Ion concentration-dependence of rat cardiac unitary L-type calcium channel conductance.

Little is known about the native properties of unitary cardiac L-type calcium currents (i(Ca)) measured with physiological calcium (Ca) ion concentration, and their role in excitation-contraction (E-C) coupling. Our goal was to chart the concentration-dependence of unitary conductance (gamma) to physiological Ca concentration and compare it to barium ion (Ba) conductance in the absence of agonists. In isolated, K-depolarized rat myocytes, i(Ca) amplitudes were measured using cell-attached patches with 2 to 70 mM Ca or 2 to 105 mM Ba in the pipette. At 0 mV, 2 mM of Ca produced 0.12 pA, and 2 mM of Ba produced 0.19 pA unitary currents. Unitary conductance was described by a Langmuir isotherm relationship with a maximum gammaCa of 5.3 +/- 0.2 pS (n = 15), and gammaBa of 15 +/- 1 pS (n = 27). The concentration producing half-maximal gamma, Kd(gamma), was not different between Ca (1.7 +/- 0.3 mM) and Ba (1.9 +/- 0.4 mM). We found that quasi-physiological concentrations of Ca produced currents that were as easily resolvable as those obtained with the traditionally used higher concentrations. This study leads to future work on the molecular basis of E-C coupling with a physiological concentration of Ca ions permeating the Ca channel.

Kinetic components of the gating currents of human cardiac L-type Ca2+ channels.

It has been reported previously that the beta subunit increases both the ionic current and the gating charge movement of the human cardiac L-type Ca2+ channel alpha1 subunit, and that steady-state measurements reveal the presence of two distinct components of the charge movement [Josephson IR, Varadi G (1996) Biophys J 70:1285-1293]. The present work identifies and characterizes the kinetic properties of the components of the human cardiac L-type Ca channel gating currents (Ig), and determines the relationship of these components to the activation of the Ca channel ionic current (ICa). Cloned human cardiac L-type alpha1+alpha2+beta3 subunits were transiently expressed in HEK293 cells and calcium channel gating currents were recorded following the addition of 5 mM Co2+. The steady-state charge integrals of the gating currents (QON-Vm) were fit by a sum of two Boltzmann components: QON1, which ranged over more negative potentials, and QON2, which ranged over more positive potentials. The kinetic components of the ON and OFF gating currents were identified using bi-exponential curve fitting. Reconstruction of the two kinetic components of charge (QONfast and QONslow) yielded distributions that were similar in their voltage dependence and relative proportion to those measured directly by steady-state integration of QON1 and QON2. Changes in the initial conditions were found to affect QON1 and QON2 differently. The time constants of the ON gating current decays were similar to those of the activation of ICa. The results suggest that: (1) the activation of the human cardiac L-type Ca channel involves the movements of at least two, functionally distinct gating structures; (2) a fast charge movement (approximately 1/4 of the total charge; QON1 or QONfast) precedes a slower charge movement (approximately 3/4 of the total charge; QON2 or QONslow); and (3) channel opening is associated with the conformational change(s) producing QONslow.

Depolarization shifts the voltage dependence of cardiac sodium channel and calcium channel gating charge movements.

In cardiac ventricular myocytes, membrane depolarization leads to the inactivation of the Na channel and Ca channel ionic currents. The inactivation of the ionic currents has been associated with a reduction of the gating charge movement ("immobilization") which governs the activation of Na channels and Ca channels. The nature of the apparent "immobilization" of the charge movement following depolarization was explored in embryonic chick ventricular myocytes using voltage protocols applied from depolarized holding potentials. It was found that although all of the charge was mobile following inactivation, the voltage dependence of its movement was shifted to more negative potentials. In addition, the shift in the distribution of the Na channel charge could be differentiated from that of the Ca channel charge on the basis of kinetic as well as steady-state criteria. These results suggest that the voltage-dependent activation of Na channel and Ca channel charge movements leads to conformational changes and charge rearrangements that differentially bias the movements of these voltage sensors, and concomitantly produce channel inactivation.

The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels.

The properties of the gating currents (nonlinear charge movements) of human cardiac L-type Ca2- channels and their relationship to the activation of the Ca2+ channel (ionic) currents were studied using a mammalian expression system. Cloned human cardiac alpha1 + rabbit alpha 2 subunits or human cardiac alpha 1 + rabbit alpha 2 + human beta 3 subunits were transiently expressed in HEK293 cells. The maximum Ca2+ current density increased from -3.9 +/- 0.9 pA/pF for the alpha 1 + alpha 2 subunits to -11.6 +/- 2.2 pA/pF for alpha 1 + alpha 2 + beta 3 subunits. Calcium channel gating currents were recorded after the addition of 5 mM Co2+, using a -P/5 protocol. The maximum nonlinear charge movement (Qmax) increased from 2.5 +/- 0.3 nC/muF for alpha 1 + alpha 2 subunit to 12.1 +/- 0.3 nC/muF for alpha 1 + alpha 2 + beta 3 subunit expression. The QON was equal to the QOFF for both subunit combinations. The QON-Vm data were fit by a sum of two Boltzmann expressions and ranged over more negative potentials, as compared with the voltage dependence for activation of the Ca2+ conductance. We conclude that 1) the beta subunit increases the number of functional alpha 1 subunits expressed in the plasma membrane of these cells and 2) the voltage-dependent activation of the human cardiac L-type calcium channel involves the movements of at least two nonidentical and functionally distinct gating structures.

Cardiac channel gating charge movements: recovery from inactivation.

The nonlinear charge movements which occur during membrane depolarization of cardiac ventricular myocytes (QON) have been previously identified and separated, by kinetic and steady-state criteria, into constituent components arising from the gating of Na channels and Ca channels. In contrast, the nature and time course of the OFF charge movements (QOFF), which follow membrane repolarization have not been as clearly established. In order to address this question cardiac QOFF was studied using small-diameter, 17-day-old embryonic chick ventricular myocytes that can be rapidly and uniformly voltage-clamped. The application of brief (5.4 ms) depolarizing steps were employed to produce Na channel inactivation but little Ca channel inactivation. Following the return of the membrane potential to -100 mV QOFF, measured as the gating current termed IgOFF, displayed two kinetic components. Double exponential fits to IgOFF yielded time constants of a few tenths of a millisecond for the fast component (IgOFFfast) and of 1-2 ms for the slower component (IgOFFslow). The time course and voltage dependence for the slower component suggested that it might be linked to the inactivation, and the recovery from inactivation, of Na channels. In order to identify these kinetic components double-pulse protocols were employed in which the duration of the prepulse and the interval separating the prepulse and test pulse were varied. The time course for the decay of IgOFFslow following a brief inactivating prepulse was similar to the time course for the recovery of the Na channel QON (QNaRecov). Both IgOFFslow and QNaRecov preceded the recovery of the Na channel (ionic) current. The recovery from inactivation of both the Na current and QNa displayed a similar voltage dependence. These experiments have helped to identify the two components of cardiac IgOFF and, therefore, will facilitate the interpretation of further biophysical and pharmacological studies concerning cardiac Na channel and Ca channel gating charge movements.

Voltage- and concentration-dependent effects of lidocaine on cardiac Na channel gating charge movements.

The effects of lidocaine, a local anesthetic and cardiac antiarrhythmic agent, were studied on cardiac nonlinear Na channel and Ca channel charge movements (gating currents) of 17-day-old embryonic chick ventricular myocytes. Gating currents were recorded following the blockade of all ionic currents and the subtraction of the linear capacity currents (-P/5). From a holding potential of -100 mV the ON charge movement (QON) displayed two kinetic components: a rapidly decaying component associated with Ca channel gating, and a slower component associated with Ca channel gating. A depolarizing prepulse to -50 mV for 125 ms reduced the fast component of QON, with little effect on the slower component. Similarly, 20 microM lidocaine also reduced the fast component of QON (Na channel charge movement) and had little effect on the slower component (Ca channel charge movement). Higher concentrations of lidocaine (125 microM) reduced both the fast and the slower components of QON. The effects of either a prepulse to -50 mV, or 20 microM lidocaine on the steady-state QON/Vm relationship were nearly identical. These results suggest that lidocaine "blocks" cardiac Na (ionic) currents by a reduction in the availability of Na channel charge movement (QON), and that this reduction is similar to that produced by voltage-dependent inactivation.

Kinetic and steady-state properties of Na+ channel and Ca2+ channel charge movements in ventricular myocytes of embryonic chick heart.

Nonlinear or asymmetric charge movement was recorded from single ventricular myocytes cultured from 17-d-old embryonic chick hearts using the whole-cell patch clamp method. The myocytes were exposed to the appropriate intracellular and extracellular solutions designed to block Na+, Ca2+, and K+ ionic currents. The linear components of the capacity and leakage currents during test voltage steps were eliminated by adding summed, hyperpolarizing control step currents. Upon depolarization from negative holding potentials the nonlinear charge movement was composed of two distinct and separable kinetic components. An early rapidly decaying component (decay time constant range: 0.12-0.50 ms) was significant at test potentials positive to -70 mV and displayed saturation above 0 mV (midpoint -35 mV; apparent valence 1.6 e-). The early ON charge was partially immobilized during brief (5 ms) depolarizing test steps and was more completely immobilized by the application of less negative holding potentials. A second slower-decaying component (decay time constant range: 0.88-3.7 ms) was activated at test potentials positive to -60 mV and showed saturation above +20 mV (midpoint -13 mV, apparent valence 1.9 e-). The second component of charge movement was immobilized by long duration (5 s) holding potentials, applied over a more positive voltage range than those that reduced the early component. The voltage dependencies for activation and inactivation of the Na+ and Ca2+ ionic currents were determined for myocytes in which these currents were not blocked. There was a positive correlation between the voltage dependence of activation and inactivation of the Na+ and Ca2+ ionic currents and the activation and immobilization of the fast and slow components of charge movement. These complementary kinetic and steady-state properties lead to the conclusion that the two components of charge movement are associated with the voltage-sensitive conformational changes that precede Na+ and Ca2+ channel openings.

Phosphorylation shifts the time-dependence of cardiac Ca++ channel gating currents.

A general mechanism for the physiological regulation of the activity of voltage-dependent Na+, Ca++, K+, and Cl channels by neurotransmitters in a variety of excitable cell types may involve a final common pathway of a cyclic AMP-dependent phosphorylation of the channel protein. The functional correlates of channel phosphorylation are known to involve a change in the probability of opening, and a negative or positive shift in the voltage dependence for activation of the conductance. The voltage dependence for activation appears to be governed by the properties of the charge movement of the voltage-sensing moiety of the channel. This study of the gating charge movement of cardiac Ca++ channels has revealed that isoproterenol or cAMP (via a presumed phosphorylation of the channel) speeds the kinetics of the Ca++ channel gating charge movement. These results suggest that the changes in the kinetics and voltage dependence of the cardiac calcium currents produced by beta-adrenergic stimulation are initiated, in part, by parallel changes in the gating charge movement.

Fast activation of cardiac Ca++ channel gating charge by the dihydropyridine agonist, BAY K 8644.

Nonlinear charge movement (gating current) was studied by the whole-cell patch clamp method using cultured 17-d-old embryonic chick heart cells. Na+ and Ca++ currents were blocked by the addition of 10 microM TTX and 3 mM CoCl2; Cs+ replaced K+ both intra- and extracellularly. Linear capacitive and leakage currents were subtracted by a P/5 procedure. The small size (15 microns in diameter) and the lack of an organized internal membrane system in these myocytes permits a rapid voltage clamp of the surface membrane. Ca++ channel gating currents were activated positive to -60 mV; the rising phase was not distorted due to the system response time. The addition of BAY K 8644 (10(-6) M) caused a shortening of the time to peak of the Ca++ gating current, and a negative shift in the isochronal Qon vs. Vm curve. Qmax was unchanged by BAY K 8644. The voltage-dependent shift produced by BAY K 8644 is similar to that produced by isoproterenol (Josephson, I.R., and N. Sperelakis. 1990. Biophys. J. 57:305a. [Abstr.]). The results suggest that the binding of BAY K 8466 to one or more of the Ca++ channel subunits alters the kinetics and shifts the voltage dependence of gating. These changes in the gating currents can explain the parallel changes in the macroscopic Ca++ currents.

Developmental increases in the inwardly-rectifying K+ current of embryonic chick ventricular myocytes.

Whole-cell and single-channel inwardly-rectifying K+ currents (IK1) of early (3-day-old) and late (17-day-old) embryonic chick ventricular myocytes were compared to ascertain whether there are developmental changes in the properties of this conductance. The magnitude of the IK1 conductance in the early myocytes was small, but it was increased about five-fold in the older embryonic myocytes. It was found that the density of inwardly-rectifying K+ channels was greater (in the surface membrane) of the 17-day than in the 3-day embryonic myocyte. In addition, the single channel conductance for 17-day myocytes was several-fold larger than for the 3-day myocytes. These results suggest that cardiac inward rectifier channels may not only proliferate in number, but may also undergo structural alterations during development.

Two types of outward K+ channel currents in early embryonic chick ventricular myocytes.

Outward K+ currents were recorded from 3-day-old embryonic chick ventricular myocytes using the patch clamp method. Two types of macroscopic outward currents were observed, one with rapid activation and de-activation time courses, and the other displaying a slower activation and long-duration tail currents. A time-dependent inactivation at positive potentials was a feature of the rapidly-activating current, allowing resolution of an early outward current. Single K+ channel currents were recorded using the outside-out patch technique. Two classes of K+ channels, which may contribute to the macroscopic currents, were differentiated on the basis of their conductances and kinetics. One class (ca 20 pS conductance) showed a rapid activation upon depolarization, and the other class (ca 60 pS) had a more delayed activation. A time-dependent inactivation of the rapid-activating, single-channel K+ current was also recorded. The two types of K+ channels contribute outward current during the plateau and promote the repolarization of the action potential, and the slowly de-activating K+ current may also be involved in the electrogenesis of automaticity observed in some of these cells.

Tetrodotoxin differentially blocks peak and steady-state sodium channel currents in early embryonic chick ventricular myocytes.

Single ventricular myocytes were dissociated from 3-day-old embryonic chick hearts and maintained in culture for 9-21 h. The whole-cell patch clamp method was used to record tetrodotoxin (TTX)-sensitive fast Na+ currents. The peak Na+ current recorded at -20 mV ranged from 10 to 70 microA/cm2. At more negative potentials, a component of the current decayed very slowly, resulting in a significant steady-state or "late" Na+ current. The origin of the late Na+ current was revealed through the examination of single Na+ channel currents recorded in outside-out membrane patches. The single Na+ channel conductance was 20 pS. A high percentage of the trials (approximately 16%) displayed multiple reopenings of a single Na+ channel, resulting in bursts of current lasting for greater than or equal to 150 ms. The frequency distributions of the Na+ channel open-times were bi-exponential. The burst-like mode of Na+ channel activity (which underlies the slowly- or non-inactivating currents recorded macroscopically), was blocked to a greater degree by TTX, compared to the peak current. The results suggest that differential blockade may occur as a result of the slow binding and increased affinity of TTX to the open Na channel.

Lidocaine blocks Na, Ca and K currents of chick ventricular myocytes.

In addition to the well-known reduction in the maximal rate of rise the antiarrhythmic agent, lidocaine, also produces a shortening in the duration of the normal ventricular action potential. The voltage-dependent currents carried by Na, Ca and K ions were examined in the absence and presence of lidocaine, in order to identify the ionic mechanism(s) underlying the action potential shortening. Whole-cell and single channel currents were studied by the patch clamp method using single ventricular myocytes from embryonic chick hearts. Exposure to lidocaine (10 to 1000 microM) produced a decrease in the magnitude of the small-magnitude, slowly-inactivating component of the Na current, and a decrease in the magnitude of the Ca current; these results provide a mechanism for the action potential shortening and suggest possible explanations for the antiarrhythmic action of this therapeutic agent. A decrease in the magnitude of the inwardly-rectifying K+ current was also found, which was attributable to a reduction in the probability of opening of single K+ channels.

Properties of inwardly rectifying K+ channels in ventricular myocytes.

The voltage- and time-dependent properties of whole-cell, multi-channel (outside-out), and single channel inwardly-rectifying K+ currents were studied using adult and neonatal rat, and embryonic chick ventricular myocytes. Inward rectification of the current-voltage relationship was found in the whole-cell and single channel measurements. The steady-state single channel probability of opening decreased with hyperpolarization from EK, as did the mean open time, thereby explaining the time-dependent inactivation of the macroscopic current. Myocytes dialysed with a Mg++-free K+ solution (to remove the property of inward rectification) displayed a quasi-linear current-voltage relationship. The outward K+ currents flowing through the modified inward rectifier channels were able to be blocked by the local anesthetic and anti-arrhythmic agent, lidocaine.

Calcium channels of amphibian stomach and mammalian aorta smooth muscle cells.

Whole-cell and single-channel calcium currents were studied using single smooth muscle cells enzymatically-isolated from stomach of Amphiuma tridactylum and from guinea-pig aorta. These cells have a high specific resistance and can sustain calcium action potentials after suppression of potassium currents. Dialyzed Amphiuma smooth muscle cells had calcium currents which were stable for several hours whereas the calcium currents of aortic cells ran down quickly. Single channel calcium currents in cell-attached patches behaved similarly for the two cell types. Calcium channel conductance in 110 mM barium was 12 pS and the mean open time was 1.4 ms at a nominal membrane potential of +10 mV. Exposure of both cell types to BAY K8644 resulted in a dramatic prolongation of the calcium channel open times and a shift in the probability of opening to more negative potentials. Low-threshold calcium channels were not identified in the extensively studied amphibian cells. High-threshold calcium channels therefore appear to be the primary pathway for the calcium influx that produces contraction in these smooth muscle cells.

Inwardly rectifying single-channel and whole cell K+ currents in rat ventricular myocytes.

The voltage-dependent properties of inwardly rectifying potassium channels were studied in adult and neonatal rat ventricular myocytes using patch voltage-clamp techniques. Inward rectification was pronounced in the single-channel current-voltage relation and outward currents were not detected at potentials positive to the calculated reversal potential for potassium (EK). Single-channel currents having at least three different conductances were observed and the middle one was predominant. Its single-channel conductance was nonlinear ranging from 20 to 40 pS. Its open-time distribution was fit by a single exponential and the time constants decreased markedly with hyperpolarization from EK. The distribution of the closed times required at least two exponentials for fitting, and their taus were related to the bursting behavior displayed at negative potentials. The steady-state probability of being open (P0) for this channel was determined from the single-channel records; in symmetrical isotonic K solutions P0 was 0.73 at -60 mV, but fell to 0.18 at -100 mV. The smaller conductance was about one-half the usual value and the open times were greatly prolonged. The large conductance was about 50 percent greater than the usual value and the open times were very brief. The P0(V) relation, the kinetics and the conductance of the predominant channel account for most of the whole cell inwardly rectifying current. The kinetics suggest that an intrinsic K+-dependent mechanism may control the gating, and the conductance of this channel. In the steady state, the opening and closing probabilities for the two smaller channels were not independent of each other, suggesting the possibility of a sub-conductance state or cooperativity between different channels.

Early outward current in rat single ventricular cells.

Voltage clamp experiments were conducted using single ventricular myocytes which had been dissociated enzymatically from adult rat hearts in order to examine further the membrane currents which contribute to the unusual plateau of the rat action potential. Membrane currents were recorded, using a single microelectrode (switching) voltage clamp circuit. From holding potentials near the resting potential (-80 to -90 mV), depolarizing clamp steps above -20 mV elicited an early outward current which overlapped in time with the slow inward current and displayed time-dependent inactivation. This is the first demonstration of a transient potassium current in an isolated ventricular myocyte. The early outward potential was voltage-inactivated at holding potentials of -50 to -40 mV and was blocked by 4-aminopyridine. The current was not dependent on Cao or ICa and was blocked by Bao. Double pulse experiments revealed that the time course for the recovery of the early outward current at -80 mV was rapid, and had a tau of 25 msec. The possible functional significance of this current is discussed.

A comparison of calcium currents in rat and guinea pig single ventricular cells.

The slow inward calcium currents were compared in rat and guinea pig heart using enzymatically dissociated, single ventricular cells. A single electrode voltage clamp was used, in which current and voltage were sampled separately using a time-sharing method. Spatial homogeneity of membrane potential during peak slow inward calcium current was assessed by measuring the potential with two microelectrodes 50 micron apart; the potentials were within 3 mV of each other. Peak current-voltage relations for slow inward calcium currents were similar for the two species, but the individual currents showed a faster time course of inactivation and a slower time course of recovery from inactivation for rat, compared with guinea pig. The potassium current blockers 4-aminopyridine and tetraethylammonium chloride did not produce significant effects on the net membrane currents recorded at the holding potentials (-50 to -40 mV) used in this study. The underlying mechanism for the inactivation of the slow inward calcium currents was explored using a double pulse procedure. In both rat and guinea pig heart cells prepulses to very positive potentials were associated with a partial restoration of the slow inward calcium current in the following test pulse. In addition, internal ethylene glycol-bis N,N,N',N'-tetraacetic acid or substitution of barium for calcium slowed the rate of inactivation of the slow inward calcium current in rat heart cells. Calcium activation of nonspecific currents was thought less likely to have produced these results due to the lack of effect of depolarizing prepulses on hyperpolarizing test pulses. A calcium-dependent component of inactivation may be responsible for the differences observed in both the inactivation and the recovery time courses of the slow inward calcium current in these species.

Effect of phenytoin on the sodium current in isolated rat ventricular cells.

The mode of action of the antiarrhythmic drug, phenytoin, on the cardiac sodium current was investigated using isolated rat ventricular cells, under voltage clamp conditions. It was found that the blocking effect of phenytoin on INa displays both voltage- and use-dependence. At a concentration of 20 mumol/l, it produced a tonic block of INa measuring 18% of control. After a train of 10 depolarizing pulses of 500 ms duration (applied at a frequency of 1 Hz), the degree of block was increased to 45% of control. Phenytoin also shifted the steady-state inactivation curve of INa to more hyperpolarized potentials by 5.4 mV. The blocking effects of phenytoin during repetitive depolarizing voltage steps suggest that phenytoin binds preferentially to inactivated channels, and that removal of this block may primarily occur from resting channels; moreover, the removal of block is strongly voltage-dependent.

Voltage- and use-dependent effects of lidocaine on sodium current in rat single ventricular cells.

We compared the blocking effects of the local anesthetics, lidocaine and benzocaine, on the sodium current, using single rat ventricular cells to obtain further information about the voltage dependence and kinetics of local anesthetic interaction with cardiac sodium channels. We used a hybrid voltage clamp system which employed a suction pipette for passing current and internal perfusion, and a microelectrode for membrane potential measurement. Lidocaine (20 microM) and benzocaine (100 microM) produced qualitatively similar effects on sodium current when test pulses were applied infrequently. Both of these agents decreased the peak sodium current without producing a shift of the current-voltage curve. They did, however, shift the inactivation curves of sodium current to hyperpolarized potentials; the V0.5 was shifted by -9.5 mV for lidocaine and by -5 mV for benzocaine. Lidocaine produced a significant use-dependent effect that was proportional to the duration of the voltage step. Benzocaine produced only minimal use-dependent effects. The characteristics of the lidocaine block suggest that this agent binds preferentially to inactivated sodium channels and that dissociation from resting channels is voltage-dependent. The differences in lipid solubility and molecular weight between lidocaine and benzocaine may explain the differences in their use-dependent blocking effects on sodium current.