Loss of Cav1.3 channels reveals the critical role of L- and BK-channel coupling in pace-making mouse adrenal chromaffin cells

Westudied wild-type (WT) and Cav1.3/mouse chromaffin cells (MCCs) with the aim to determine the isoform of L-type Ca2channel (LTCC) and BK channels that underlie the pacemaker current controlling spontaneous firing. Most WT-MCCs (80%) were spontaneously active (1.5 Hz) and highly sensitive to nifedipine and BayK-8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3- pyridinecarboxylic acid, methyl ester). Nifedipine blocked the firing, whereas BayK-8644 increased threefold the firing rate. The two dihydropyridines and theBKchannel blocker paxilline altered the shape of action potentials (APs), suggesting close coupling of LTCCs to BKchannels.WT-MCCsexpressed equal fractions of functionally active Cav1.2 and Cav1.3 channels. Cav1.3 channel deficiency decreased the number of normally firing MCCs (30%; 2.0 Hz), suggesting a critical role of these channels on firing, which derived from their slow inactivation rate, sizeable activation at subthreshold potentials, and close coupling to fast inactivating BK channels as determined by using EGTA and BAPTA Ca2 buffering. By means of the action potential clamp, in TTX-treated WT-MCCs, we found that the interpulse pacemaker current was always net inward and dominated by LTCCs. Fast inactivating and non-inactivatingBKcurrents sustained mainly the afterhyperpolarization of the short APs (2–3 ms) and only partially the pacemaker current during the long interspike (300 –500 ms). Deletion of Cav1.3 channels reduced drastically the inward Ca2 current and the corresponding Ca2-activated BK current during spikes. Our data highlight the role of Cav1.3, and to a minor degree of Cav1.2, as subthreshold pacemaker channels in MCCs and open new interesting features about their role in the control of firing and catecholamine secretion at rest and during sustained stimulations matching acute stress.