Density of apamin-sensitive Ca(2+)-dependent K+ channels in bovine chromaffin cells: relevance to secretion

Three objectives were defined when planning this study: (i) to identify binding sites for [125I]-apamin in intact bovine adrenal medulla chromaffin cells and to estimate their density and selectivity; (ii) to determine whether apamin modified the release of catecholamines evoked by brief pulses of dimethylphenylpiperazinium (DMPP, 1 or 5 microM for 10 sec), histamine (10 microM for 10 sec) or high K+ (20, 35 or 70 mM for 10 sec) applied to superfused cells; and (iii) to test whether apamin affected the profiles of the changes in cytosolic Ca2+ concentrations [Ca2+]i obtained in suspensions of cells loaded with fura-2 and stimulated with DMPP or histamine. At equilibrium, increasing concentrations of [125I]-apamin gave a saturation curve whose Scatchard transformation produced a Kd of 132 pM and a Bmax of 0.72 fmol/10(6) cells. Quinine, tetraethylammonium, charybdotoxin or glibenclamide (blockers of various subtypes of K+ channels) did not inhibit [125I]apamin binding. Binding was blocked by apamin and by d-tubocurarine, two blockers of small-conductance Ca(2+)-activated K+ channels (SK channels). The number of binding sites for [125I]apamin amounted to approx. 900 per single chromaffin cell, 0.72 sites per micron 2 surface area. Apamin (1 microM) enhanced the secretory response to histamine (10 microM), DMPP (1 or 5 microM) and high K+ (20 or 35 mM) by 2-3-fold. The response to 70 mM K+, however, was unaffected. Apamin also enhanced the peak [Ca2+]i increase produced by DMPP or histamine by approx. 30%. Overall, these results strongly support the hypothesis that under physiological conditions, SK channels control some of the electrical activity of chromaffin cells and indirectly, the opening of voltage-dependent Ca2+ channels, the access of Ca2+ to the secretory machinery and the rate of catecholamine release to the circulation from the intact adrenal gland.