller somatic spikes. Experimentally, blocking the A-type K+ current (IK,A) results in broadening of somatic spikes. Similarly, in a compartmental model of the photoreceptor, reducing the maximum A-type K+ conductance (gK,Amax) results in broadening of somatic spikes. However, simulations predict that little or no broadening of axonal spikes occurs when gK,Amax is reduced. The results can be explained by the voltage-dependent properties of IK,A and the different potential ranges that the somatic and axonal spike traverse. Because of the steeper I-V curve and faster activation of the K+ channels at higher potentials, the recruitment of additional K+ channels in the axon is able to compensate for the decrease in K+ conductance, yielding less spike broadening. These results also support the idea that spike duration in the axon may not be reliably inferred based upon recordings collected from the soma. Action Editor: Jonathan D. Victor  相似文献   

9.
A voltage-dependent but Ca2+-independent regulation of N-methyl-D-aspartate (NMDA) receptor outward activity was studied at the single channel level using outside-out patches of cultured mouse cortical neurons. Unlike the inward activity associated with Ca2+ and Na+ influx, the NMDA receptor outward K+ conductance was unaffected by changes in Ca2+ concentration. Following a depolarizing pre-pulse, the single channel open probability (NP o), amplitude, and open duration of the NMDA inward current decreased, whereas the same pre-depolarization increased those parameters of the NMDA outward current (pre-pulse facilitation). The outward NP o was increased by the pre-pulse facilitation, disregarding Ca2+ changes. The voltage–current relationships of the inward and outward currents were shifted by the pre-depolarization toward opposite directions. The Src family kinase inhibitor, PP1, and the Src kinase antibody, but not the anti-Fyn antibody, blocked the pre-pulse facilitation of the NMDA outward activity. On the other hand, a hyperpolarizing pre-pulse showed no effect on NMDA inward currents but inhibited outward currents (pre-pulse depression). Application of Src kinase, but not Fyn kinase, prevented the pre-pulse depression. We additionally showed that a depolarization pre-pulse potentiated miniature excitatory synaptic currents (mEPSCs). The effect was blocked by application of the NMDA receptor antagonist AP-5 during depolarization. These data suggest a voltage-sensitive regulation of NMDA receptor channels mediated by Src kinase. The selective changes in the NMDA receptor-mediated K+ efflux may represent a physiological and pathophysiological plasticity at the receptor level in response to dynamic changes in the membrane potential of central neurons.  相似文献   

10.
To quantitatively understand intracellular Na+ and Cl homeostasis as well as roles of Na+/K+ pump and cystic fibrosis transmembrane conductance regulator Cl channel (ICFTR) during the β1-adrenergic stimulation in cardiac myocyte, we constructed a computer model of β1-adrenergic signaling and implemented it into an excitation-contraction coupling model of the guinea-pig ventricular cell, which can reproduce membrane excitation, intracellular ion changes (Na+, K+, Ca2+ and Cl), contraction, cell volume, and oxidative phosphorylation. An application of isoproterenol to the model cell resulted in the shortening of action potential duration (APD) after a transient prolongation, the increases in both Ca2+ transient and cell shortening, and the decreases in both Cl concentration and cell volume. These results are consistent with experimental data. Increasing the density of ICFTR shortened APD and augmented the peak amplitudes of the L-type Ca2+ current (ICaL) and the Ca2+ transient during the β1-adrenergic stimulation. This indirect inotropic effect was elucidated by the increase in the driving force of ICaL via a decrease in plateau potential. Our model reproduced the experimental data demonstrating the decrease in intracellular Na+ during the β-adrenergic stimulation at 0 or 0.5 Hz electrical stimulation. The decrease is attributable to the increase in Na+ affinity of Na+/K+ pump by protein kinase A. However it was predicted that Na+ increases at higher beating rate because of larger Na+ influx through forward Na+/Ca2+ exchange. It was demonstrated that dynamic changes in Na+ and Cl fluxes remarkably affect the inotropic action of isoproterenol in the ventricular myocytes.  相似文献   

11.
Although a neurotoxic role has been postulated for the β-amyloid protein (βAP), which accumulates in brain tissues in Alzheimer's disease, a precise mechanism underlying this toxicity has not been identified. The peptide fragment consisting of amino acid residues 25 through 35 (βAP25-35), in particular, has been reported to be toxic in cultured neurons. We report that βAP25-35, applied to rat hippocampal neurons in culture, caused reversible and repeatable increases in the intracellular Ca2+ concentration ([Ca2+]i), as measured by fura 2 fluorimetry. Furthermore, βAP25-35 induced bursts of excitatory potentials and action potential firing in individual neurons studied with whole cell current clamp recordings. The βAP25-35–induced [Ca2+]i elevations and electrical activity were enhanced by removal of extracellular Mg2+, and they could be blocked by tetrodotoxin, by non-N-methyl-D -aspartate (NMDA) and NMDA glutamate receptor antagonists, and by the L-type Ca2+ channel antagonist nimodipine. Similar responses of bursts of action potentials and [Ca2+]i increases were evoked by βAP1-40. Responses to βAP25-35 were not prevented by pretreatment with pertussis toxin. Excitatory responses and [Ca2+]i elevations were not observed in cerebellar neuron cultures in which inhibitory synapses predominate. Although the effects of βAP25-35 depended on the activation of glutamatergic synapses, there was no enhancement of kainate- or NMDA-induced currents by βAP25-35 in voltage-clamp studies. We conclude that βAP25-35 enhances excitatory activity in glutamatergic synaptic networks, causing excitatory potentials and Ca2+ influx. This property may explain the toxicity of βAP25–35. © 1995 John Wiley & Sons, Inc.  相似文献   

12.
Ca-calmodulin-dependent protein kinase II (CaMKII) was recently shown to alter Na+ channel gating and recapitulate a human Na+ channel genetic mutation that causes an unusual combined arrhythmogenic phenotype in patients: simultaneous long QT syndrome and Brugada syndrome. CaMKII is upregulated in heart failure where arrhythmias are common, and CaMKII inhibition can reduce arrhythmias. Thus, CaMKII-dependent channel modulation may contribute to acquired arrhythmic disease. We developed a Markovian Na+ channel model including CaMKII-dependent changes, and incorporated it into a comprehensive myocyte action potential (AP) model with Na+ and Ca2+ transport. CaMKII shifts Na+ current (INa) availability to more negative voltage, enhances intermediate inactivation, and slows recovery from inactivation (all loss-of-function effects), but also enhances late noninactivating INa (gain of function). At slow heart rates, with long diastolic time for INa recovery, late INa is the predominant effect, leading to AP prolongation (long QT syndrome). At fast heart rates, where recovery time is limited and APs are shorter, there is little effect on AP duration, but reduced availability decreases INa, AP upstroke velocity, and conduction (Brugada syndrome). CaMKII also increases cardiac Ca2+ and K+ currents (ICa and Ito), complicating CaMKII-dependent AP changes. Incorporating ICa and Ito effects individually prolongs and shortens AP duration. Combining INa, ICa, and Ito effects results in shortening of AP duration with CaMKII. With transmural heterogeneity of Ito and Ito downregulation in heart failure, CaMKII may accentuate dispersion of repolarization. This provides a useful initial framework to consider pathways by which CaMKII may contribute to arrhythmogenesis.  相似文献   

13.
Neuronal ion channels of different types often do not function independently but will inhibit or potentiate the activity of other types of channels, a process called cross-talk. The N-methyl-D-aspartate receptor (NMDA receptor) and the γ-aminobutyric acid type A receptor (GABAA receptor) are important excitatory and inhibitory receptors in the central nervous system, respectively. Currently, cross-talk between the NMDA receptor and the GABAA receptor, particularly in the central auditory system, is not well understood. In the present study, we investigated functional interactions between the NMDA receptor and the GABAA receptor using whole-cell patch-clamp techniques in cultured neurons from the inferior colliculus, which is an important nucleus in the central auditory system. We found that the currents induced by aspartate at 100 μmol L−1 were suppressed by the pre-perfusion of GABA at 100 μmol L−1, indicating cross-inhibition of NMDA receptors by activation of GABAA receptors. Moreover, we found that the currents induced by GABA at 100 μmol L−1 (I GABA) were not suppressed by the pre-perfusion of 100 μmol L−1 aspartate, but those induced by GABA at 3 μmol L−1 were suppressed, indicating concentration-dependent cross-inhibition of GABAA receptors by activation of NMDA receptors. In addition, inhibition of IGABA by aspartate was not affected by blockade of voltage-dependent Ca2+ channels with CdCl2 in a solution that contained Ca2+, however, CdCl2 effectively attenuated the inhibition of I GABA by aspartate when it was perfused in a solution that contained Ba2+ instead of Ca2+ or a solution that contained Ca2+ and 10 mmol L−1 BAPTA, a membrane-permeable Ca2+ chelator, suggesting that this inhibition is mediated by Ca2+ influx through NMDA receptors, rather than voltage-dependent Ca2+ channels. Finally, KN-62, a potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII), reduced the inhibition of I GABA by aspartate, indicating the involvement of CaMKII in this cross-inhibition. Our study demonstrates a functional interaction between NMDA and GABAA receptors in the inferior colliculus of rats. The presence of cross-talk between these receptors suggests that the mechanisms underlying information processing in the central auditory system may be more complex than previously believed.  相似文献   

14.
Several mathematical models of rabbit ventricular action potential (AP) have been proposed to investigate mechanisms of arrhythmias and excitation-contraction coupling. Our study aims at systematically characterizing how ionic current properties modulate the main cellular biomarkers of arrhythmic risk using two widely-used rabbit ventricular models, and comparing simulation results using the two models with experimental data available for rabbit. A sensitivity analysis of AP properties, Ca2+ and Na+ dynamics, and their rate dependence to variations (±15% and ±30%) in the main transmembrane current conductances and kinetics was performed using the Shannon et al. (2004) and the [Mahajan et?al., 2008a] and [Mahajan et?al., 2008b] AP rabbit models. The effects of severe transmembrane current blocks (up to 100%) on steady-state AP and calcium transients, and AP duration (APD) restitution curves were also simulated using both models. Our simulations show that, in both virtual rabbit cardiomyocytes, APD is significantly modified by most repolarization currents, AP triangulation is regulated mostly by the inward rectifier K+ current (IK1) whereas APD rate adaptation as well as [Na+]i rate dependence is influenced by the Na+/K+ pump current (INaK). In addition, steady-state [Ca2+]i levels, APD restitution properties and [Ca2+]i rate dependence are strongly dependent on INaK, the L-Type Ca2+ current (ICaL) and the Na+/Ca2+ exchanger current (INaCa), although the relative role of these currents is markedly model dependent. Furthermore, our results show that simulations using both models agree with many experimentally-reported electrophysiological characteristics. However, our study shows that the Shannon et al. model mimics rabbit electrophysiology more accurately at normal pacing rates, whereas Mahajan et al. model behaves more appropriately at faster rates. Our results reinforce the usefulness of sensitivity analysis for further understanding of cellular electrophysiology and validation of cardiac AP models.  相似文献   

15.
Calcium (Ca2+)-activated chloride (Cl) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca2+-activated Cl currents (ICl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate ICl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. ICl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. ICl(Ca) was abolished by treatment with the Ca2+ channel blocker Co2+, the L-type Ca2+ channel blocker nifedipine, and the Cl channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16Ainh-A01, and a neutralizing antibody against ANO1 inhibited ICl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of ICl(Ca) by using NPPB and T16Ainh-A01 caused a prolonged Ca2+ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers ICl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.  相似文献   

16.
Dopamine (DA) released from the hypothalamus tonically inhibits pituitary lactotrophs. DA (at micromolar concentration) opens potassium channels, hyperpolarizing the lactotrophs and thus preventing the calcium influx that triggers prolactin hormone release. Surprisingly, at concentrations ∼1000 lower, DA can stimulate prolactin secretion. Here, we investigated whether an increase in a K+ current could mediate this stimulatory effect. We considered the fast K+ currents flowing through large-conductance BK channels and through A-type channels. We developed a minimal lactotroph model to investigate the effects of these two currents. Both I BK and I A could transform the electrical pattern of activity from spiking to bursting, but through distinct mechanisms. I BK always increased the intracellular Ca2+ concentration, while I A could either increase or decrease it. Thus, the stimulatory effects of DA could be mediated by a fast K+ conductance which converts tonically spiking cells to bursters. In addition, the study illustrates that a heterogeneous distribution of fast K+ conductances could cause heterogeneous lactotroph firing patterns. Action Editor: Christiane Linster  相似文献   

17.
Summary The whole-cell patch-clamp method has been used to measure Ca2+ influx through otherwise K+-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K+ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K+ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K+ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca2+ is more negative than or equal to the K+ equilibrium potential (–47 mV), indicating that the current is due to Ca2+ influx through the K+ channels prior to their closure. Decreasing internal [Ca2+] (Ca i ) from 200 to 2 nm or increasing the external [Ca2+] (Ca o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca2+ influx also decrease the amplitude of time-activated current, indicating that Ca2+ permeation is slower than K+ permeation, and so causes a partial block. Increasing Ca o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K+ channels in Zea mays guard cells also appear to have a Ca i -, and Ca o -dependent ability to mediate Ca2+ influx. We suggest that the inwardly rectiying K+ channels are part of a regulatory mechanism for Ca i . Changes in Ca o and (associated) changes in Ca i regulate a variety of intracellular processes and ion fluxes, including the K+ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.  相似文献   

18.
Summary The voltage- and time-dependent K+ current,I K + out , elicited by depolarization of corn protoplasts, was inhibited by the addition of calcium channel antagonists (nitrendipine, nifedipine, verapamil, methoxyverapamil, bepridil, but not La3+) to the extracellular medium. These results suggested that the influx of external Ca2+ was necessary for K+ current activation. The IC50, concentration of inhibitor that caused 50% reduction of the current, for nitrendipine was 1 m at a test potential of +60 mV following a 20-min incubation period.In order to test whether intracellular Ca2+ actuated the K+ current, we altered either the Ca2+ buffering capacity or the free Ca2+ concentration of the intracellular medium (pipette filling solution). By these means,I K + out could be varied over a 10-fold range. Increasing the free Ca2+ concentration from 40 to 400nm also shifted the activation of the K+ current toward more negative potentials. Maintaining cytoplasmic Ca2+ at 500nm with 40nm EGTA resulted in a more rapid activation of the K+ current. Thus the normal rate of activation of this current may reflect changes in cytoplasmic Ca2+ on depolarization. Increasing intracellular Ca2+ to 500nm or 1 m also led to inactivation of the K+ current within a few minutes. It is concluded thatI K + out is regulated by cytosolic Ca2+, which is in turn controlled by Ca2+ influx through dihydropyridine-, and phenylalkylamine-sensitive channels.  相似文献   

19.
Dysregulation of intracellular Ca2+ homeostasis is associated with various pathological conditions and arrhythmogenesis of the heart. The objective of this study was to investigate the effects of an acute increase in intracellular Ca2+ concentration ([Ca2+]i) on the electrophysiology of ventricular myocytes by mimicking intracellular Ca2+ overload. The [Ca2+]i was clamped to either a controlled (65–100 nmol L−1) or increased (1 μmol L−1) level. The transmembrane action potentials and ionic currents were recorded using whole-cell patch clamp techniques. We found that the acute increase in [Ca2+]i shortened the action potential duration, reduced the action potential amplitude, maximum depolarization velocity and resting membrane potential, caused delayed after-depolarizations (DADs), and triggered activity—compared with these parameters in the control. The increased [Ca2+]i augmented late I Na in a time-dependent manner, reduced I CaL and I K1, and increased I Kr but not I Ks. The results of this study can be used to explain calcium overload-induced ventricular arrhythmias.  相似文献   

20.
The influence of cytosolic pH (pHi) in controlling K+-channel activity and its interaction with cytosolic-free Ca2+ concentration ([Ca2+]i) was examined in stomatal guard cells ofVicia faba L. Intact guard cells were impaled with multibarrelled microelectrodes and K+-channel currents were recorded under voltage clamp while pHi or [Ca2+]i was monitored concurrently by fluorescence ratio photometry using the fluorescent dyes 2,7-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Fura-2. In 10 mM external K+ concentration, current through inward-rectifying K+ channels (IK,in) was evoked on stepping the membrane from a holding potential of –100 mV to voltages from –120 to –250 mV. Challenge with 0.3-30 mM Na+-butyrate and Na+-acetate outside imposed acid loads, lowering pHi from a mean resting value of 7.64 ± 0.03 (n = 25) to values from 7.5 to 6.7. The effect on pHi was independent of the weak acid used, and indicated a H+-buffering capacity which rose from 90 mM H+/pH unit near 7.5 to 160 mM H+/pH unit near pHi 7.0. With acid-going pHi, (IK,in) was promoted in scalar fashion, the current increasing in magnitude with the acid load, but without significant effect on the current relaxation kinetics at voltages negative of –150 mV or the voltage-dependence for channel gating. Washout of the weak acid was followed by transient rise in pHi lasting 3–5 min and was accompanied by a reduction in (IK,in) before recovery of the initial resting pHi and current amplitude. The pHi-sensitivity of the current was consistent with a single, titratable site for H+ binding with a pKa near 6.3. Acid pHi loads also affected current through the outward-rectifying K+ channels (IK,out) in a manner antiparallel to (IK,in) The effect on IK, out was also scalar, but showed an apparent pKa of 7.4 and was best accommodated by a cooperative binding of two H+. Parallel measurements showed that Na+-butyrate loads were generally without significant effect on [Ca2+]i, except when pHi was reduced to 7.0 and below. Extreme acid loads evoked reversible increases in [Ca2+]i in roughly half the cells measured, although the effect was generally delayed with respect to the time course of pHi changes and K+-channel responses. The action on [Ca2+]i coincided with a greater variability in (IK,in) stimulation evident at pHi values around 7.0 and below, and with negative displacements in the voltage-dependence of (IK,in) gating. These results distinguish the actions of pHi and [Ca2+]i in modulating (IK,in) they delimit the effect of pHi to changes in current amplitude without influence on the voltage-dependence of channel gating; and they support a role for pHi as a second messenger capable of acting in parallel with, but independent of [Ca2+]i in controlling the K+ channels.Abbreviations BCECF 2,7-bis (2-carboxyethyl)-5(6)-carboxy fluorescein - [Ca2+]i cytosolic free Ca2+ concentration - gK ensemble (steady-state) K+-channel conductance - IK,out, IK,in outward-, inward-rectifying K+ channel (current) - IN current-voltage (relation) - Mes 2-(N-morpholinolethanesulfonic acid - pHi cytosolic pH - V membrane potential  相似文献   

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