Single calcium channels in rat and guinea-pig hippocampal neurons

Abstract

1. Calcium (Ca2+) channels were studied under whole-cell clamp and in cell-attached patch recordings from acutely isolated and tissue-cultured rat hippocampal neurons. Under whole-cell voltage clamp of tissue-cultured neurons the current-voltage plot for Ca2+ channel current was biphasic, with a 'hump' on the descending phase of the plot when the cell was held at -80 mV and stepped to various command potentials. When determined from a holding potential of -40 mV the plot was no longer biphasic. 2. In cell-attached patch experiments extracellular isotonic potassium gluconate was used to zero the cell membrane potential. When the patch electrode solution contained monovalent cations (150 mM-Li+ in most experiments) and no Ca2+, a small channel (approximately 13 pA) could be clearly distinguished in tissue-cultured neurons from a large dihydropyridine-sensitive channel (approximately 36 pS). With Ba2+ as the charge carrier it was more difficult to resolve small channel openings. The small channel was activated during voltage steps from negative holding potentials (-80 to -100 mV) over a range of potentials (-70 mV and less negative). The channels inactivated rapidly (time constants of 20-40 ms) during the voltage step. Steady-state inactivation and activation functions were well fitted by single Boltzmann equations of the form y = 1/[1 + exp [V-V0.5)/k)] and y = 1/[1 + exp [V0.5-V)/k)], where V0.5 = -82.9 +/- 2.4 and -55.2 +/- 1.5 mV and k = 4.5 +/- 0.6 and 4.9 +/- 0.6 mV, respectively. These small channels were not found in acutely isolated adult cells. 3. Ensemble averages of small-channel activity in numerous sweeps were very similar in time course to the T currents recorded in the whole-cell mode. Often small channels occurred in clusters, with many channels in a single patch. In these multichannel patches the voltage dependence of the kinetics of the channel was clearly revealed. 4. Small channels were insensitive to the dihydropyridine nifedipine (20 microM) and to TTX (1-5 microM), but the Li+ current through this channel was readily blocked by including 2 mM-Ca2+ in the recording pipette. Small-channel activity persisted for minutes in off-cell patches, and in cell-attached patches was not affected by phorbol esters. 5. The large channel was studied with electrodes filled with 110 mM-Ca2+ or Ba2+ (single-channel conductance approximately 24 pS in Ba2+). Activity of this channel was dramatically increased by the dihydropyridine Bay K 8644 and suppressed by nifedipine.(ABSTRACT TRUNCATED AT 400 WORDS)