Studies of calcium channels in rat clonal pituitary cells with patch electrode voltage clamp

Abstract

The properties of the Ca channel in tissue cultured clonal cells (GH3) isolated from a rat anterior pituitary tumor were studied with the patch electrode voltage-clamp technique. To isolate the current through the Ca channel, the currents through the Na+ channel, the delayed K channel and the Ca2+ induced K channel were minimized by replacing the external Na+ with tetraethylammonium and adding ethylene glycol-bis(.beta.-aminoethylether)N,N,N'',N''-tetraacetic acid to the K-free solution inside the patch electrode. The selectivity ratios through the Ca channel with different cations were 2.7 (Ba2+):1.6 (Sr2+):1.0 (Ca2+), and the m2 form of the activation kinetics, and the relationships between the time constant and the membrane potential were common to the 3 divalent cations. The amplitude of the Ba2+ current increased linearly with [Ba2+]o up to 25 mM and thereafter tended to show saturation. The current-voltage relation showed a positive shift along the voltage axis as [Ba2+]o increased, probably due to the screening effect of Ba2+ on the negative surface charges. The time constant of activation as a function of the membrane potential showed a parallel shift as [Ba2+]o was increased, suggesting that the activation kinetics were independent of the permeant ion concentration. The time constant of the tail current was consistent with m2 kinetics for opening and closing of the Ca channel. The extrapolated ''instantaneous'' tail current rapidly increased as the activating membrane potential became more positive and reached an apparent saturation at membrane potentials substantially more positive than the potential that gave the maximum peak inward current, and suggested that the single channel has a sigmoidal current-voltage relationship. The power density spectrum obtained during the steady-state inward Ba2+ current had a cut-off frequency which was nearly voltage independent; this is expected if the fluctuation of the current originates from m2 activation kinetics. The results of noise analysis suggest that the amplitude of the single Ca channel current was .apprx. 0.2 pA at 25 mM-Ba2+ and 0.7 pA at 100 mM-Ba2+ for membrane potentials in the vicinity of the maximum inward current.