Functional and molecular identification of ERG channels in murine portal vein myocytes

Ion channels encoded byether-à-go-go-related genes (ERG) have been implicatedin repolarization of the cardiac action potential and also ascomponents of the resting membrane conductance in various cells. Theaim of the present study was to determine whether ERG channels wereexpressed in smooth muscle cells isolated from portal vein. RT-PCRdemonstrated the expression of murine ERG (mERG), and real-timequantitative PCR showed that the mERG1b isoform predominated over themERG1a, mERG2, and mERG3 in portal vein. Single myocytes from portalvein displayed membrane staining with an ERG1-specific antibody. Wholecell voltage-clamp experiments were performed to determine whetherportal vein myocytes expressed functional ERG channels. Large inwardcurrents with distinctive kinetics were elicited that were inhibitedrapidly by E-4031 (mean amplitude of the E-4031-sensitive current at120 mV was 205 ± 24 pA; n = 14). Deactivationof the E-4031-sensitive current was voltage dependent (mean timeconstants at 80 and 120 mV were 103 ± 9 and 33 ± 2 ms,respectively; n = 13). Because of the rapid kinetics ofmERG currents at more negative potentials, there was a substantialnoninactivating "window" current that reached a maximum of66 ± 10 pA at 70 mV. Complete portal veins exhibitedspontaneous contractile activity in isometric tension experiments, andthis activity was modified significantly by E-4031. These data showthat ERG channels are expressed in murine portal vein myocytes that maycontribute to the resting membrane conductance.

     

Characterization of the effects of ryanodine, TTX, E-4031 and 4-AP on the sinoatrial and atrioventricular nodes

AIMS: To characterize the effects of inhibition of Ryanodine receptor (RyR), TTX-sensitive neuronal Na+ current (iNa), "rapidly activating" delayed rectifier K+ current (iKr) and ultrarapid delayed rectifier potassium current (IKur) on the pacemaker activity of the sinoatrial node (SAN) and the atrioventricular node (AVN) in the mouse. METHODS: The structure of mouse AVN was studied by histology and immunolabelling of Cx43 and hyperpolarization-activated, cyclic nucleotide-binding channels (HCN). The effects of Ryanodine, TTX, E-4031 and 4-AP on pacemaker activities recorded from mouse intact SAN and AVN preparations have been investigated. RESULTS: Immuno-histological characterization delineated the structure of the AVN showing the similar molecular phenotype of the SAN. The effects of these inhibitors on the cycle length (CL) of the spontaneous pacemaker activity of the SAN and the AVN were characterized. Inhibition of RyR by 0.2 and 2 microM Ryanodine prolonged CL by 42+/-12.3% and 64+/-18.1% in SAN preparations by 163+/-72.3% and 241+/-91.2% in AVN preparations. Inhibition of TTX-sensitive iNa by 100 nM TTX prolonged CL by 22+/-6.0% in SAN preparations and 53+/-13.6% in the AVN preparations. Block of iKr by E-4031 prolonged CL by 68+/-12.5% in SAN preparations and 28+/-3.4% in AVN preparations. Inhibition of iKur by 50 microM 4-AP prolonged CL by 20+/-3.4% in SAN preparations and 18+/-3.0% in AVN preparations. CONCLUSION: Mouse SAN and AVN showed distinct different response to the inhibition of RyR, TTX-sensitive INa, IKr and iKur, which reflects the variation in contribution of these currents to the pacemaker function of the cardiac nodes in the mouse. Our data provide valuable information for developing virtual tissue models of mouse SAN and AVN.     

Activation and inactivation kinetics of an E-4031-sensitive current from single ferret atrial myocytes.

Ferret atrial myocytes can display an E-4031-sensitive current (IKr) that is similar to that previously described for guinea pig cardiac myocytes. We examined the ferret atrial IKr as the E-4031-sensitive component of current using the amphotericin B perforated patch-clamp technique. Steady-state IKr during depolarizing pulses showed characteristic inward rectification. Activation time constants during a single pulse were voltage dependent, consistent with previous studies. However, for potentials positive to +30 mV, IKr time course became complex and included a brief transient component. We examined the envelope of tails of the drug-sensitive current for activation in the range -10 to +50 mV and found that the tail currents for IKr do not activate with the same time course as the current during the depolarizing pulse. The activation time course determined from tail currents was relatively voltage insensitive over the range +30 to +50 mV (n = 5), but was voltage sensitive for potentials between -10 and +30 mV and appeared to show some sigmoidicity in this range. These data indicate that activation of IKr occurs in at least two steps, one voltage sensitive and one voltage insensitive, the latter of which becomes rate limiting at positive potentials. We also examined the rapid time-dependent inactivation process that mediates rectification at positive potentials. The time constants for this process were only weakly voltage dependent over the range of potentials from -50 to +60 mV. From these data we constructed a simple linear four-state model that reproduces the general features of ferret IKr, including the initial transient at positive potentials and the apparent discrepancy between the currents during the initial depolarizing pulse and the tail current.     

Two components of cardiac delayed rectifier K+ current. Differential sensitivity to block by class III antiarrhythmic agents

An envelope of tails test was used to show that the delayed rectifier K+ current (IK) of guinea pig ventricular myocytes results from the activation of two outward K+ currents. One current was specifically blocked by the benzenesulfonamide antiarrhythmic agent, E-4031 (IC50 = 397 nM). The drug-sensitive current, "IKr" exhibits prominent rectification and activates very rapidly relative to the slowly activating drug-insensitive current, "IKs." IKs was characterized by a delayed onset of activation that occurs over a voltage range typical of the classically described cardiac IK. Fully activated IKs, measured as tail current after 7.5-s test pulses, was 11.4 times larger than the fully activated IKr. IKr was also blocked by d-sotalol (100 microM), a less potent benzenesulfonamide Class III antiarrhythmic agent. The activation curve of IKr had a steep slope (+7.5 mV) and a negative half-point (-21.5 mV) relative to the activation curve of IKs (slope = +12.7 mV, half-point = +15.7 mV). The reversal potential (Erev) of IKr (-93 mV) was similar to EK (-94 mV for [K+]o = 4 mM), whereas Erev of IKs was -77 mV. The time constants for activation and deactivation of IKr made up a bell-shaped function of membrane potential, peaking between -30 and -40 mV (170 ms). The slope conductance of the linear portion of the fully activated IKr-V relation was 22.5 S/F. Inward rectification of this relation occurred at potentials greater than -50 mV, resulting in a voltage-dependent decrease in peak IKr at test potentials greater than 0 mV. Peak IKr at 0 mV averaged 0.8 pA/pF (n = 21). Although the magnitude of IKr was small relative to fully activated IKs, the two currents were of similar magnitude when measured during a relatively short pulse protocol (225 ms) at membrane potentials (-20 to +20 mV) typical of the plateau phase of cardiac action potentials.     

Ether-a-go-go-related gene 3 is the main candidate for the E-4031-sensitive potassium current in the pacemaker interstitial cells of Cajal

The interstitial cells of Cajal (ICC), as pacemaker cells of the gut, contribute to rhythmic peristalsis and muscle excitability through initiation of slow-wave activity, which subsequently actively propagates into the musculature. An E-4031-sensitive K(+) current makes a critical contribution to membrane potential in ICC. This study provides novel features of this current in ICC in physiological solutions and seeks to identify the channel isoform. In situ hybridization and Kit immunohistochemistry were combined to assess ether-a-go-go-related gene (ERG) mRNA expression in ICC in mouse jejunum, while the translated message was assessed by immunofluorescence colocalization of ERG and Kit proteins. E-4031-sensitive currents in cultured ICC were studied by the whole cell patch-clamp method, with physiological K(+) concentration in the bath and the pipette. In situ hybridization combined with Kit immunohistochemistry detected m-erg1 and m-erg3, but not m-erg2, mRNA in ICC. ERG3 protein was colocalized with Kit-immunoreactive ICC in jejunum sections, but ERG1 protein was visualized only in the smooth muscle cells. At physiological K(+) concentration, the E-4031-sensitive outward current in ICC was different from its counterpart in cardiac and gut smooth muscle cells. In particular, inactivation upon depolarization and recovery from inactivation by hyperpolarization were modest in ICC. In summary, the E-4031-sensitive currents influence the kinetics of the pacemaker activity in ICC and contribute to maintenance of the resting membrane potential in smooth muscle cells, which together constitute a marked effect on tissue excitability. Whereas this current is mediated by ERG1 in smooth muscle, it is primarily mediated by ERG3 in ICC.     

ERG K+ currents regulate pacemaker activity in ICC

Ether-à-go-go-related gene (ERG) K channels have been implicated in the generation of pacemaker activities in the heart. To study the presence and function of ERG K channels in the pacemaker cells of the small intestine [the interstitial cells of Cajal (ICC)], a combination of patch-clamp techniques, tissue and live cell immunohistochemistry, RT-PCR, and in vitro functional studies were performed. Nonenzymatically isolated ICC in culture were identified by vital staining and presence of rhythmic inward currents. RT-PCR showed the presence of ERG mRNA in the intestinal musculature, and immunohistochemistry on tissue and cultured cells demonstrated that protein similar to human ERG was concentrated on ICC in the Auerbach's plexus region. Whole cell ERG K+ currents were evoked on hyperpolarization from 0 mV (but not from -70 mV) up to -120 mV and showed strong inward rectification. The currents were inhibited by E-4031, cisapride, La3+, and Gd3+ but not by 50 microM Ba2+. The ERG K+ inward current had a typical transient component with fast activation and inactivation kinetics followed by significant steady-state current. E-4031 also inhibited tetraethylammonium (TEA)-insensitive outward current indicating that the ERG K+ current is operating at depolarizing potentials. In contrast to TEA, blockers of the ERG K+ currents caused marked increase in tissue excitability as reflected by an increase in slow-wave duration and an increase in superimposed action potential activity. In summary, ERG K channels in ICC contribute to the membrane potential and play a role in regulation of pacemaker activity of the small intestine.     

Postnatal development of E-4031-sensitive potassium current in rat carotid chemoreceptor cells.

The O2 sensitivity of dissociated type I cells from rat carotid body increases with age until approximately 14-16 days. Hypoxia-induced depolarization appears to be mediated by an O2-sensitive K+ current, but other K+ currents may modulate depolarization. We hypothesized that membrane potential may be stabilized in newborn type I cells by human ether-a-go-go-related gene (HERG)-like K+ currents that inhibit hypoxia-induced depolarization and that a decrease in this current with age could underlie, in part, the developmental increase in type I cell depolarization response to hypoxia. In dissociated type I cells from 0- to 1- and 11- to 16-day-old rats, using perforated patch-clamp and 70 mM K+ extracellular solution, we measured repolarization-induced inward K+ tail currents in the absence and presence of E-4031, a specific HERG channel blocker. This allowed isolation of the E-4031-sensitive HERG-like current. E-4031-sensitive peak currents in type I cells from 0- to- 1-day-old rats were 2.5-fold larger than in cells from 11- to 16-day-old rats. E-4031-sensitive current density in newborn type I cells was twofold greater than in cells from 11- to 16-day-old rats. Under current clamp conditions, E-4031 enhanced hypoxia-induced depolarization in type I cells from 0- to- 1-day-old but not 11- to 16-day-old rats. With use of fura 2 to measure intracellular Ca2+, E-4031 increased the cytosolic Ca2+ concentration response to anoxia in cells from 0- to- 1-day-old but not cells from 11- to 16-day-old rats. E-4031-sensitive K+ currents are present in newborn carotid body type I cells and decline with age. These findings are consistent with a role for E-4031-sensitive K+ current, and possibly HERG-like K+ currents, in the type I cell hypoxia response maturation.     

Clotrimazole-sensitive K+ currents regulate pacemaker activity in interstitial cells of Cajal

Interstitial cells of Cajal (ICC) are pacemaker cells for gut peristaltic motor activity. Compared with cardiac pacemaker cells, little is known about mechanisms that regulate ICC excitability. The objective of the present study was to investigate a potential role for clotrimazole (CTL)-sensitive K currents (I(CTL)) in the regulation of ICC excitability and pacemaker activity. ICC were studied in situ and in short-term culture by using the whole cell patch-clamp configuration. In situ, ICC exhibited spontaneous transient inward currents followed by transient outward currents. CTL blocked outward currents, thereby increasing the net inward currents, and depolarized ICC, thereby establishing CTL-sensitive channels as regulators of ICC pacemaker activity. In short-term culture, a I(CTL) was identified that showed increased conductance when depolarized from the resting membrane potential to 0 mV and subsequent inward rectification at further depolarized potentials. The I(CTL) markedly increased with increasing intracellular calcium and was insensitive to the ether-à-go-go-related K channel blocker E-4031 and the large-conductance calcium-activated K channel blocker iberiotoxin. I(CTL) contributed 3-9 nS to the whole cell conductance at 0 mV membrane potential under physiological conditions; it was fast activating (tau = 88 ms), showed little time-dependent inactivation, and exhibited a deactivation time constant of 38 ms. The nitric oxide donor sodium nitroprusside (SNP) increased I(CTL). Single-channel activity, activated by calcium and SNP, was inhibited by CTL, with a single-channel conductance of approximately 38 pS. In summary, ICC generate a I(CTL) on depolarization through an intermediate-conductance calcium-activated K channel that regulates pacemaker activity and ICC excitability.     

Role of dual pacemaker mechanisms in sinoatrial node discharge

We investigated whether in the sinoatrial node (SAN) there are two different pacemaker mechanisms and whether either one can maintain spontaneous discharge. These questions were studied by means of an electrophysiological technique and of blockers of different diastolic currents in rabbit and guinea pig isolated SAN. In SAN subsidiary pacemakers of both species, Cs(+) (5-10 mM) or high [K(+)](o) (10-12 mM) decreased the maximum diastolic potential, abolished diastolic depolarization (DD) at polarized levels (subsidiary DD), unmasked a U-shaped dominant DD at depolarized levels, but did not stop the SAN. In rabbit SAN, E4031 (1 microM) and d-sotalol (100 microM) did not stop discharge, but did so after block of subsidiary DD by high [K(+)](o) or Cs(+). In guinea pig SAN, in Tyrode solution E4031, d-sotalol or indapamide (100 microM) did not stop SAN discharge. In the presence of Cs(+) or high [K(+)](o) indapamide (but not E4031 or d-sotalol) stopped the SAN. Ba(2+) (1-5 mM) led to stoppage of discharge both in Tyrode solution and in high [K(+)](o) or Cs(+). Depolarization by blockers of DD unmasked sinusoidal fluctuations, which during recovery were responsible for resumption of discharge. We conclude that in rabbit and guinea pig SAN, two different pacemaker mechanisms (Cs(+)- and K(+)-sensitive subsidiary DD, and Cs(+)- and K(+)-insensitive dominant DD) can independently sustain discharge, but block of both mechanisms leads to quiescence. Abolition of dominant DD by blockers of I(K) is consistent with a decay of I(K) as the dominant pacemaking mechanism, I(Kr) being more important in rabbit and I(Ks) in guinea pig. Sinusoidal fluctuations appear to be an essential component of the pacemaking process.     

Pharmacological and biophysical isolation of K+ currents encoded by ether-a-go-go-related genes in murine hepatic portal vein smooth muscle cells

Previous studies have shown that murine portal vein myocytes express ether-à-go-go related genes (ERGs) and exhibit distinctive currents when recorded under symmetrical K⁺ conditions. The aim of the present study was to characterize ERG channel currents evoked from a negative holding potential under conditions more pertinent to a physiological scenario to assess the possible functional impact of this conductance. Currents were recorded with ruptured or perforated patch variants of the whole cell technique from a holding potential of –60 mV. Application of three structurally distinct and selective ERG channel blockers, E-4031, dofetilide, and the peptide toxin BeKM-1, all inhibited a significant proportion of the outward current and abolished inward currents with distinctive "hooked" kinetics recorded on repolarization. Dofetilide-sensitive currents at negative potentials evoked by depolarization to +40 mV had a voltage-dependent time to peak and rate of decay characteristic of ERG channels. Application of the novel ERG channel activator PD-118057 (1–10 µM) markedly enhanced the hooked inward currents evoked by membrane depolarization and hyperpolarized the resting membrane potential recorded by current clamp and the perforated patch configuration by 20 mV. In contrast, ERG channel blockade by dofetilide (1 µM) depolarized the resting membrane potential by 8 mV. These data are the first record of ERG channel currents in smooth muscle cells under quasi-physiological conditions that suggest that ERG channels contribute to the resting membrane potential in these cells. vascular smooth muscle; voltage-dependent K⁺ current; membrane excitability     

Sodium nitroprusside increases pacemaker rhythm of sinoatrial nodes via nitric oxide-cGMP pathway

Effects of sodium nitroprusside (SNP), a nitric oxide donor, on the action potential in isolated guinea-pig sinoatrial nodes and ventricular papillary muscles were investigated. In the driven ventricular papillary muscle, SNP (10(-10)-10(-3) M) decreased the twitch tension in a concentration-dependent manner without significantly changing the configuration of action potential and the maximal velocity of depolarizing upstroke. In isolated sinoatrial nodes, SNP (10(-8)-10(-3) M) increased the pacemaker rhythm in a concentration-dependent manner. At 10(-5) M SNP, the pacemaker activity increased from 197.2+/-6.1 to 221.4+/-9.7 bpm. Changes of configuration of the action potential included a decrease of the duration of repolarization, i.e., from peak to the maximal diastolic potential (MDP), from 141.4+/-6.4 to 130.0+/-7.0 ms and an increase of the slope of the diastolic membrane potential from 101.6+/-5.3 to 116.5+/-7.3 mV/s (n=6, p<0.05). However, MDP and threshold potential were not significantly changed. Methylene blue (MB, 10(-5) M), a guanylate cyclase inhibitor, significantly decreased the pacemaker activity of the sinoatrial node by increasing the durations of repolarization and diastolic depolarization. After pretreatment with 10(-5) M MB, the effect of SNP was inhibited. The results indicate that nitric oxide, released from SNP, increases the pacemaker activity by enhancing the rates of repolarization and diastolic depolarization. These effects are possibly due to increases in delayed-rectifier K+ and diastolic slow inward currents, which are involved in a mechanism associated with the NO-cGMP pathway.     

Role of HERG-like K+ currents in opossum esophageal circular smooth muscle

An inwardlyrectifying K⁺ conductance closelyresembling the human ether-a-go-go-related gene (HERG) current wasidentified in single smooth muscle cells of opossum esophageal circularmuscle. When cells were voltage clamped at 0 mV, in isotonicK⁺ solution (140 mM), stephyperpolarizations to 120 mV in 10-mV increments resulted inlarge inward currents that activated rapidly and then declined slowly(inactivated) during the test pulse in a time- and voltage- dependentfashion. The HERG K⁺ channelblockers E-4031 (1 µM), cisapride (1 µM), andLa³⁺ (100 µM) strongly inhibitedthese currents as did millimolar concentrations ofBa²⁺. Immunoflourescence stainingwith anti-HERG antibody in single cells resulted in punctate stainingat the sarcolemma. At membrane potentials near the resting membranepotential (50 to 70 mV), thisK⁺ conductance did not inactivatecompletely. In conventional microelectrode recordings, both E-4031 andcisapride depolarized tissue strips by 10 mV and also induced phasiccontractions. In combination, these results provide direct experimentalevidence for expression of HERG-likeK⁺ currents in gastrointestinalsmooth muscle cells and suggest that HERG plays an important role inmodulating the resting membrane potential.

     

KvLQT1 modulates the distribution and biophysical properties of HERG. A novel alpha-subunit interaction between delayed rectifier currents

Cardiac repolarization is under joint control of the slow (IKs) and rapid (IKr) delayed rectifier currents. Experimental and clinical evidence indicates important functional interactions between these components. We hypothesized that there might be more direct interactions between the KvLQT1 and HERG alpha-subunits of IKs and IKr and tested this notion with a combination of biophysical and biochemical techniques. Co-expression of KvLQT1 with HERG in a mammalian expression system significantly accelerated HERG current deactivation at physiologically relevant potentials by increasing the contribution of the fast component (e.g. upon repolarization from +20 mV to -50 mV: from 20 +/- 3 to 32 +/- 5%, p < 0.05), making HERG current more like native IKr. In addition, HERG current density was approximately doubled (e.g. tail current after a step to +10 mV: 18 +/- 3 versus 39 +/- 7 pA/picofarad, p < 0.01) by co-expression with KvLQT1. KvLQT1 co-expression also increased the membrane immunolocalization of HERG by approximately 2-fold (p < 0.05). HERG and KvLQT1 co-immunolocalized in canine ventricular myocytes and co-immunoprecipitated in cultured Chinese hamster ovary cells as well as in native cardiac tissue, indicating physical interactions between HERG and KvLQT1 proteins in vitro and in vivo. Protein interaction assays also demonstrated binding of KvLQT1 (but not another K+ channel alpha-subunit, Kv3.4) to a C-terminal HERG glutathione S-transferase fusion protein. Co-expression with HERG did not affect the membrane localization or ionic current properties of KvLQT1. This study shows that the alpha-subunit of IKs can interact with and modify the localization and current-carrying properties of the alpha-subunit of IKr, providing potentially novel insights into the molecular function of the delayed rectifier current system.     

Characterization of two distinct depolarization-activated K+ currents in isolated adult rat ventricular myocytes

Depolarization-activated outward K+ currents in isolated adult rat ventricular myocytes were characterized using the whole-cell variation of the patch-clamp recording technique. During brief depolarizations to potentials positive to -40 mV, Ca(2+)-independent outward K+ currents in these cells rise to a transient peak, followed by a slower decay to an apparent plateau. The analyses completed here reveal that the observed outward current waveforms result from the activation of two kinetically distinct voltage-dependent K+ currents: one that activates and inactivates rapidly, and one that activates and inactivates slowly, on membrane depolarization. These currents are referred to here as Ito (transient outward) and IK (delayed rectifier), respectively, because their properties are similar (although not identical) to these K+ current types in other cells. Although the voltage dependences of Ito and IK activation are similar, Ito activates approximately 10-fold and inactivates approximately 30-fold more rapidly than IK at all test potentials. In the composite current waveforms measured during brief depolarizations, therefore, the peak current predominantly reflects Ito, whereas IK is the primary determinant of the plateau. There are also marked differences in the voltage dependences of steady-state inactivation of these two K+ currents: IK undergoes steady-state inactivation at all potentials positive to -120 mV, and is 50% inactivated at -69 mV; Ito, in contrast, is insensitive to steady-state inactivation at membrane potentials negative to -50 mV. In addition, Ito recovers from steady-state inactivation faster than IK: at -90 mV, for example, approximately 70% recovery from the inactivation produced at -20 mV is observed within 20 ms for Ito; IK recovers approximately 25-fold more slowly. The pharmacological properties of Ito and IK are also distinct: 4-aminopyridine preferentially attenuates Ito, and tetraethylammonium suppresses predominantly IK. The voltage- and time-dependent properties of these currents are interpreted here in terms of a model in which Ito underlies the initial, rapid repolarization phase of the action potential (AP), and IK is responsible for the slower phase of AP repolarization back to the resting membrane potential, in adult rat ventricular myocytes.     

Ventricular automaticity induced by bromobenzoyl-methyladamantylamine (BMA) in different membrane potential ranges in guinea pig heart

The electrophysiological effects of bromobenzoyl - methyladamantylamine ( BMA ) were investigated in isolated electrically driven right ventricular papillary muscles of guinea pigs using conventional glass-microelectrode technique. BMA markedly increased the action potential duration, depolarized the membrane, reduced the maximum rate of depolarization (Vmax) and induced pacemaker-like action potentials. In ventricular myocardium depolarized partially (up to --40 mV) by incubation with 26 mM K+-Krebs solution, BMA induced slow action potentials. In these preparations, BMA was also able to evoke automaticity. Since the pacemaker activity occurring in the voltage range of --90 mV to --60 mV has been attributed to the deactivation of a pacemaker K+ current labelled IK2, and that occurring in the plateau range (from --40 mV to +10 mV) has been attributed to the deactivation of an outward plateau K+ current labelled IX1 , it can be concluded that BMA may inhibit both IK2 and IX1 currents.     

兔主动脉前庭自律细胞与窦房结电生理特性的比较

为进一步阐明左心室流出道(主动脉前庭)自律细胞的特性,及其与窦房结细胞的异同,本实验利用常规的玻璃微电极细胞内记录技术,观察了一些离子通道阻断剂分别对离体兔窦房结起搏细胞与左心室流出道慢反应自律细胞的电生理特性的影响,重点探讨了这两种自律细胞的0期、4期去极离子流的异同。结果表明:(1)用1μmol/L维拉帕米(verapamil,VER)灌流后,窦房结及主动脉前庭自律细胞的动作电位幅值(APA)、0相最大除极速率(V_(max))、最大舒张电位(MDP)绝对值、舒张期除极速率(VDD)、自发放电频率(RPF)均明显下降,复极90％时间(APD_(90))延长(P<0.05)。(2)用180μmol/L氯化镍(NiCl_2)灌流,两自律细胞的VDD均明显下降;APA、V_(max)和RPF也显著降低,且窦房结细胞的APD_(90)明显延长。(3)给予2 mmol/L 4-氨基吡啶(4-AP)后,窦房结及主动脉前庭自律细胞的VDD均明显增快,MDP绝对值、APA和V_(max)显著下降,APD_(90)明显延长(P<0.05)。(4)给予2 mmol/L氯化铯(CsCl),两自律细胞的VDD及RPF均明显变慢。结果提示:(1)主动脉前庭自发慢反应电位的0相、4相去极离子流及复极离子流均与窦房结优势起搏细胞相似。(2)主动脉前庭起搏细胞Ca~(2+)内流为其0相主要去极离子流,复极过程主要由K~+外流引起,4相自动除极以K~+外流衰减为主,另外     

Properties of aconitine-modified sodium channels in single cells of mouse ventricular myocardium

Effects of the plant alkaloid Aconitine on the kinetics of sodium channels were studied in enzymatically isolated single cells of the mouse ventricular myocardium. Aconitine (1 mumol/l) induced a prolongation of the 90% repolarization of action potentials from 52.4 +/- 3.7 ms to 217.0 +/- 12.5 ms. Delayed terminal repolarization and oscillatory afterpotentials preceded spontaneous activity with high frequencies. Peak sodium currents were diminished from 28.0 +/- 9.0 to 14.0 +/- 6.0 nA. The reversal potential of the sodium current was shifted from 16.0 +/- 11.0 to -8.0 +/- 6.0 mV (52.5 mmol/l extracellular sodium concentration) suggesting a decreased selectivity of the Aconitine-modified Na channels. The m-affinity-curves were shifted 31 mV towards more negative potentials at a constant slope. The h affinity-curves were shifted in the same direction by 13 mV. The slope parameter of the h affinity-voltage relationship was enlarged from 9.1 +/- 2.2 mV to 15.6 +/- 4.4 mV. Shifts in m affinity and h affinity resulted in an increased "window". The alkaloid modified channels inactivated extremely slowly at potentials negative to -40 mV, but showed a fast and complete inactivation at potentials positive to -40 mV.