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The effects of changing the intracellular Ca concentration ([Ca]i) on the calcium current (iCa) were studied in isolated single ventricular cells of the guinea-pig. [Ca]i was varied by an intracellular perfusion technique using a suction pipette. iCa measured from internally perfused cells at a pCa lower than 9.0 was dependent on the extracellular Ca concentration ([Ca]o). Increasing [Ca]o from 1.8 to 5.4 mM increased the amplitude of iCa, and reduction of [Ca]o from 1.8 to 0.01 mM decreased the amplitude. The inactivation time course of iCa became faster as [Ca]o was increased and slower as [Ca]o was reduced. By decreasing the pCa of the internal perfusate from 9.0 to 6.8, the amplitude of iCa was decreased markedly, but no significant change was observed in its time course. Analysis of the I-V curve led to the conclusion that a change in the driving force was not a major factor in the reduction of iCa. The "steady state inactivation" of iCa was measured by a double-pulse method. The amplitude of iCa elicited by the test pulse was smaller at pCa 7.4 than at pCa 9.0 at potentials of between -27 and +33 mV. By normalizing the iCa amplitude, however, the "steady state inactivation" curve in the control and at high [Ca]i overlapped. Similar results were obtained in Sr-Tyrode solution. The degree of "steady state inactivation" of iCa at the potentials positive to +10 mV was larger in Ca-Tyrode than in Sr-Tyrode solution. It is proposed that the reduction in amplitude of iCa at higher [Ca]i is caused by a reduction of the maximum conductance of iCa (gCa) and that Ca ions passing through iCa channels have a remarkable effect on its inactivation.

The dihydropyridine derivatives CGP 28392 and BAY K 8644 exert a strong positive inotropic effect in mammalian cardiac muscle, presumably by increasing Ca influx during the action potential. Analysis of the drug effects at the level of single Ca channels by means of the patch-clamp method revealed complicated changes in the kinetics of channel gating. The prevailing effect is a prolongation of the mean open time of Ca channels. In addition, the intervals between channel openings can be slightly prolonged. Ensemble averages of single-channel traces showed a concentration-dependent increase in the mean current amplitude by the drugs. In contrast to beta-adrenoceptor agonists, the increase in Ca current by the dihydropyridine derivatives is not associated with an increase in the intracellular cyclic AMP level.

Voltage-dependent Ca channels were studied in living, tissue cultured rat heart cells using patch clamp analysis of single channels and radioligand binding studies at different membrane potentials. Ca channel activating and blocking dihydropyridines (DHP) show cooperative interaction and, therefore, bind to at least two different binding sites on the channel protein. Cooperative interactions between activating and blocking DHP, between DHP and verapamil and between DHP and diltiazem are all voltage dependent. The type of interaction in polarized cells and hence, the possible effects in vivo cannot be predicted from studies in cell homogenates.

The properties of voltage-gated Cl channels of cultured smooth muscle cell prepared from embryonic rat aorta were studied. In the excised patch (inside-out configuration), we observed the activity of channels, opening and closing spontaneously, when the membrane potential was held at around 0 mV. The channels were active at a potential range between +10 and -10 mV. A step change of the membrane potential from the active potential range in either a positive or a negative direction closed the channel to an apparently inactivated state. The time course of this inactivation process became faster as the amplitude of the step change was increased. Returning the membrane potential to 0 mV allowed the channel to recover from the inactivated state. The channel had at least two open conductance states. Ca ions at the cytoplasmic face were not required for the activation of the channel. Adenosine nucleotides at the same side of the membrane had no effect on channel activity. The channels were selective to anions rather than cations, and they had a large single channel conductance of 340.5 +/- 20.4 pS in symmetrical 150 mM TEA-Cl. The reversal potential of the channel was shifted by -15.2 +/- 2.6 and 17.0 +/- 1.7 mV, when the Cl concentration at the intracellular side was changed to 75 mM or 300 mM, respectively. The permeability sequence of halides was I- greater than Br- greater than Cl- greater than F- (1.4:1.3:1.0:0.7), whereas the conductance sequence was Cl- greater than Br- greater than F- greater than I- (1.00:0.89:0.86:0.83).(ABSTRACT TRUNCATED AT 250 WORDS)

Voltage-dependent calcium channels are widely distributed in excitable membranes and are involved in the regulation of many cellular functions. These channels can be modulated by neurotransmitters and drugs. There is one particular type of calcium channel in cardiac cells (L-type) whose gating is affected in different ways by beta-adrenoceptor and 1,4-dihydropyridine agonists. We have analysed single calcium channel currents (i) in myocytes from rat hearts in the absence and presence of isoproterenol or 8-bromo-cAMP. We have found that both compounds have similar effects on calcium channel properties. They increase the overall open state probability (po) of individual calcium channels while i remains unaffected. Analysis of the gating kinetics of calcium channels showed: a slight increase in the mean open times of calcium channels, a reduction in time intervals between bursts of channel openings, an increase in burst length and a prominent reduction in failures of calcium channels to open upon depolarization. These kinetic changes caused by isoproterenol and 8-bromo-cAMP can account for the increase in po. Since the macroscopic calcium current, ICa, can be described by ICa = N X po X i, the increase in po accounts for the well-known increase in ICa by beta-adrenergic catecholamines. Cyclic AMP-dependent phosphorylation of calcium channels is a likely metabolic step involved in this modulation. Another class of drug that modulates calcium channel gating is the 1,4-dihydropyridines which can either enhance or reduce ICa, either by prolonging the open state of the channels or by facilitating the inactivated state. Both effects depend strongly on membrane potential and are independent of cyclic AMP-dependent phosphorylation reactions.

1. The clusters composed of 10-20 cells of the rabbit sinoatrial node were prepared by coronary perfusion of collagenase and their response to ionophoretic application of ACh was recorded. The hyperpolarizing response showed a sigmoidal onset without any clear latency when the ACh pipette was positioned close to the cell surface. 2. Equations describing the kinetics and amplitude of the ACh-induced K current were determined based on the reported experimental results obtained by relaxation and noise analysis. 3. The kinetics of the ACh-induced K current simulated well the time course of the ACh response and also the effect on the spontaneous action potential when data on the kinetics was incorporated into the S-A node pacemaker mathematical model.

Primary cardiac cell cultures were prepared from the hearts of neonatal rats. The patch-clamp method (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was applied for studying whole-cell Na+ currents and single-channel Na+ currents, respectively. Whole-cell recordings yielded voltage- and time-dependent Na+ currents which could be blocked by tetrodotoxin. Single-channel Na+ currents were directly compared in cell-attached patches and in inside-out patches. In cell-attached patches the elementary current was about -1 pA at -10 mV and the slope conductance over a 50 mV voltage range was 15.1 +/- 1.6 pS (mean +/- S.D.). Inactivation during depolarization and after conditioning clamp steps, in the steady state, resulted from a reduced opening probability of Na+ channels. In inside-out patches, with identical solutions at both membrane surfaces, there was a large (40-50 mV) shift of channel opening and inactivation kinetics towards more negative potentials. However, for levels of comparable opening probabilities, mean open times of Na+ channels were similar in cell-attached and inside-out patches. Tetrodotoxin (10-20 microM) had no effect on Na+ channels when applied from the inside, but blocked them completely after application to the outside membrane surface.

We have investigated the effects of two oppositely acting enantiomers of the 1,4-dihydropyridine derivative 202-791 on voltage-dependent Ca channels by combining electrophysiological techniques and binding studies. The (S)-enantiomer of 202-791 promoting prolonged openings of single Ca channels, and thereby increasing transmembrane Ba currents, was classified as channel activator. The (R)-enantiomer favoring a closed state of the channel, and thereby reducing Ba currents, was classified as a channel blocker. Both compounds shifted the steady state current inactivation curve toward more negative potentials. At holding potentials positive to -20 mV, the Ca channel-activating effect of the (S)-enantiomer turned over into a blocking effect. In cells with normal resting potential the combination of the two enantiomers revealed a possible positive cooperative effect resulting in an enhancement of the open state probability of the channels. At depolarized holding potentials the activator enhanced the inhibitory effect of the blocker. Binding studies in intact cells were performed by using the radiolabeled channel-blocking dihydropyridine 3H-(+)-PN 200-110. The results showed a strong increase in binding affinity but no change in binding capacity when the cells were depolarized. Analysis of the interactions of (S)- and (R)-202-791 with this radioligand indicated stimulation of 3H-(+)-PN 200-110 binding by the (S)-enantiomer in polarized cells (membrane potential -38 +/- 4 mV). This effect could be attributed to an increase in binding affinity. The (R)-enantiomer had no such positive cooperative effect, but acted as a purely competitive ligand. Depolarization to 0 mV increased the apparent affinity of both enantiomers by factors of 38 (blocker) and 12 (activator), but abolished the cooperative effect of (S)-202-791 on the binding of the radioligand. Ca ions had little effect on the binding of 3H-(+)-PN 200-110 in polarized cells. However, in the presence of the activating (S)-enantiomer, Ca transformed the usual hyperbolic binding isotherm of the radioligand into a strongly sigmoid curve. Sigmoidicity was minimal with 3-5 microM Ca and maximal with 0.5 mM Ca. Together these data demonstrate homotropic and heterotropic cooperative interactions between channel activator and channel blocker. They indicate that at least two high affinity binding sites for dihydropyridines are associated with voltage-dependent Ca channels. Voltage dependence of both--binding affinity and cooperativity--suggests that these binding sites are located close to a structural component of the channel which is involved in the potential-sensitive gating process.

Modulation of ion channels is of increasing interest as it is an important step in the regulation of cellular functions. We have analysed the effect of 8-bromocyclic AMP on Ca2+ channels in cultured cardiac cells by the patch-clamp method and report here that there was a large increase in the probability of opening of the channels. On the basis of a recently proposed kinetic reaction scheme we suggest that cyclic AMP-dependent phosphorylation of Ca2+ channels primarily promotes the forward rate constants which lead to the open state of a Ca2+ channel during depolarization.

We studied voltage-dependent binding and action of 1,4-dihydropyridine enantiomers (Sandoz (+)-(S) & (-)-(R)-202-791) in intact cardiac cells. Drug action was studied by patch clamp method. (+)-enantiomer primarily prolonged open time of Ca channel, thus enhancing Ca currents, while (-)-enantiomer predominantly favored closed state of the channel, reducing the currents. Electrophysiological study also revealed that steady-state inactivation was greatly enhanced by both enantiomers. Therefore, it seems that both enantiomers have a capability of activating and blocking effects. Strongly voltage-dependent binding affinity of these enantiomers was revealed by displacement of 3H-(+)-PN200-110 with each enantiomers. Binding affinity of both enantiomers in depolarized cells was much higher than in polarized cells. These results indicate that both enantiomers have higher affinity when Ca channels are inactivated.