Origin of the background sodium current and effects of sodium removal in cultured embryonic cardiac cells

Cardiac automaticity is partly due to a diastolic sodium current. Possible mediators of this include tetrodotoxin-sensitive "fast" channels, cesium-sensitive time-dependent pacemaker current channels, calcium-gated nonspecific channels, and electrogenic sodium-calcium exchange. We have studied the effects of abrupt sodium removal on membrane current and conductance in voltage-clamped chick embryonic myocardial cell aggregates, in the presence of various sodium flux inhibitors. Total replacement of sodium by lithium, Tris, or tetraethylammonium ions in aggregates clamped in the pacemaker range caused a brief outward current followed by a sustained net inward current. The outward current reached a peak value of 1.1 +/- 0.5 microA/cm2 at a mean latency of 5.4 +/- 1.2 sec. (n = 6; V = -70.5 +/- 8.9 mV; Tris). Conductance often decreased during the outward current. The inward current developed exponentially (t = 19 +/- 5 sec) and reached a steady state value of -1.6 +/- 0.4 microA/cm2. This current was reversed by depolarization (mean reversal potential = -13 +/- 13 mV), and was accompanied by increased conductance and spontaneous mechanical activity. Neither of the sodium-removal currents was affected by 20 microM tetrodotoxin. Cesium (up to 20 mM) had no effect on the late inward current or the mechanical activity, but decreased the early outward current by 80 +/- 12%. Manganese (25 mM), which blocks sodium-calcium exchange, abolished the late inward current and the mechanical activity. Manganese also reduced the early outward current by 27 +/- 10%. Manganese and cesium together blocked all the effects of sodium removal. We conclude that removal of extracellular sodium interrupts a cesium-sensitive "background" current, that may be related to the time-dependent pacemaker current, If. Sodium removal also causes gradual activation of a nonspecific conductance, which can ultimately depolarize the cells, and which may be gated by cytoplasmic calcium.     

Inositol trisphosphate promotes Na-Ca exchange current by releasing calcium from sarcoplasmic reticulum in cardiac myocytes

An early inward tail current evoked by membrane depolarization (from -80 to -40 mV) sufficient to activate sodium but not calcium current was studied in single voltage-clamped ventricular myocytes isolated from guinea pig hearts. Like forward-mode Na-Ca exchange, this early inward tail current required [Na+]o and [Ca2+]i and is thought to follow earlier reverse-mode Na-Ca exchange that triggers Ca2+ release from sarcoplasmic reticulum. The dependence of the early inward tail current on [Ca2+]i was supported by the ability of small (+10 mV) and large (+80 mV) voltage jumps from -40 mV to decrease and increase, respectively, the size of early inward tail currents evoked by subsequent voltage steps from -80 to -40 mV. As expected, tetrodotoxin selectively inhibited the early inward tail current but not the late inward tail current that followed voltage jumps to +40 mV test potentials. Although tetrodotoxin also blocked the fast Na+ current, replacement of extracellular Na+ by Li+ sustained the fast Na+ current. However, Li+, which does not support Na-Ca exchange, reversibly suppressed both the early and late inward tail currents. Inhibitors (ryanodine and caffeine) and promoters (intracellularly dialyzed inositol 1,4,5-trisphosphate) of sarcoplasmic reticulum Ca2+ release decreased and increased, respectively, the magnitude of the early inward tail current. The results substantiate the hypothesis that Ca2+ release from the sarcoplasmic reticulum participates in early Na-Ca exchange current and demonstrate that inositol 1,4,5-trisphosphate, by releasing Ca2+ from the sarcoplasmic reticulum, can promote Na-Ca exchange across the plasma membrane.     

Potassium current suppression by quinidine reveals additional calcium currents in neuroblastoma cells.

Quinine and quinidine have been evaluated with regard to their effects on the electrical activity of neuroblastoma cells. Under voltage-clamp conditions, we have found that quinine and quinidine block both the voltage-dependent and Ca2+-dependent K+ conductances. Blockage of the voltage-dependent K+ channel is manifest as an increase in the amplitude and in the duration of the action potential. Blockage of the Ca2+-dependent K+ channel in Na+-free (replaced by Tris) solutions containing 6.8 mM Ca2+ and tetraethylammonium ion or 4-aminopyridine (to block the voltage-dependent K+ current) is seen as a further prolongation of the Ca2+ action potential and diminution of the after-hyperpolarization. A critical role of the Ca2+-dependent K+ conductance in modulation of the rate and duration of trains of Ca2+ action potentials is shown by the use of low concentrations (5-40 microM) of quinine or quinidine, which diminish the Ca2+-dependent K+ conductance in a graded manner. After complete blockade of K+ currents, the peak Ca2+ currents are enhanced at all voltages, especially at values more positive than -30 mV, where a steady-state inward current appears as well. In this same voltage range, the decay of the Ca2+ current exhibits two time constants--that of the transient inward current, which is about 20 msec, and a much slower (approximately 2000 msec) component. It is suggested that neuroblastoma cells have two types of calcium channels--one which generates the Ca2+ action potential and a second, distinguished by activation at more depolarized levels and by a slow rate of inactivation, which underlies the calcium entry necessary to activate the Ca2+-dependent K+ conductance.     

Egg-laying hormone of Aplysia induces a voltage-dependent slow inward current carried by Na+ in an identified motoneuron.

This report presents studies on ionic currents in Aplysia motoneuron B16 that are modulated by the neuropeptide egg-laying hormone (ELH) of Aplysia. ELH induces an inward current that persists in the presence of the peptide and that decays slowly after ELH is removed from the bath. The effect is not due to a decrease in the delayed potassium current, the calcium-activated potassium current, or the transient potassium current. Current-voltage measurements indicate that ELH produces increased inward currents from -80 mV to approximately equal to 0 mV. The effect is particularly enhanced in the region from -40 mV to -25 mV where a negative slope conductance due to voltage-dependent slow inward current is observed. The slow inward current and the response to ELH persist in saline solutions in which Ca2+ is replaced with Co2+ but are eliminated when Na+ is replaced with equimolar concentrations of either Tris or N-methyl-D-glucamine. The response to ELH is unaffected by replacing chloride with equimolar acetate; by increasing the potassium concentration; or by adding tetraethylammonium chloride, CsCl, 4-amino-pyridine, or tetrodotoxin to the saline bath. In addition, the reversal potentials for the ELH response (range, -28 to +46 mV), obtained from difference current-voltage relationships, are consistent with an increase in the Na+-dependent slow inward current. We conclude that at least one of the effects of ELH on B16 is to increase a slow inward current carried by Na+.     

Two levels of resting potential in canine cardiac Purkinje fibers exposed to sodium-free solutions.

Canine cardiac Purkinje fibers exposed to sodium-free solutions containing 16 mM CaCl2, 20 mM tetraethylammonium chloride, 108 mM tetramethylammonium chloride, and 2.7 mM KCl may be quiescent at a resting potential of either -50 mV or -90 mV. The membrane potential of these fibers can be switched from -50 mV to -90 mV by a hyperpolarizing current pulse and from -90 mV to -50 mV by a depolarizing current pulse. The transition from -50 mV to -90 mV depends on a voltage-dependent increase in potassium conductance, that conductance being low at -50 mV and high at -90 mV. A reduction in potassium conductance causes the fiber to depolarize from -90 mV to -50 mV because of the presence of an inward current which apparently is carried mainly by Ca. Fibers that show a high resting potential cannot be excited except by depolarizing stimuli strong enough to move the membrane from -90 mV to a threshold potential of about -40 mV. Fibers that show a low resting potential are more easily excited and may show rhythmic activity sustained by afterpotentials that appear only if the low membrane potential is accompanied by a low potassium conductance. Slow changes in membrane potential also are seen; these changes may result from movements of chloride.     

Outward potassium currents in freshly isolated smooth muscle cell of dog coronary arteries

Outward membrane currents were characterized in single coronary smooth muscle cells of adult beagle dogs. The cells averaged 96.4 x 7.1 microns and had a resting potential of -50.7 mV, an input resistance of 307.9 M omega, a capacitance of 32.3 pF, and a calculated membrane surface area of 4,037 microns2. The cells contracted in response to external application of acetylcholine or high K+. In voltage clamp by use of the suction pipette method, outward current began to appear at -50 mV and reached 15.2 nA at 50 mV with a current density of 376.5 microA/cm2. The current was reduced by external tetraethylammonium, Ba2+, and internal Cs+, and its reversal potential had a Nernst relation to external K+ concentration. Elevation of external Ca2+ (Ca2+o) from 0 to 0.3 mM increased total K+ current by up to 300%; elevation of internal Ca2+ (Ca2+i) to 5 x 10(-7) M by internal perfusion increased total outward current to a similar extent, suggesting a large difference in Ca2+ transmembrane sensitivity. Total whole-cell K+ current consisted of two components: an initial time-independent current (Ii) followed by a time-dependent current (It). Ii and It were through separate K+ channels based on differences in a) sensitivity to Ca2+09b) modulation by an inward Ca2+ current, c) current amplitudes and activation kinetics, and d) responses to pharmacological agents. It was the largest component, measuring 4.5 nA in 0 mM Ca2+o but increasing to 11.9 nA in 0.3 mM Ca2+o with a steep 2.5 power function. It activated with a biexponential time course; in Ca2+o-free solution, its time course was relatively insensitive to voltage changes but became voltage sensitive in the presence of Ca2+o. Further, such sensitivity was abolished or enhanced by Co2+ or Bay K 8644, respectively. We concluded that there are two types of Ca2+-sensitive K+ currents, Ii and It, in coronary smooth muscle cells. Via an inward Ca2+ channel Ca2+o strongly modulates It, both in amplitude and kinetics.     

Electrophysiological properties of cultured dog myocytes obtained by endomyocardial biopsy

Right ventricular cardiac tissue (10-20 mg wet weight) was obtained from anesthetized adult dogs by endomyocardial biopsy. The biopsy could be repeated in one dog every 2 weeks for up to 3 months. Fifty to 200 cardiomyocytes, dispersed with collagenase and trypsin, were collected by centrifugation of the cells with 50% polysucrose-sodium diatrizoate solution (Ficoll-Paque). Single cardiomyocytes were suspended in a minimum essential medium containing 20% fetal bovine serum and 8-bromoadenosine 3': 5'-cyclic monophosphate (0.1 mM) for up to 3 weeks. Approximately 70-80% of the cultured cardiomyocytes were rod shaped after 24 hours (10-20% after 7 days). Cytoplasmic organelles of the cultured cells, examined with a transmission electron microscope, were within the normal range of canine heart morphology in vivo. Resting membrane potential of the cells was about -80 mV when superfused with a Krebs' solution containing 4.7 mM potassium ions. The action potential lasted for 300 msec and had a peak amplitude of about 120 mV. Voltage-clamp experiments demonstrated the presence of an inward calcium current (congruent to 0.9 nA at +9 mV), which was facilitated by isoproterenol (0.1-1 microMs). The background potassium current showed typical inward rectification at potentials more negative than -80 mV. The results indicate that morphological, electrophysiological, and pharmacological properties of the cultured cardiomyocytes were intact. We propose that the culture techniques we have developed can be useful for repeated investigation on functional aspects of cardiac muscles in myocardial disease.     

Ionic basis for the antagonism between adenosine and isoproterenol on isolated mammalian ventricular myocytes

We studied the effects of adenosine and isoproterenol on membrane currents of isolated bovine and guinea pig ventricular myocytes with a two-microelectrode voltage clamp technique. Adenosine (50 microM to 0.2 mM) alone had no effect on any of the membrane currents measured, but it antagonized the effects induced by 10 nM isoproterenol. Peak calcium membrane current was augmented by isoproterenol from a control of 4.8 +/- 0.6 to 8.6 +/- 0.8 nA and adenosine reduced it to 5.7 +/- 0.7 nA (mean +/- SEM of six cells). The inactivation time constant was not altered by isoproterenol alone or isoproterenol plus adenosine, and neither was the voltage dependence of peak calcium membrane current. Thus, the changes caused by isoproterenol could be described as an increase in maximal calcium conductance from 0.86 +/- 0.7 to 1.55 +/- 0.04 mS/cm2 and partially antagonized by adenosine to 0.97 +/- 0.04 mS/cm2. Isoproterenol also increased the non-inactivating component of calcium membrane current from 17 +/- 1 to 24 +/- 4%, and adenosine reduced it to 18 +/- 2% (n = 4). The steady state activation and inactivation variables remained unchanged. Consistent with these effects on calcium membrane current, adenosine completely antagonized the isoproterenol-induced increase of the slow action potentials obtained in sodium-free medium. Isoproterenol increased the steady state outward currents at potentials between -90 and -30 mV (i.e., probable iK1). Adenosine alone had no effect on potassium membrane current, but it antagonized the effects of isoproterenol. Slow action potentials in 25 mM potassium were enhanced by isoproterenol, but were only moderately attenuated by adenosine. Accordingly, in 25 mM potassium the isoproterenol-induced changes in membrane currents were not antagonized by adenosine. This lack of inhibition by adenosine of the isoproterenol effects in 25 mM potassium could not be mimicked by 1-minute-long conditioning prepulses to -45 mV. The results indicate that adenosine by itself (absence of isoproterenol) has no effect on maximal calcium conductance, that the isoproterenol-induced increase in cyclic adenosine 3',5'-monophosphate, which leads to an increase in maximal calcium conductance, is antagonized by adenosine, and that such action can account for the ability of adenosine to attenuate the stimulatory effects of isoproterenol.     

Gated currents in isolated olfactory receptor neurons of the larval tiger salamander.

The electrical properties of enzymatically isolated olfactory receptor cells were studied with whole-cell patch clamp. Voltage-dependent currents could be separated into three ionic components: a transient inward sodium current, a sustained inward calcium current, and an outward potassium current. Three components of the outward current could be identified by their gating and kinetics: a calcium-dependent potassium current [IK(Ca)], a voltage-dependent potassium current [IK(V)], and a transient potassium current (Ia). Typical resting potentials were near -54 mV, and typical input resistance was 3-6 G omega. Thus, only 3 pA of injected current was required to depolarize the cell to spike threshold near -45 mV. The response to a current step consisted of either a single spike regardless of stimulus strength, or a train of less than 8 spikes, decrementing in amplitude and frequency over approximately equal to 250 msec. Thus, the receptor response cannot be finely graded with stimulus intensity.     

Unblock of the slow inward current induces the arrhythmogenic transient inward current in isolated guinea-pig myocytes.

This study investigated whether abrupt changes in extracellular Ca2+ concentration or washout of the Ca2+ antagonists Mn2+ or verapamil, could induce transient inward current (ITI) in enzymatically disaggregated guinea-pig myocytes. Single electrode voltage-clamp techniques were used. ITI was elicited upon repolarization to various voltage steps from an activating step to +20 mV. The holding potential was -80 mV. Slow inward current (ICa) was induced by steps to -10 mV. Continuous exposure to either 2.5 or 6.0 mM Ca2+ did not induce ITI; however, following exposure of cells to 0.5 mM Ca2+ for 20 min which decreased ICa, return to 2.5 or 6.0 mM Ca2+ induced ITI. ITI could be observed for 10 to 20 min following sudden elevations of Ca2+. Similar effects also were seen when Ca2+ was increased from 2.5 to 6.0 mM. Exposure to 2.0 mM Mn2+ or 2.0 microM verapamil blocked ICa. Washout of either blocker induced ITI, particularly in 6.0 mM Ca2+. Peak ITI occurred upon repolarization at c. -70 mV; a reversal potential could not be demonstrated. Thus, abrupt changes in Ca2+ influx, produced either by sudden changes in external Ca2+ or by washout of Ca2+ antagonists, induced ITI with characteristics similar to those described for ITI induced by toxic concentrations of cardiac glycosides.     

Calcium currents in GH3 cultured pituitary cells under whole-cell voltage-clamp: inhibition by voltage-dependent potassium currents.

To isolate inward Ca2+ currents in GH3 rat pituitary cells, an inward Na+ current as well as two outward K+ currents, a transient voltage-dependent current (IKV) and a slowly rising Ca2+-activated current (IKCa), must be suppressed. Blockage of these outward currents, usually achieved by replacement of intracellular K+ with Cs+, reveals sustained inward currents. Selective blockage of either K+ current can be accomplished in the presence of intracellular K+ by use of quaternary ammonium ions. When IKCa and Na+ currents are blocked, the net current elicited by stepping the membrane potential (Vm) from -60 to 0 mV is inward first, becomes outward and peaks in 10-30 msec, and finally becomes inward again. Under this condition, in which both IKV and Ca2+ currents should be present throughout the duration of the voltage step, the Ca2+ current was not detected at the time of peak outward current. That is, plots of peak outward current vs. Vm are monotonic and are not modified by nisoldipine or low external Ca2+ as would be expected if Ca2+ currents were present. However, similar plots at times other than at peak current are not monotonic and are altered by nisoldipine or low Ca2+ (i.e., inward currents decrease and plots become monotonic). When K+ channels are first inactivated by holding Vm at -30 mV, a sustained Ca2+ current is always observed upon stepping Vm to 0 mV. Furthermore, substitution of Ba2+ for Ca2+ causes blockage of IKV and inhibition of this current results in inward Ba2+ currents with square wave kinetics. These data indicate that the Ca2+ current is completely inhibited at peak outward IKV and that Ca2+ conductance is progressively disinhibited as the transient K+ current declines due to channel inactivation. This suggests that in GH3 cells Ca2+ channels are regulated by IKV.     

Patch-clamp study of the ionic currents underlying action potentials in cultured frog pituitary melanotrophs

The ionic conductance mechanisms underlying the action potential behaviour of frog melanotrophs in primary culture were studied by using the patch-clamp technique in whole-cell configuration. The action potentials spontaneously generated by these cells were predominantly sodium spikes with a calcium component. Voltage-dependent sodium, calcium, potassium and calcium-activated potassium currents were identified and analysed separately. The voltage-dependent sodium current was characterized by its fast kinetic, its low-threshold activation, its voltage-dependent inactivation and a tetrodotoxin sensitivity. Calcium currents were identified on the basis of their ionic selectivity to divalent cations (Ba2+, Ca2+, Co2+) and their time course. Only two of the three well-documented calcium currents could be detected in frog melanotrophs. A sustained calcium current (ICaS) and an inactivating calcium current (ICaN) were elicited by step depolarizations up to -20 mV. ICaN inactivated for membrane potentials more positive than -50 mV; its inactivation appeared to be both voltage- and calcium-dependent. Transient calcium current (ICaT) has never been observed. Two types of potassium currents were identified: voltage-dependent potassium (IKV) and calcium-activated potassium currents, (IK[Ca]). They were both suppressed by tetraethylammonium chloride, whereas only IK(Ca) was blocked by cobalt. These major ionic currents underlying spontaneous electrical activity are assumed to be involved in the process of alpha-melanocyte-stimulating hormone release. The present study provides the ground for future investigations regarding the relationships between the electrical and secretory activities in amphibian pars intermedia cells.     

Ionic currents in single smooth muscle cells of the canine renal artery.

Membrane currents from single smooth muscle cells enzymatically isolated from canine renal artery were recorded using the patch-clamp technique in the whole-cell and cell-attached configurations. These cells exhibited a mean resting potential, input resistance, membrane time constant, and cell capacitance of -51.8 +/- 2.1 mV, 5.2 +/- 0.98 G omega, 116.2 +/- 16.4 msec, and 29.1 +/- 2.0 pF, respectively. Inward current, when elicited from a holding potential of -80 mV, activated near -50 mV, reached a maximum near 0 mV and was sensitive to the dihydropyridine agonist Bay K 8644 and dihydropyridine antagonist nisoldipine. Two components of macroscopic outward current were identified from voltage-step and ramp depolarizations. The predominant charge carrier of the net outward current was identified as K+ by tail-current experiments (reversal potential, -61.0 +/- 0.8 mV in 10.8 mM [K+]o 0 mM [K+]i). The first component was a small, low-noise, voltage- and time-dependent current that activated between -40 and -30 mV (IK(dr)), and the second component was a larger, noisier, voltage- and time-dependent current that activated at potentials positive to +10 mV (IK(Ca)). Both IK(dr) and IK(Ca) displayed little inactivation during long (4-second) voltage steps. IK(Ca) and IK(dr) could be pharmacologically separated by using various Ca2+ and K+ channel blockers. IK(Ca) was substantially inhibited by external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), tetraethylammonium (Ki at +60 mV, 307 microM), and charybdotoxin (100 nM) but was insensitive to 4-aminopyridine (0.1-10 mM). IK(dr) was inhibited by 4-aminopyridine (Ki at +10 mV, 723 microM) and tetraethylammonium (Ki at +10 mV, 908 microM) but was insensitive to external NiCl2 (500 microM), CdCl2 (300 microM), EGTA (5 mM), and charybdotoxin (100 nM). Two types of single K+ channels were identified in cell-attached patches. The most abundant K+ channel that was recorded exhibited voltage-dependent activation, was blocked by external tetraethylammonium (250 microM), and had a large single-channel conductance (232 +/- 12 pS with 150 mM K+ in the patch pipette, 130 +/- 17 pS with 5.4 mM K+ in the patch pipette). The second channel was also voltage dependent, was blocked by 4-aminopyridine (5 mM), and exhibited a smaller single-channel conductance (104 +/- 8 pS with 150 mM K+ in the patch pipette, 57 +/- 6 pS with 5.4 mM K+ in the patch pipette). These results suggest that depolarization of canine renal artery cells opens dihydropyridine-sensitive Ca2+ channels and at least two K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of tetrandrine on calcium and potassium currents in isolated rat hepatocytes

AIM: To study the effects of tetrandrine (Tet) on calciumrelease-activated calcium current (ICRAC), delayed rectifierpotassium current (IK), and inward rectifier potassiumcurrents (IK1) in isolated rat hepatocytes.METHODS: Hepatocytes of rat were isolated by usingperfusion method. Whole cell patch-clamp techniques wereused in our experiment.RESULTS: The peak amplitude.of ICRAC was -508±115 pA(n＝15), its reversal potential of ICRAC was about 0 mV. At thepotential of -100 mV, Tet inhibited the peak amplitude ofICRAC from -521±95 pA to -338±85 pA (P＜0.01 vs control,n＝5), with the inhibitory rate of 35 % at 10 μmol/L andfrom -504±87 pA to -247±82 pA (P＜0.01 vscontrol, n＝5),with the inhibitory rate of 49 % at 100 μmol/L, withoutaffecting its reversal potential. The amplitude of ICRAC wasdependent on extracellular Ca2+ concentration. The peakamplitude of ICRAC was -205±105 pA (n＝3) in tyrode's solutionwith Ca2+ 1.8 mmol/L (P＜0.01 vs the peak amplitude ofICRAC in external solution with Ca2+ 10 mmol/L). Tet at theconcentration of 10 and 100 μmol/L did not markedly changethe peak amplitude of delayed rectifier potassium currentand inward rectifier potassium current (P＞0.05 vs control).CONCLUSION: Tet protects hepatocytes by inhibiting ICRAC,which is not related to I K and IK1.     

An inward rectifier and a voltage-dependent K+ current in single, cultured pericytes from bovine heart

OBJECTIVE: The purpose of this study was to describe passive electrical properties and major membrane currents in coronary pericytes. METHODS: 78 single, cultured bovine pericytes were studied with the patch-clamp technique in the whole-cell mode. RESULTS: The membrane potential of the cells was -48.9+/-9.6 mV (mean+/-S.D.) with 5 mM and -23.2+/-2.2 mV with 60 mM extracellular K+. The membrane capacitance was 150.2+/-123.2 pF. The current-voltage relation of the pericytes was dominated by an inward current at hyperpolarized potentials and an outward current at depolarized potentials. Increasing extracellular K+ from 5 to 60 mM led to an increase of the inward current and to a shift of this current to more depolarized potentials. The inward current was very sensitive to extracellular barium (50 microM). The maximum slope conductance of the cells at hyperpolarized potentials was 2.9+/-2.8 nS. Inward rectification of whole-cell currents was steep (slope factor = 6.8 mV). With elevated external K+ the outward current reversed near the potassium equilibrium potential. Onset of the outward current was sigmoid and inactivation of this current was monoexponential, slow (time constant = 12.8 s) and incomplete. Voltage-dependence of outward current steady-state activation was steep (slope factor = 4.6 mV). The outward current was very sensitive to 4-aminopyridine (dissociation constant = 0.1 mM). The maximum slope conductance at depolarized potentials was 16.6+/-15.6 nS. CONCLUSION: We report for the first time, patch-clamp recordings from coronary pericytes. An inward rectifier and a voltage-dependent K+ current were identified and characterized. Regulation of these currents may influence coronary blood flow.     

Funnel-web spider venom and a toxin fraction block calcium current expressed from rat brain mRNA in Xenopus oocytes.

Injection of rat brain mRNA into Xenopus oocytes has been shown to induce a calcium current (ICa) that is insensitive to dihydropyridine and omega-conotoxin. We examined the effect of funnel-web spider venom on two aspects of this expressed ICa: (i) the calcium-activated chloride current [ICl(Ca)] and (ii) the currents carried by barium ions through calcium channels (IBa). In the presence of 1.8 mM extracellular calcium, ICl(Ca) tail current became detectable between -30 and -40 mV from a holding potential of -80 mV and reached a maximal amplitude between 0 and +10 mV. Total spider venom partially (83%) and reversibly blocked the calcium-activated chloride current without changing its voltage sensitivity. A chromatographic toxin fraction from the venom also blocked this current (64%). The venom had a minimal effect on INa and IK. Direct investigation of inward current mediated by calcium channels was carried out in high-barium solution. IBa had a higher threshold of activation (-30 to -20 mV) and reached its maximal amplitude at about +20 mV. Total venom or a partly purified chromatographic toxic fraction blocked IBa partially and reversibly without changing its current-voltage characteristics. Furthermore, the extent of the total venom block depended on the concentration of extracellular barium. Only 35% of the IBa was blocked in 60 mM Ba2+, whereas the block increased to 65% and 71%, respectively, for 40 and 20 mM Ba2+. On the basis of these results, we propose that the calcium channels expressed from rat brain mRNA in Xenopus oocytes is similar to the recently discovered P-type channels.     

酚噻嗪对豚鼠心室肌细胞动作电位时程和跨膜离子电流作用的研究

通过观察酚噻嗪对豚鼠单心室肌细胞动作电位时程 (APD)及动作电位形成过程中主要离子通道的作用 ,探讨其诱发长QT间期 (LQT)综合征及室性心律失常的可能机制。采用膜片钳技术中的Nystatin 破膜法观察酚噻嗪对豚鼠心室肌细胞APD的作用 ,采用全细胞技术研究酚噻嗪对心室肌细胞动作电位形成过程中主要离子电流的作用。结果 :①在 5Hz剌激频率时 ,10 0 μmol/L酚噻嗪使APD90 延长 2 5 .8%± 3.8% (n =10 ,P <0 .0 1) ,作用呈部分可逆性 ;②在分别持续 2 5 0ms和 10 0 0ms至不同膜电位水平去极化的实验中 ,酚噻嗪对延迟整流钾电流 (IK)尾电流有明显的抑制作用 ,在 +6 0mV持续 2 5 0ms的去极化时 ,5 0 .6 4 %± 6 .4 6 %的IK尾电流受抑制 (n =7,P <0 .0 5 ) ,这一抑制作用呈浓度依赖性 ,半抑制浓度 (IC50 ) =2 5 .98μmol/L ,Hill常数为 0 .75。当采用IK快速激活成分 (IKr)的特异性阻断剂E 4 0 31阻断IKr后 ,酚噻嗪对IK尾电流的阻断作用消失。 30 0 μmol/L酚噻嗪对IKr的抑制作用仍弱于 0 .5mmol/LE 4 0 31。在 10 0 0ms,至 +2 0mV去极化的情况下 ,酚噻嗪对IK尾电流的IC50 为 2 5 .98μmol/L ,这一抑制作用可部分洗脱。受酚噻嗪抑制的电流与IK中IKr具有相似的通道动力学特性 ;③未发现酚噻嗪对IK稳态电流、IK?     

Adenosine-activated potassium conductance in cultured striatal neurons.

We have examined the effect of adenosine on the membrane properties of cultured embryonic mouse striatal neurons using patch electrode techniques. Adenosine at 50 microM effectively blocked spontaneous action potential activity. Adenosine or 2-chloroadenosine caused a slow hyperpolarization of the membrane potential and, under voltage clamp, an outward current that was blocked by 1 mM theophylline. ATP also caused a hyperpolarization that was slower and weaker than the adenosine response and could be blocked by 1 mM theophylline. The current induced by adenosine appears to be carried by potassium since (i) an inward current was generated by adenosine when the cells were internally perfused with cesium salts and (ii) the reversal potential of the outward current shifted 57 mV with a 10-fold change in extracellular potassium concentration. The adenosine response is voltage dependent in that the current evoked by adenosine is reduced at holding potentials more positive than -55 mV, despite a larger driving force. Though calcium influx is not required for adenosine to activate the potassium conductance, some components of the cytosol may be essential, since the response is lost during intracellular perfusion.     

Blocking and isolation of a calcium channel from neurons in mammals and cephalopods utilizing a toxin fraction (FTX) from funnel-web spider poison

A Ca2+-channel blocker derived from funnel-web spider toxin (FTX) has made it possible to define and study the ionic channels responsible for the Ca2+ conductance in mammalian Purkinje cell neurons and the preterminal in squid giant synapse. In cerebellar slices, FTX blocked Ca2+-dependent spikes in Purkinje cells, reduced the spike afterpotential hyperpolarization, and increased the Na+-dependent plateau potential. In the squid giant synapse, FTX blocked synaptic transmission without affecting the presynaptic action potential. Presynaptic voltage-clamp results show blockage of the inward Ca2+ current and of transmitter release. FTX was used to isolate channels from cerebellum and squid optic lobe. The isolated product was incorporated into black lipid membranes and was analyzed by using patch-clamp techniques. The channel from cerebellum exhibited a 10- to 12-pS conductance in 80 mM Ba2+ and 5-8 pS in 100 mM Ca2+ with voltage-dependent open probabilities and kinetics. High Ba2+ concentrations at the cytoplasmic side of the channel increased the average open time from 1 to 3 msec to more than 1 sec. A similar channel was also isolated from squid optic lobe. However, its conductance was higher in Ba2+, and the maximum opening probability was about half of that derived from cerebellar tissue and also was sensitive to high cytoplasmic Ba2+. Both channels were blocked by FTX, Cd2+, and Co2+ but were not blocked by omega-conotoxin or dihydropyridines. These results suggest that one of the main Ca2+ conductances in mammalian neurons and in the squid preterminal represents the activation of a previously undefined class of Ca2+ channel. We propose that it be termed the "P" channel, as it was first described in Purkinje cells.     

Endothelin induces a nonselective cation current in vascular smooth muscle cells.

The potent vasoconstrictor endothelin leads to smooth muscle cell depolarization and increases in intracellular Ca2+. Although effects of endothelin on calcium channels have been described, it also has been speculated that endothelim may activate additional ion channels. The purpose of the present study was to identify an alternative ion current that could play a role in depolarizing cells in response to vasoconstrictors like endothelin and vasopressin. The effects of endothelin, vasopressin, sarafotoxin S6b, and phenylephrine were assessed using whole-cell patch-clamp recordings from primary dissociated rat aortic or mesenteric arterial smooth muscle cells cultured for 24-72 hours. From the usual resting potentials of these cells of -50 to -60 mV, endothelin (1-100 nM) induced a depolarization via an increase in membrane conductance. This depolarization was phasic, oscillating repeatedly from the resting potential to a relatively depolarized level and back to the resting potential. From a holding potential of -60 mV, endothelin-1, endothelin-3, vasopressin, or sarafotoxin S6b (but not phenylephrine) induced transient inward currents that also could be phasic. In external sodium, lithium, or cesium (but not Tris) and in internal potassium or cesium, these currents reversed near 0 mV. Although nifedipine-insensitive, the inward currents were absent in zero calcium, barium, or strontium, or in the presence of cobalt or nickel. These results represent the first report of a nonselective cation current in primary vascular smooth muscle cells that is calcium dependent and that could be responsible for the depolarizations induced from the resting potential by vasoconstrictors such as endothelin.