Faculty Bibliography

Cardiac calcium channel activity is markedly increased by beta-adrenergic agents or calcium agonists such as Bay K 8644. The molecular mechanisms underlying these important modulatory effects have been studied with patch clamp techniques by several groups. This paper presents new experiments and reviews published evidence from fluctuation analysis of whole cell calcium current and unitary recordings of single calcium channel activity. Two different factors underlie the enhancement of calcium channel activity seen with beta-stimulation or cyclic AMP: (1) increased availability of calcium channels, expressed in whole cell recordings as an increase in the number of functional channels and in single channel recordings as an increase in the proportion of non-blank sweeps. (2) changes in opening probability, due to alteration of the fast kinetics of channel opening and closing. Both factors contribute to the beta-adrenergic enhancement in frog, rat, and guinea-pig ventricular cells although their quantitative importance is somewhat variable. Unlike beta-adrenergic agents, calcium agonists such as Bay K 8644 promote a mode of channel gating that is characterized by long openings and short closings, seen only rarely in control or with beta-stimulation

Electrophysiological recordings were used to analyze single calcium channels in planar lipid bilayers after membranes from bovine cardiac sarcolemmal vesicles had been incorporated into the bilayer. In these cell-free conditions, channels in the bilayer showed unitary barium or calcium conductances, gating kinetics, and pharmacological responses that were similar to dihydropyridine-sensitive calcium channels in intact cells. The open channel current varied in a nonlinear manner with voltage under asymmetric (that is, physiological) ionic conditions. However, with identical solutions on both sides of the bilayer, the current-voltage relation was linear. In matched experiments, calcium channels from skeletal muscle T-tubules differed significantly from cardiac calcium channels in their conductance properties and gating kinetics

We used the patch clamp technique to record unitary calcium (Ca2+) channel activity in freshly dissociated ventricular myocytes from adult guinea pigs. We found two types of Ca2+ channels with distinct permeation and gating properties and different sensitivity to pharmacological agents. One channel (T-type) requires negative membrane potentials to remove inactivation. It gives rise to a transient mean current and is not affected by dihydropyridines or isoproterenol. The other Ca2+ channel (L-type) has a larger unitary barium-conductance, activates at more positive potentials and its averaged current decays much more slowly. It shows a distinct gating pattern with different gating modes, the proportion of which is drastically altered by dihydropyridine Ca2+-channel agonists and antagonists. L-type channel activity is modulated by beta-adrenergic stimulation by a mechanism of action which differs from that of dihydropyridine Ca2+-channel agonists

Ca channel currents were recorded in Cs-loaded calf cardiac Purkinje fibres and Cs-dialysed myocytes from guinea-pig ventricle to evaluate the dependence of Ca channel inactivation on membrane depolarization and intracellular free Ca concentration ([Ca]i). The decay of Ca channel current during a maintained depolarization was slowed when external Ca was replaced by Sr or Ba. The decay reflected a genuine inactivation of Ca channel conductance, as assessed by the decreased amplitude of inward tail currents following progressively longer depolarizing pulses in ventricular cells. Increasing depolarization slowed inward current inactivation in the presence of extracellular Ca concentration ([Ca]o), but speeded inactivation in the presence of extracellular Ba concentration ([Ba]o), suggesting the participation of fundamentally different mechanisms. Ca channel currents were recorded in Ca-free external solutions to study 'voltage-dependent inactivation'. Inactivation of outward Ca channel current due to Cs efflux was seen with external Ba or in the absence of any permeant divalent cation. With Ca as the charge carrier, increasing [Ca]o speeded the rate of inactivation as expected for [Ca]i-dependent inactivation. The relationship between inactivation and the intracellular Ca transient was assessed by double-pulse experiments. Conditioning pulses that produced maximal inward Ca current and contractile tension left behind more inactivation than either stronger or weaker depolarizations. The agreement between maximal inward current and maximal inactivation remained close when their voltage dependence was shifted along the voltage axis by elevation of [Ca]o. We conclude that inactivation of cardiac Ca channels is both [Ca]i dependent and voltage dependent. The [Ca]i-dependent process may serve as a negative feed-back mechanism for regulating Ca entry into heart cells; the voltage-dependent mechanism may prevent a secondary rise in Ca channel current when intracellular Ca falls during maintained depolarization of cardiac cells

How many types of calcium channels exist in neurones? This question is fundamental to understanding how calcium entry contributes to diverse neuronal functions such as transmitter release, neurite extension, spike initiation and rhythmic firing. There is considerable evidence for the presence of more than one type of Ca conductance in neurones and other cells. However, little is known about single-channel properties of diverse neuronal Ca channels, or their responsiveness to dihydropyridines, compounds widely used as labels in Ca channel purification. Here we report evidence for the coexistence of three types of Ca channel in sensory neurones of the chick dorsal root ganglion. In addition to a large conductance channel that contributes long-lasting current at strong depolarizations (L), and a relatively tiny conductance that underlies a transient current activated at weak depolarizations (T), we find a third type of unitary activity (N) that is neither T nor L. N-type Ca channels require strongly negative potentials for complete removal of inactivation (unlike L) and strong depolarizations for activation (unlike T). The dihydropyridine Ca agonist Bay K 8644 strongly increases the opening probability of L-, but not T- or N-type channels

Calcium influx is vital for several aspects of cardiac activity, so it is important to ask if heart cells possess a single or multiple types of Ca channel. Only one Ca channel type has been identified in patch-clamp studies of unitary current, despite suggestions to the contrary from whole-cell recordings in heart cells and unitary recordings from other cells. Here we describe a novel type of cardiac Ca channel with several properties that distinguish it from the hitherto-identified Ca channel in heart cells. Its conductance in isotonic Ba is small (8 pS), and is no larger in Ba than in Ca. It activates and inactivates at relatively negative potentials and remains functional long after patch excision. It is insensitive to dihydropyridines such as nimodipine and the Ca agonist Bay K 8644, and is more resistant to block by external Cd than the previously described type of cardiac Ca channel

A large-conductance calcium channel in chicken dorsal root ganglion neurons was studied with patch-clamp recordings of unitary currents. In addition to the conventional pattern of Ca-channel gating previously described in neurons ('mode 1'), we observed a different form of gating behavior ('mode 2'). Unlike the brief (approximately equal to 1 ms) openings in mode 1, mode 2 openings tend to be longer (greater than 10 ms) and often outlast the test pulse. In mode 2, the probability of channel openness (P) is high at relatively negative potentials where P in mode 1 is low. Mode 2 activity appears much less often than mode 1 activity in the absence of drug. However, the balance is strongly shifted in favor of mode 2 by the dihydropyridine Ca agonist Bay K 8644, an effect that underlies a marked enhancement of Ca-channel activity. This is the first evidence for dihydropyridine control of neuronal Ca-channel function at the single-channel level. Sweeps showing mode 1 or mode 2 gating appeared interspersed with sweeps with no openings, during which the channel was unavailable for opening ('null mode' or 'mode 0'). Two approaches showed that switching between all three modes occurred on a time scale of seconds: (i) channels tended to remain in the same mode from one sweep to the next, with pulses at 0.25 Hz; and (ii) steady depolarizations in Bay K 8644 produced clusters of mode 2 openings lasting several seconds. Changes in the rates of switching might be important in neurochemical modulation of Ca channels. Bay K 8644 and other dihydropyridine Ca agonists might be useful experimental tools for manipulating transmitter release, neurite extension, and other neuronal functions dependent on intracellular Ca

Single cardiac transmembranous Ca channels have three modes of gating behaviour in the absence of drugs, expressed as current records with brief openings (mode 1), with no openings because of channel unavailability (mode 0 or null mode) and with long-lasting openings and very brief closings that appear only rarely (mode 2). The dihydropyridine Ca agonist Bay K 8644 enhances Ca channel current by promoting mode 2, while the Ca antagonists nitrendipine and nimodipine inhibit the current by favouring mode 0

Membrane currents and action potentials were recorded in single ventricular cells obtained from guinea-pig hearts by enzymatic dissociation. Ca2+ channel currents carried by Ba2+ or Ca2+ were recorded with a suction pipette (5-10 microns diameter) for voltage clamp and internal dialysis. Currents through Na+, K+ and non-selective monovalent cation channels were suppressed by suitable holding potentials and external and internal solutions. The dialysis method allowed exchange within minutes of alkali metal cations (e.g. Cs+) and small molecules (e.g. quaternary derivatives of lidocaine and verapamil). Nevertheless, Ca2+ channels remained functional for considerable periods, typically 20 min and sometimes more than 1 h. With Ba2+ outside and Cs+ inside, current flow through Ca2+ channels changed from inward to outward at strongly positive levels beyond a clear-cut reversal potential Erev. Several methods for defining Erev were in close agreement: (1) zero-crossing of leak-subtracted peak current, (2) inversion of time-dependent current changes during channel activation or inactivation, (3) inversion of drug-sensitive current as defined by channel blockers such as Cd2+ or D-600. Erev varied with external Ba2+ or internal Cs+. Erev increased by 29 mV per 10-fold increase in Ba2+. Interpreted with constant-field theory, Erev values correspond to PBa/PCs of approximately 1360. With 5 mM-Ca2+ outside and 151 mM-Cs+ inside, Ca2+ channel current reversed near + 75 mV, corresponding to PCa/PCs approximately 6000. Earlier measurements of Erev (Lee & Tsien, 1982) suggest that PCa/PK greater than 1000. At strongly positive membrane potentials where channel activation is maximal, the Ca2+ channel current-voltage relationship is strongly non-linear, with conductance increasing on either side of an inflexion point near Erev. Activation of inward or outward currents through Ca2+ channels follows a sigmoid time course, as expected if activation were a multi-step process