Macroscopic and unitary properties of physiological ion flux through L-type Ca2+ channels in guinea-pig heart cells

1. We investigated the currents through L-type Ca²⁺ channels when Ca²⁺ (1-10 mM) was the charge carrier, as is the case physiologically. 2. Na⁺ was removed from both the external and internal solutions to eliminate currents through Na⁺ channels and Na⁺-Ca²⁺ exchange. 3. From a holding potential of -50 mV only L-type channels were available to open with depolarization. Macroscopic L-type currents were maximal during depolarizing pulses to + 10 mV (peak current density of 4.7 ± 0.3 nA nF^-1 4. During depolarizing steps as long as 180 ms, the decay of current through L-type channels was incomplete, in contrast to that of T-type current. 5. Unitary currents recorded with comparable ionic conditions and voltage protocols exhibited a single-channel conductance of 6.9 pS in 10 mM Ca²⁺. Ensemble average currents reproduced accurately the features of whole-cell L-type current, including the maintained component. 6. Convolution analysis was employed to clarify the single-channel basis of the complex current waveform of L-type channels. First openings underlie the peak, while the maintained pedestal is generated by multiple re-openings. As with T-type channels, single openings are brief and contribute little to the time course of the average current. 7. The prominent maintained component of macroscopic and ensemble average L-type current cannot be explained by simple Markov models in which current decay reflects the progressive entry of channels into an absorbing inactivated state. 8. We considered the possibility that the maintained component of current arises from the existence of multiple distinct gating patterns, one of which lacks inactivation. Individual sweeps were sorted among three patterns of gating (no openings, active-early and active-late). Patterns of activity are not randomly distributed; instead, they tend to cluster over time. 9. Most of the maintained current is attributable to the 'active-late' pattern of gating. Considered separately, this pattern can be well described by a simple Markov chain lacking an inactivated state. The 'active-early' gating pattern accounts entirely for the initial current transient, and for about one-third of the maintained component; thus, inactivation, even when present, must be reversible rather than absorbing. 10. The unitary current amplitudes and peak open probabilities measured for single L-type channels, when compared to the average macroscopic L-type current density, predict 170 functional channels per picofarad, or 28 000 L-type channels per typical ventricular myocyte. The number of predicted channels is large relative to previous electrophysiological estimates which did not consider the low probability of opening for L-type channels, but agrees fairly well with the estimated density of dihydropyridine binding sites in the heart.