Voltage inactivation of Ca 2+ entry and secretion associated with N‐ and P/Q‐type but not L‐type Ca 2+ channels of bovine chromaffin cells

1 In this study we pose the question of why the bovine adrenal medullary chromaffin cell needs various subtypes (L, N, P, Q) of the neuronal high‐voltage activated Ca 2+ channels to control a given physiological function, i.e. the exocytotic release of catecholamines. One plausible hypothesis is that Ca 2+ channel subtypes undergo different patterns of inactivation during cell depolarization. 2 The net Ca 2+ uptake (measured using 45 Ca 2+ ) into hyperpolarized cells (bathed in a nominally Ca 2+ ‐free solution containing 1·2 mM K + ) after application of a Ca 2+ pulse (5 s exposure to 100 mM K + and 2 mM Ca 2+ ), amounted to 0·65 ± 0·02 fmol cell −1 ; in depolarized cells (bathed in nominally Ca 2+ ‐free solution containing 100 mM K + ) the net Ca 2+ uptake was 0·16 ± 0·01 fmol cell −1 . 3 This was paralleled by a dramatic reduction of the increase in the cytosolic Ca 2+ concentration, [Ca 2+ ] i , caused by Ca 2+ pulses applied to fura‐2‐loaded single cells, from 1181 ± 104 nM in hyperpolarized cells to 115 ± 9 nM in depolarized cells. 4 A similar decrease was observed when studying catecholamine release. Secretion was decreased when K + concentration was increased from 1·2 to 100 mM; the Ca 2+ pulse caused, when comparing the extreme conditions, the secretion of 807 ± 35 nA of catecholamines in hyperpolarized cells and 220 ± 19 nA in depolarized cells. 5 The inactivation by depolarization of Ca 2+ entry and secretion occluded the blocking effects of combined ω‐conotoxin GVIA (1 μM) and ω‐agatoxin IVA (2 μM), thus suggesting that depolarization caused a selective inactivation of the N‐ and P/Q‐type Ca 2+ channels. 6 This was strengthened by two additional findings: (i) nifedipine (3 μM), an L‐type Ca 2+ channel blocker, suppressed the fraction of Ca 2+ entry (24 %) and secretion (27 %) left unblocked by depolarization; (ii) FPL64176 (3 μM), an L‐type Ca 2+ channel ‘activator’, dramatically enhanced the entry of Ca 2+ and the secretory response in depolarized cells. 7 In voltage‐clamped cells, switching the holding potential from ‐80 to ‐40 mV promoted the loss of 80 % of the whole‐cell inward Ca 2+ channel current carried by 10 mM Ba 2+ ( I Ba ). The residual current was blocked by 80 % upon addition of 3 μM nifedipine and dramatically enhanced by 3 μM FPL64176. 8 Thus, it seems that the N‐ and P/Q‐subtypes of calcium channels are more prone to inactivation at depolarizing voltages than the L‐subtype. We propose that this different inactivation might occur physiologically during different patterns of action potential firing, triggered by endogenously released acetylcholine under various stressful conditions.