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Voltage Dependence (voltage + dependence)
Selected AbstractsGating of the expressed T-type Cav3.1 calcium channels is modulated by Ca2+ACTA PHYSIOLOGICA, Issue 4 2006L. Lacinová Abstract Aim:, We have investigated the influence of Ca2+ ions on the basic biophysical properties of T-type calcium channels. Methods:, The Cav3.1 calcium channel was transiently expressed in HEK 293 cells. Current was measured using the whole cell patch clamp technique. Ca2+ or Na+ ions were used as charge carriers. The intracellular Ca2+ was either decreased by the addition of 10 mm ethyleneglycoltetraacetic acid (EGTA) or increased by the addition of 200 ,m Ca2+ into the non-buffered intracellular solution. Various combinations of extra- and intracellular solutions yielded high, intermediate or low intracellular Ca2+ levels. Results:, The amplitude of the calcium current was independent of intracellular Ca2+ concentrations. High levels of intracellular Ca2+ accelerated significantly both the inactivation and the activation time constants of the current. The replacement of extracellular Ca2+ by Na+ as charge carrier did not affect the absolute value of the activation and inactivation time constants, but significantly enhanced the slope factor of the voltage dependence of the inactivation time constant. Slope factors of voltage dependencies of channel activation and inactivation were significantly enhanced. The recovery from inactivation was faster when Ca2+ was a charge carrier. The number of available channels saturated for membrane voltages more negative than ,100 mV for the Ca2+ current, but did not reach steady state even at ,150 mV for the Na+ current. Conclusions:, Ca2+ ions facilitate transitions of Cav3.1 channel from open into closed and inactivated states as well as backwards transition from inactivated into closed state, possibly by interacting with its voltage sensor. [source] Cloning and functional characterization of a novel connexin expressed in somites of Xenopus laevisDEVELOPMENTAL DYNAMICS, Issue 3 2005Teun P. De Boer Abstract Connexin-containing gap junctions play an essential role in vertebrate development. More than 20 connexin isoforms have been identified in mammals. However, the number identified in Xenopus trails with only six isoforms described. Here, identification of a new connexin isoform from Xenopus laevis is described. Connexin40.4 was found by screening expressed sequence tag databases and carrying out polymerase chain reaction on genomic DNA. This new connexin has limited amino acid identity with mammalian (<50%) connexins, but conservation is higher (,62%) with fish. During Xenopus laevis development, connexin40.4 was first expressed after the mid-blastula transition. There was prominent expression in the presomitic paraxial mesoderm and later in the developing somites. In adult frogs, expression was detected in kidney and stomach as well as in brain, heart, and skeletal muscle. Ectopic expression of connexin40.4 in HEK293 cells, resulted in formation of gap junction like structures at the cell interfaces. Similar ectopic expression in neural N2A cells resulted in functional electrical coupling, displaying mild, asymmetric voltage dependence. We thus cloned a novel connexin from Xenopus laevis, strongly expressed in developing somites, with no apparent orthologue in mammals. Developmental Dynamics 233:864,871, 2005. © 2005 Wiley-Liss, Inc. [source] Proteolytic cleavage of the voltage-gated Ca2+ channel ,2, subunit: structural and functional featuresEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007Arturo Andrade Abstract By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (,1) and three auxiliary (,2,, , and ,) subunits. The ,2, subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, ,2, must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular ,2 from its transmembrane , domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature ,2,, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of ,2, was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. [source] Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Paola Imbrici Abstract Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker -like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kv,1.x subunits. Here we show that the human Kv,1.1 and Kv,1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kv,1.1 and Kv,1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements. [source] Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerabilityEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2005E. Avignone Abstract Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+ -free medium, NMDA (10 µm; 3,5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 µm) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mm) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition. [source] Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 1 2004Takahiro Yasuda Abstract The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel , and ,2,, subunits on expression levels and biophysical properties of three different types (Cav1.2, Cav2.1 and Cav2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Cav1.2 and Cav2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel , subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel ,1 subunit, and ,3 subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the ,2,, subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Cav2 channel family, appears to act synergistically with , subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by , and by ,2,, subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the ,2,,1 subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes. [source] No evidence for calcium electrogenic exchanger in frog semicircular canal hair cellsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2002M. Martini Abstract We investigated the possibility that, in hair cells mechanically isolated from frog semicircular canals, Ca2+ extrusion occurs via a Na+ : Ca2+ (cardiac type) or a Na+ : Ca2+,K+ (retinal type) exchanger. Cells concurrently imaged during whole-cell patch-clamp recordings using the Ca2+ sensitive fluorescent dye Oregon Green 488 BAPTA-1 (100 µm) showed no voltage dependence of Ca2+ clearance dynamics following a Ca2+ load through voltage-gated Ca2+ channels. Reverse exchange was probed in hair cells dialyzed with a Ca2+ - and K+ -free solution, containing a Na+ concentration that saturates the exchanger, after zeroing the contribution to the whole-cell current from Ca2+ and K+ conductances. In these conditions, no reverse exchange current was detected upon switching from a Ca2+ -free external solution to a solution containing concentrations of Ca2+ alone, or Ca2+ + K+ that saturated the exchanger. By contrast, the same experimental protocol elicited peak exchange currents exceeding 100 pA in gecko rod photoreceptors, used as positive controls. In both cell types, we also probed the forward mode of the exchanger by rapidly increasing the intracellular Ca2+ concentration using flash photolysis of two novel caged Ca2+ complexes, calcium 2,2,-{[1-(2-nitrophenyl)ethane-1,2-diyl]bis(oxy)}bis(acetate) and calcium 2,2,-{[1-(4,5-dimethoxy-2-nitrophenyl)ethane-1,2-diyl]bis(oxy)} bis(acetate), in the presence of internal K+ and external Na+. No currents were evoked by UV-triggered Ca2+ jumps in hair cells, whereas exchanger conformational currents up to 400 pA, followed by saturating forward exchange currents up to 40 pA, were recorded in rod photoreceptors subjected to the same experimental conditions. We conclude that no functional electrogenic exchanger is present in this hair cell population, which leaves the abundant plasma membrane Ca2+ -ATPases as the primary contributors to Ca2+ extrusion. [source] Cardiac L-type calcium current is increased in a model of hyperaldosteronism in the ratEXPERIMENTAL PHYSIOLOGY, Issue 6 2009Beatriz Martin-Fernandez Accumulating evidence supports the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. It has been postulated that aldosterone could contribute to ventricular arrhythmogeneity by modulation of cardiac ionic channels. The aim of this study was to analyse ex vivo the electrophysiological characteristics of the L-type cardiac calcium current (ICaL) in a model of hyperaldosteronism in the rat. Aldosterone was administered for 3 weeks, and cardiac collagen deposition and haemodynamic parameters were analysed. In addition, RT-PCR and patch-clamp techniques were applied to study cardiac L-type Ca2+ channels in isolated cardiomyocytes. Administration of aldosterone induced maladaptive cardiac remodelling that was related to increased collagen deposition, diastolic dysfunction and cardiac hypertrophy. In addition, ventricular myocytes isolated from the aldosterone-treated group showed increased ICaL density and conductance and prolongation of the action potential duration. No changes in kinetics or in voltage dependence of activation and inactivation of ICaL were observed, but relative expression of CaV1.2 mRNA levels was higher in cardiomyocytes isolated from the aldosterone-treated group. The present study demonstrates that aldosterone treatment induces myocardial fibrosis, cardiac hypertrophy, increase of ICaL density, upregulation of L-type Ca2+ channels and prolongation of action potential duration. It could be proposed that aldosterone, through these mechanisms, might exert pro-arrhythmic effects in the pathological heart. [source] The Janus-faced atracotoxins are specific blockers of invertebrate KCa channelsFEBS JOURNAL, Issue 16 2008Simon J. Gunning The Janus-faced atracotoxins are a unique family of excitatory peptide toxins that contain a rare vicinal disulfide bridge. Although lethal to a wide range of invertebrates, their molecular target has remained enigmatic for almost a decade. We demonstrate here that these toxins are selective, high-affinity blockers of invertebrate Ca2+ -activated K+ (KCa) channels. Janus-faced atracotoxin (J-ACTX)-Hv1c, the prototypic member of this toxin family, selectively blocked KCa channels in cockroach unpaired dorsal median neurons with an IC50 of 2 nm, but it did not significantly affect a wide range of other voltage-activated K+, Ca2+ or Na+ channel subtypes. J-ACTX-Hv1c blocked heterologously expressed cockroach large-conductance Ca2+ -activated K+ (pSlo) channels without a significant shift in the voltage dependence of activation. However, the block was voltage-dependent, indicating that the toxin probably acts as a pore blocker rather than a gating modifier. The molecular basis of the insect selectivity of J-ACTX-Hv1c was established by its failure to significantly inhibit mouse mSlo currents (IC50 , 10 ,m) and its lack of activity on rat dorsal root ganglion neuron KCa channel currents. This study establishes the Janus-faced atracotoxins as valuable tools for the study of invertebrate KCa channels and suggests that KCa channels might be potential insecticide targets. [source] Regulation of L-type Ca++ currents and process morphology in white matter oligodendrocyte precursor cells by golli-myelin proteinsGLIA, Issue 11 2010Daniel Fulton Abstract The golli myelin basic proteins are expressed in oligodendroglial precursor cells (OPCs) where they play a role in regulating Ca2+ homeostasis. During depolarization, they influence process outgrowth and migration through their action on voltage-operated Ca2+ channels (VOCCs). To identify ion channels that are modulated by golli, we examined the electrophysiological properties of VOCCs in OPCs in the white matter of golli knock-out and control mice. OPCs exhibited two distinct Ca2+ channels, which were distinguished by their voltage dependence and pharmacological profiles and which exhibited many of the hallmarks of LVA/T-type and HVA/L-type Ca2+ channels. The density of high-voltage-activated (HVA) currents was reduced in OPCs recorded in golli-KO tissue, while low-voltage-activated (LVA) currents remained unaltered in these cells. These data indicate that golli exerts an exclusive influence on L-type Ca2+ channels in OPCs. Oligodendrocytes (OLs) also displayed LVA and HVA currents, although the density of these currents was much reduced at this developmental stage. These currents were not altered in golli-KO OLs showing the influence of golli on L-type Ca2+ channels is restricted to a specific time-window during the course of oligodendroglial development. The actions of golli on OPC L-type Ca2+ channels were accompanied by changes in process morphology, including a reduction in process complexity and the appearance of enlarged varicosities that decorated these cellular processes. These data on L-type Ca2+ channels and process development provide in situ evidence for the influence of golli on VOCCs, and offer an explanation for the hypomyelination observed in the brains of golli-KO mice. © 2010 Wiley-Liss, Inc. [source] Block of HERG-Carried K+ Currents by the New Repolarization Delaying Agent H 345/52JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 6 2003Gregory J. Amos M.D. Ph.D. Introduction: The aim of this study was to analyze the block of HERG-carried membrane currents caused by H 345/52, a new antiarrhythmic compound with low proarrhythmic activity, in transfected mouse fibroblasts. Methods and Results: Using the whole-cell configuration of the voltage patch clamp technique, it was demonstrated that H 345/52 concentration-dependently blocked HERG-carried currents with an IC50 of 230 nM. H 345/52 preferentially bound to the open channel with unusually rapid kinetics and was trapped by channel closure. Voltage-independent behavior of H 345/52 was observed during both square-pulse and action potential clamp protocols. In contrast, the Class III agents dofetilide (10 nM) and almokalant (250 nM) demonstrated significant membrane potential-dependent effects during square-pulse clamp protocols. When using action potential clamp protocols, voltage dependence was seen with dofetilide but not with almokalant. Mathematical simulations of human ventricular action potentials predicted that the different voltage-dependent behaviors would not produce marked variations in action potential duration prolongation patterns. Conclusion: We propose that block of IKr is the principal mechanism by which H 345/52 delays repolarization in human myocardium. The voltage independence of HERG/IKr block is unlikely to underlie the low proarrhythmic potential, and ancillary effects on other membrane currents must be considered. (J Cardiovasc Electrophysiol, Vol. 14, pp. 651-658, June 2003) [source] The action of Lambert,Eaton myasthenic syndrome immunoglobulin G on cloned human voltage-gated calcium channelsMUSCLE AND NERVE, Issue 5 2002Ashwin Pinto MRCP, DPhil Abstract In the Lambert,Eaton myasthenic syndrome (LEMS), immunoglobulin G (IgG) autoantibodies to presynaptic voltage-gated calcium channels (VGCCs) at the neuromuscular junction lead to a reduction in nerve-evoked release of neurotransmitter and muscle weakness. We have examined the action of LEMS IgGs on cloned human VGCCs stably expressed in transfected human embryonic kidney (HEK293) cell lines: 10,13 (,1A-2, ,2b,, ,4a) and C2D7 (,1B-1 , ,2b,, ,1b). All LEMS IgGs studied showed surface binding to [125I]-,-CTx-MVIIC-labeled VGCCs in the ,1A cell line and two of six IgGs showed surface binding to [125I]-,-CTx-GVIA-labeled VGCCs in the ,1B cell line. We next studied the effect of LEMS IgGs (2 mg/ml) on whole-cell calcium currents in the ,1A and ,1B cell lines. Overnight treatment of ,1A (10,13) cells with LEMS IgGs led to a significant reduction in peak current density without alteration of the current,voltage relationship or the voltage dependence of steady-state inactivation. In contrast, LEMS IgGs did not reduce peak current density in the ,1B cell line. Overall these data demonstrate the specificity of LEMS IgGs for the ,1A cell line and suggest that LEMS IgGs bind to and downregulate VGCCs in this cell line. Although several LEMS IgGs can be shown to bind to the ,1B (C2D7) cell line, no functional effects were seen on this channel. © 2002 Wiley Periodicals, Inc. Muscle Nerve 25: 000,000, 2002 [source] The molecular interactions of pyrethroid insecticides with insect and mammalian sodium channels,PEST MANAGEMENT SCIENCE (FORMERLY: PESTICIDE SCIENCE), Issue 10 2001Horia Vais Abstract Recent progress in the cloning of , (para) and , (TipE) Na channel sub-units from Drosophila melanogaster (fruit fly) and Musca domestica (housefly) have facilitated functional expression studies of insect Na channels in Xenopus laevis oocytes, assayed by voltage clamp techniques. The effects of Type I and Type II pyrethroids on the biophysical properties of these channels are critically reviewed. Pyrethroid resistance mutations (termed kdr and super-kdr) that reduce the sensitivity of the insect Na channel to pyrethroids have been identified in a range of insect species. Some of these mutations (eg L1014F, M918T and T929I) have been incorporated into the para Na channel of Drosophila, either individually or in combination, to investigate their effects on the sensitivity of this channel to pyrethroids. The kdr mutation (L1014F) shifts the voltage dependence of both activation and steady-state inactivation by ,5,mV towards more positive potentials and facilitates Na channel inactivation. Incorporation of the super-kdr mutation (M918T) into the Drosophila Na channel also increases channel inactivation and causes a >100-fold reduction in deltamethrin sensitivity. These effects are shared by T929I, an alternative mutation that confers super-kdr -like resistance. Parallel studies have been undertaken using the rat IIA Na channel to investigate the molecular basis for the low sensitivity of mammalian brain Na channels to pyrethroids. Rat IIA channels containing the mutation L1014F exhibit a shift in their mid-point potential for Na activation, but their overall sensitivity to permethrin remains similar to that of the wild-type rat channel (ie both are 1000-fold less sensitive than the wild-type insect channel). Mammalian neuronal Na channels have an isoleucine rather than a methionine at the position (874) corresponding to the super-kdr (M918) residue of the insect channel. Replacement of the isoleucine of the wild-type rat IIA Na channel with a methionine (I874M) increases deltamethrin sensitivity 100-fold. In this way, studies of wild-type and mutant Na channels of insects and mammals are providing a molecular understanding of kdr and super-kdr resistance in insects, and of the low pyrethroid sensitivity of most mammalian Na channels. They are also giving valuable insights into the binding sites for pyrethroids on these channels. © 2001 Society of Chemical Industry [source] Effect of 1,1,-dibenzyl-4,4,-bipyridyl dichloride (DBD) on charge-conduction process and photovoltaic response of a polypyrrole (PPy) thin-film devicePOLYMER INTERNATIONAL, Issue 4 2002S Roy Abstract The present communication deals with analysing the effect of 1,1,-dibenzyl-4,4,-bipyridyl dichloride (DBD) substitution at the N -position of 2,5-polypyrrole (PPy), on electrical, impedance and photovoltaic properties. The thin-film device was fabricated by sandwiching DBD-substituted PPy between indium tin oxide (ITO) and aluminium (Al) electrodes. The formation of a Schottky barrier with Al and ohmic contact with ITO are explained in terms of p-type semiconducting behaviour of DBD-substituted PPy. In the low-voltage region, Ohm's law is followed, while in the high-voltage region, a space-charge-limited conduction (SCLC) controlled by the exponential-trap distribution was observed. DBD substitution causes shifting of the Fermi level towards the valence-band edge and an increase in charge-carrier mobility. A remarkable change in dark electrical conductivity of the order of five has been observed in DBD-substituted PPy. The electrical and impedance measurements of an ITO/PPy:DBD/Al device confirms the formation of a Schottky barrier at the DBD-substituted PPy/Al interface. Additionally, it can be modelled by a simple equivalent circuit of two resistance,capacitance (RC) elements in series representing the bulk and a junction-region. At low frequency, the device capacitance follows a pronounced voltage dependence. From a detailed analysis of the J,V and C,V characteristics, the ionized acceptor concentration (Na), width of depletion layer (W) and potential barrier height (,b) have been evaluated. We observed a significant enhancement in photocurrent on DBD substitution. The increase in photocurrent is explained by the efficient charge separation induced by the intermolecular transfer of photo-excited electrons from PPy to DBD. The substitution also causes a reduction in the trapping centres in the material. © 2002 Society of Chemical Industry [source] Permeant anions contribute to voltage dependence of ClC-2 chloride channel by interacting with the protopore gateTHE JOURNAL OF PHYSIOLOGY, Issue 14 2010Jorge E. Sánchez-Rodríguez It has been shown that the voltage (Vm) dependence of ClC Cl, channels is conferred by interaction of the protopore gate with H+ ions. However, in this paper we present evidence which indicates that permeant Cl, ions contribute to Vm -dependent gating of the broadly distributed ClC-2 Cl, channel. The apparent open probability (PA) of ClC-2 was enhanced either by changing the [Cl,]i from 10 to 200 mm or by keeping the [Cl,]i low (10 mm) and then raising [Cl,]o from 10 to 140 mm. Additionally, these changes in [Cl,] slowed down channel closing at positive Vm suggesting that high [Cl,] increased pore occupancy thus hindering closing of the protopore gate. The identity of the permeant anion was also important since the PA(Vm) curves were nearly identical with Cl, or Br, but shifted to negative voltages in the presence of SCN, ions. In addition, gating, closing rate and reversal potential displayed anomalous mole fraction behaviour in a SCN,/Cl, mixture in agreement with the idea that pore occupancy by different permeant anions modifies the Vm dependence ClC-2 gating. Based on the ec1-ClC anion pathway, we hypothesized that opening of the protopore gate is facilitated when Cl, ions dwell in the central binding site. In contrast, when Cl, ions dwell in the external binding site they prevent the gate from closing. Finally, this Cl, -dependent gating in ClC-2 channels is of physiological relevance since an increase in [Cl,]o enhances channel opening when the [Cl,]i is in the physiological range. [source] Voltage-dependent and -independent titration of specific residues accounts for complex gating of a ClC chloride channel by extracellular protonsTHE JOURNAL OF PHYSIOLOGY, Issue 7 2009María Isabel Niemeyer The ClC transport protein family comprises both Cl, ion channel and H+/Cl, and H+/NO3, exchanger members. Structural studies on a bacterial ClC transporter reveal a pore obstructed at its external opening by a glutamate side-chain which acts as a gate for Cl, passage and in addition serves as a staging post for H+ exchange. This same conserved glutamate acts as a gate to regulate Cl, flow in ClC channels. The activity of ClC-2, a genuine Cl, channel, has a biphasic response to extracellular pH with activation by moderate acidification followed by abrupt channel closure at pH values lower than ,7. We have now investigated the molecular basis of this complex gating behaviour. First, we identify a sensor that couples extracellular acidification to complete closure of the channel. This is extracellularly-facing histidine 532 at the N-terminus of transmembrane helix Q whose neutralisation leads to channel closure in a cooperative manner. We go on to show that acidification-dependent activation of ClC-2 is voltage dependent and probably mediated by protonation of pore gate glutamate 207. Intracellular Cl, acts as a voltage-independent modulator, as though regulating the pKa of the protonatable residue. Our results suggest that voltage dependence of ClC-2 is given by hyperpolarisation-dependent penetration of protons from the extracellular side to neutralise the glutamate gate deep within the channel, which allows Cl, efflux. This is reminiscent of a partial exchanger cycle, suggesting that the ClC-2 channel evolved from its transporter counterparts. [source] In vitro characterization of HCN channel kinetics and frequency dependence in myocytes predicts biological pacemaker functionalityTHE JOURNAL OF PHYSIOLOGY, Issue 7 2009Xin Zhao The pacemaker current, mediated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, contributes to the initiation and regulation of cardiac rhythm. Previous experiments creating HCN-based biological pacemakers in vivo found that an engineered HCN2/HCN1 chimeric channel (HCN212) resulted in significantly faster rates than HCN2, interrupted by 1,5 s pauses. To elucidate the mechanisms underlying the differences in HCN212 and HCN2 in vivo functionality as biological pacemakers, we studied newborn rat ventricular myocytes over-expressing either HCN2 or HCN212 channels. The HCN2- and HCN212-over-expressing myocytes manifest similar voltage dependence, current density and sensitivity to saturating cAMP concentrations, but HCN212 has faster activation/deactivation kinetics. Compared with HCN2, myocytes expressing HCN212 exhibit a faster spontaneous rate and greater incidence of irregular rhythms (i.e. periods of rapid spontaneous rate followed by pauses). To explore these rhythm differences further, we imposed consecutive pacing and found that activation kinetics of the two channels are slower at faster pacing frequencies. As a result, time-dependent HCN current flowing during diastole decreases for both constructs during a train of stimuli at a rapid frequency, with the effect more pronounced for HCN2. In addition, the slower deactivation kinetics of HCN2 contributes to more pronounced instantaneous current at a slower frequency. As a result of the frequency dependence of both instantaneous and time-dependent current, HCN2 exhibits more robust negative feedback than HCN212, contributing to the maintenance of a stable pacing rhythm. These results illustrate the benefit of screening HCN constructs in spontaneously active myocyte cultures and may provide the basis for future optimization of HCN-based biological pacemakers. [source] Mechanisms by which atrial fibrillation-associated mutations in the S1 domain of KCNQ1 slow deactivation of IKs channelsTHE JOURNAL OF PHYSIOLOGY, Issue 17 2008Lioara Restier The slow delayed rectifier K+ current (IKs) is a major determinant of action potential repolarization in the heart. IKs channels are formed by coassembly of pore-forming KCNQ1 ,-subunits and ancillary KCNE1 ,-subunits. Two gain of function mutations in KCNQ1 subunits (S140G and V141M) have been associated with atrial fibrillation (AF). Previous heterologous expression studies found that both mutations caused IKs to be instantaneously activated, presumably by preventing channel closure. The purpose of this study was to refine our understanding of the channel gating defects caused by these two mutations located in the S1 domain of KCNQ1. Site-directed mutagenesis was used to replace S140 or V141 with several other natural amino acids. Wild-type and mutant channels were heterologously expressed in Xenopus oocytes and channel function was assessed with the two-microelectrode voltage clamp technique. Long intervals between voltage clamp pulses revealed that S140G and V141M KCNQ1-KCNE1 channels are not constitutively active as previously reported, but instead exhibit extremely slow deactivation. The slow component of IKs deactivation was decreased 62-fold by S140G and 140-fold by the V141M mutation. In addition, the half-point for activation of these mutant IKs channels was ,50 mV more negative than wild-type channels. Other substitutions of S140 or V141 in KCNQ1 caused variable shifts in the voltage dependence of activation, but slowed IKs deactivation to a much lesser extent than the AF-associated mutations. Based on a published structural model of KCNQ1, S140 and V141 are located near E160 in S2 and R237 in S4, two charged residues that could form a salt bridge when the channel is in the open state. In support of this model, mutational exchange of E160 and R237 residues produced a constitutively open channel. Together our findings suggest that altered charge-pair interactions within the voltage sensor module of KCNQ1 subunits may account for slowed IKs deactivation induced by S140 or V141. [source] Hyperpolarization-activated cyclic nucleotide-modulated ,HCN' channels confer regular and faster rhythmicity to beating mouse embryonic stem cellsTHE JOURNAL OF PHYSIOLOGY, Issue 3 2008Yang Qu The hyperpolarization-activated cation current (If), and the hyperpolarization-activated cyclic nucleotide-modulated ,HCN' subunits that underlie it, are important components of spontaneous activity in the embryonic mouse heart, but whether they contribute to this activity in mouse embryonic stem cell-derived cardiomyocytes has not been investigated. We address this issue in spontaneously beating cells derived from mouse embryonic stem cells (mESCs) over the course of development in culture. If and action potentials were recorded from single beating cells at early, intermediate and late development stages using perforated whole-cell voltage- and current-clamp techniques. Our data show that the proportion of cells expressing If, and the density of If in these cells, increased during development and correlated with action potential frequency and the rate of diastolic depolarization. The If blocker ZD7288 (0.3 ,m) reduced If and the beating rate of embryoid bodies. Taken together, the activation kinetics of If and results from Western blots are consistent with the presence of the HCN2 and HCN3 isoforms. At all stages of development, isoproterenol (isoprenaline) and acetylcholine shifted the voltage dependence of If to more positive and negative voltages, respectively, and they also increased and decreased the beating rate of embryonic cell bodies, respectively. Together, the data suggest that current through HCN2 and HCN3 channels confers regular and faster rhythmicity to mESCs, which mirrors the developing embryonic mouse heart, and contributes to modulation of rhythmicity by autonomic stimulation. [source] Activity-dependent regulation of voltage-gated Na+ channel expression in Mat-LyLu rat prostate cancer cell lineTHE JOURNAL OF PHYSIOLOGY, Issue 2 2006William J. Brackenbury We have shown previously that voltage-gated Na+ channels (VGSCs) are up-regulated in human metastatic disease (prostate, breast and small-cell lung cancers), and that VGSC activity potentiates metastatic cell behaviours. However, the mechanism(s) regulating functional VGSC expression in cancer cells remains unknown. We investigated the possibility of activity-dependent (auto)regulation of VGSC functional expression in the strongly metastatic Mat-LyLu model of rat prostate cancer. Pretreatment with tetrodotoxin (TTX) for 24,72 h subsequently suppressed peak VGSC current density without affecting voltage dependence. The hypothesis was tested that the VGSC auto-regulation occurred via VGSC-mediated Na+ influx and subsequent activation of protein kinase A (PKA). Indeed, TTX pretreatment reduced the level of phosphorylated PKA, and the PKA inhibitor KT5720 decreased, whilst the adenylate cyclase activator forskolin and the Na+ ionophore monensin both increased the peak VGSC current density. TTX reduced the mRNA level of Nav1.7, predominant in these cells, and VGSC protein expression at the plasma membrane, although the total VGSC protein level remained unchanged. TTX pretreatment eliminated the VGSC-dependent component of the cells' migration in Transwell assays. We concluded that the VGSC activity in Mat-LyLu rat prostate cancer cells was up-regulated in steady-state via a positive feedback mechanism involving PKA, and this enhanced the cells' migratory potential. [source] Kinetic and functional analysis of transient, persistent and resurgent sodium currents in rat cerebellar granule cells in situ: an electrophysiological and modelling studyTHE JOURNAL OF PHYSIOLOGY, Issue 1 2006Jacopo Magistretti Cerebellar neurones show complex and differentiated mechanisms of action potential generation that have been proposed to depend on peculiar properties of their voltage-dependent Na+ currents. In this study we analysed voltage-dependent Na+ currents of rat cerebellar granule cells (GCs) by performing whole-cell, patch-clamp experiments in acute rat cerebellar slices. A transient Na+ current (INaT) was always present and had the properties of a typical fast-activating/inactivating Na+ current. In addition to INaT, robust persistent (INaP) and resurgent (INaR) Na+ currents were observed. INaP peaked at ,,40 mV, showed half-maximal activation at ,,55 mV, and its maximal amplitude was about 1.5% of that of INaT. INaR was elicited by repolarizing pulses applied following step depolarizations able to activate/inactivate INaT, and showed voltage- and time-dependent activation and voltage-dependent decay kinetics. The conductance underlying INaR showed a bell-shaped voltage dependence, with peak at ,35 mV. A significant correlation was found between GC INaR and INaT peak amplitudes; however, GCs expressing INaT of similar size showed marked variability in terms of INaR amplitude, and in a fraction of cells INaR was undetectable. INaT, INaP and INaR could be accounted for by a 13-state kinetic scheme comprising closed, open, inactivated and blocked states. Current-clamp experiments carried out to identify possible functional correlates of INaP and/or INaR revealed that in GCs single action potentials were followed by depolarizing afterpotentials (DAPs). In a majority of cells, DAPs showed properties consistent with INaR playing a role in their generation. Computer modelling showed that INaR promotes DAP generation and enhances high-frequency firing, whereas INaP boosts near-threshold firing activity. Our findings suggest that special properties of voltage-dependent Na+ currents provides GCs with mechanisms suitable for shaping activity patterns, with potentially important consequences for cerebellar information transfer and computation. [source] Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cellsTHE JOURNAL OF PHYSIOLOGY, Issue 2 2004Juan Shi We investigated, by using the patch clamp technique, Ca2+ -mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 ,m)-activated TRPC6 currents (ITRPC6) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2o+) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2o+ solely inhibited TRPC7 currents (ITRPC7). Vigorous buffering of intracellular Ca2+ (Ca2i+) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2o+, disclosing fast potentiation (EC50: ,0.4 mm) and inhibition (IC50: ,4 mm) of ITRPC6 and fast inhibition (IC50: ,0.4 mm) of ITRPC7. This inhibition of ITRPC6 and ITRPC7 seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of ITRPC6 showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of ITRPC6 or its acceleration by Ca2o+ probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 ,m), coexpression of Ca2+ -insesentive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2i+ via CaM and conversely enhanced by IP3. In addition, the inactivation time course of ITRPC6 was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+,CaM-dependent and -independent mechanisms. [source] Changes in extracellular K+ concentration modulate contractility of rat and rabbit cardiac myocytes via the inward rectifier K+ current IK1THE JOURNAL OF PHYSIOLOGY, Issue 3 2004Ron Bouchard The mechanisms underlying the inotropic effect of reductions in [K+]o were studied using recordings of membrane potential, membrane current, cell shortening and [Ca2+]i in single, isolated cardiac myocytes. Three types of mammalian myocytes were chosen, based on differences in the current density and intrinsic voltage dependence of the inwardly rectifying background K+ current IK1 in each cell type. Rabbit ventricular myocytes had a relatively large IK1 with a prominent negative slope conductance whereas rabbit atrial cells expressed much smaller IK1, with little or no negative slope conductance. IK1 in rat ventricle was intermediate in both current density and slope conductance. Action potential duration is relatively short in both rabbit atrial and rat ventricular myocytes, and consequently both cell types spend much of the duty cycle at or near the resting membrane potential. Rapid increases or decreases of [K+]o elicited significantly different inotropic effects in rat and rabbit atrial and ventricular myocytes. Voltage-clamp and current-clamp experiments showed that the effects on cell shortening and [Ca2+]i following changes in [K+]o were primarily the result of the effects of alterations in IK1, which changed resting membrane potential and action potential waveform. This in turn differentially altered the balance of Ca2+ efflux via the sarcolemmal Na+,Ca2+ exchanger, Ca2+ influx via voltage-dependant Ca2+ channels and sarcoplasmic reticulum (SR) Ca2+ release in each cell type. These results support the hypothesis that the inotropic effect of alterations of [K+]o in the heart is due to significant non-linear changes in the current,voltage relation for IK1 and the resulting modulation of the resting membrane potential and action potential waveform. [source] Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytesTHE JOURNAL OF PHYSIOLOGY, Issue 2 2002Gregory C. Amberg A rapidly inactivating K+ current (A-type current; IA) present in murine colonic myocytes is important in maintaining physiological patterns of slow wave electrical activity. The kinetic profile of colonic IA resembles that of Kv4-derived currents. We examined the contribution of Kv4 ,-subunits to IA in the murine colon using pharmacological, molecular and immunohistochemical approaches. The divalent cation Cd2+ decreased peak IA and shifted the voltage dependence of activation and inactivation to more depolarized potentials. Similar results were observed with La3+. Colonic IA was sensitive to low micromolar concentrations of flecainide (IC50= 11 ,M). Quantitative PCR indicated that in colonic and jejunal tissue, Kv4.3 transcripts demonstrate greater relative abundance than transcripts encoding Kv4.1 or Kv4.2. Antibodies revealed greater Kv4.3-like immunoreactivity than Kv4.2-like immunoreactivity in colonic myocytes. Kv4-like immunoreactivity was less evident in jejunal myocytes. To address this finding, we examined the expression of K+ channel-interacting proteins (KChIPs), which act as positive modulators of Kv4-mediated currents. Qualitative PCR identified transcripts encoding the four known members of the KChIP family in isolated colonic and jejunal myocytes. However, the relative abundance of KChIP transcript was 2.6-fold greater in colon tissue than in jejunum, as assessed by quantitative PCR, with KChIP1 showing predominance. This observation is in accordance with the amplitude of the A-type current present in these two tissues, where colonic myocytes possess densities twice that of jejunal myocytes. From this we conclude that Kv4.3, in association with KChIP1, is the major molecular determinant of IA in murine colonic myocytes. [source] High cortical spreading depression susceptibility and migraine-associated symptoms in Cav2.1 S218L miceANNALS OF NEUROLOGY, Issue 1 2010Arn M. J. M. van den Maagdenberg PhD Objective The CACNA1A gene encodes the pore-forming subunit of neuronal CaV2.1 Ca2+ channels. In patients, the S218L CACNA1A mutation causes a dramatic hemiplegic migraine syndrome that is associated with ataxia, seizures, and severe, sometimes fatal, brain edema often triggered by only a mild head trauma. Methods We introduced the S218L mutation into the mouse Cacna1a gene and studied the mechanisms for the S218L syndrome by analyzing the phenotypic, molecular, and electrophysiological consequences. Results Cacna1aS218L mice faithfully mimic the associated clinical features of the human S218L syndrome. S218L neurons exhibit a gene dosage,dependent negative shift in voltage dependence of CaV2.1 channel activation, resulting in enhanced neurotransmitter release at the neuromuscular junction. Cacna1aS218L mice also display an exquisite sensitivity to cortical spreading depression (CSD), with a vastly reduced triggering threshold, an increased propagation velocity, and frequently multiple CSD events after a single stimulus. In contrast, mice bearing the R192Q CACNA1A mutation, which in humans causes a milder form of hemiplegic migraine, typically exhibit only a single CSD event after one triggering stimulus. Interpretation The particularly low CSD threshold and the strong tendency to respond with multiple CSD events make the S218L cortex highly vulnerable to weak stimuli and may provide a mechanistic basis for the dramatic phenotype seen in S218L mice and patients. Thus, the S218L mouse model may prove a valuable tool to further elucidate mechanisms underlying migraine, seizures, ataxia, and trauma-triggered cerebral edema. ANN NEUROL 2010;67:85,98 [source] Heat opens axon initial segment sodium channels: A febrile seizure mechanism?,ANNALS OF NEUROLOGY, Issue 2 2009Evan A. Thomas PhD Objective A number of hypotheses have been put forward as to why humans respond to fever by seizing. The current leading hypotheses are that respiratory alkalosis produces an as yet unidentified change in neural excitability or that inflammatory mediators potentiate excitatory synaptic transmission. However, it is well known that ion channel gating rates increase with increased temperature. Furthermore, skeletal and cardiac sodium channel activation can be temperature sensitive in some situations. We measured the temperature sensitivity of the brain sodium channel, NaV1.2, to determine whether febrile temperatures might produce a direct increase in neuronal excitability. Methods The effect of temperature on NaV1.2 electrophysiological properties was measured in a transfected mammalian cell line. The subcellular location of NaV1.2 in the mouse brain was ascertained using antibodies against NaV1.2 and ankyrin-G. Computer simulation of a hippocampal granule cell model was used to predict the effect of temperature on action potential firing. Results As well as the expected increase in gating rates, the voltage dependence of activation became 7.6mV more negative when the temperature was increased from 37°C to 41°C. NaV1.2 was localized to the axon initial segment in hippocampal and cortical neurons. Computer simulation showed that increased gating rates and the more negative activation dramatically increase neuronal excitability. Interpretation The direct effect of heat on ion channels localized to the site of action potential initiation potentially causes a profound increase in neuronal excitability. This is likely to contribute to febrile seizure genesis. Ann Neurol 2009;66:219,226 [source] A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia,ANNALS OF NEUROLOGY, Issue 6 2009Tanya Z. Fischer MD Objective Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ). Methods We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents. Results Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7. Interpretation Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders. Ann Neurol 2009;65:733,741 [source] Quercetin as a novel activator of L-type Ca2+ channels in rat tail artery smooth muscle cellsBRITISH JOURNAL OF PHARMACOLOGY, Issue 7 2002Simona Saponara The aim of this study was to investigate the effects of quercetin, a natural polyphenolic flavonoid, on voltage-dependent Ca2+ channels of smooth muscle cells freshly isolated from the rat tail artery, using either the conventional or the amphotericin B-perforated whole-cell patch-clamp method. Quercetin increased L-type Ca2+ current [ICa(L)] in a concentration- (pEC50=5.09±0.05) and voltage-dependent manner and shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction, without, however, modifying the threshold and the equilibrium potential for Ca2+. Quercetin-induced ICa(L) stimulation was reversible upon wash-out. T-type Ca2+ current was not affected by quercetin. Quercetin shifted the voltage dependence of the steady-state inactivation and activation curves to more negative potentials by about 5.5 and 7.5 mV respectively, in the mid-potential of the curves as well as increasing the slope of activation. Quercetin slowed both the activation and the deactivation kinetics of the ICa(L). The inactivation time course was also slowed but only at voltages higher than 10 mV. Moreover quercetin slowed the rate of recovery from inactivation. These results prove quercetin to be a naturally-occurring L-type Ca2+ channel activator. British Journal of Pharmacology (2002) 135, 1819,1827; doi:10.1038/sj.bjp.0704631 [source] BLOCK OF Na+ AND K+ CURRENTS IN RAT VENTRICULAR MYOCYTES BY QUINACAINOL AND QUINIDINECLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 1-2 2005Michael K Pugsley SUMMARY 1.,The electrophysiological actions of quinacainol were investigated on sodium (INa), transient outward (ito) and sustained-outward plateau (iKsus) potassium currents in rat isolated cardiac myocytes using the whole-cell patch-clamp technique and compared with quinidine. 2.,Quinacainol blocked sodium currents in a concentration-dependent manner and with a potency similar to that of quinidine (mean (±SEM) EC50 50 ± 12 vs 95 ± 25 µmol/L for quinidine and quinacainol, respectively). However, quinacainol had a considerably prolonged onset and recovery from block compared with quinidine. 3.,Neither quinacainol nor quinidine significantly changed the steady state voltage dependence of activation of sodium currents. Quinidine produced a hyperpolarizing shift in the voltage dependence for sodium current inactivation, but no such shift was observed with quinacainol at doses that produced a substantial current block. 4.,Although quinacainol did not effectively block voltage-dependent potassium currents, even at concentrations as high as 1.5 mmol/L, quinidine, at a half-maximal sodium channel-blocking concentration, reduced peak ito current amplitude, increased the rate of inactivation of ito and blocked iKsus. 5.,These results indicate that quinacainol, a quinidine analogue, blocks sodium currents in cardiac myocytes with little effect on ito or iKsus potassium currents, which suggests that quinacainol may be exerting class 1c anti-arrhythmic actions. [source] Heterogeneity Of The Properties Of INa in Epicardial And Endocardial Cells Of Rat VentricleCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 3 2002Bonny N Honen SUMMARY 1. Ventricular INa heterogeneity was investigated in adult rat hearts. Differences in transient outward potassium current (Ito) were used to confirm isolation of subepicardial and subendocardial cells. Mean peak Ito was 6.0 ± 0.7 and 1.6 ± 0.45 pA/pF in epicardial and endocardial cells, respectively (P < < 0.01). 2. Maximum sodium conductance was smaller in subendocardial cells compared with subepicardial cells (2.39 ± 0.11 vs 2.78 ± 0.12 nS/pF, respectively; n = 17 for both; 0.01 < P < 0.05) and 50% activation occurred at a slightly more negative potential (,47.6 ± 0.8 vs,44.9 ± 0.9 mV, respectively; n = 10 for both; 0.01 < P < 0.05). 3. The potential for 50% inactivation was not significantly different in subepicardial compared with subendocardial cells (72.2 ± 1.0 vs 72.8 ± 2.2 mV, respectively; n = 17 for both; NS). 4. Persistent sodium current density appeared smaller in subendocardial (n = 19) compared with subepicardial (n = 11) cells (at a test potential of ,25 mV current, density was 0.118 ± 0.041 vs 0.144 ± 0.085 pA/pF, respectively), although this was not statistically significant due to large variability between cells. 5. Mathematical modelling of the cardiac action potential indicated that the combined effects of differences in current density and voltage dependence of sodium currents are unlikely to contribute to ventricular action potential heterogeneity between epicardial and endocardial cells. [source] | |||||||||||||||||||||||||||||||