Loss of Cav1.3 channels reveals the critical role of L-type and BK channel coupling in pacemaking mouse adrenal chromaffin cells.
We studied wild-type (WT) and Cav1.3(-/-) mouse chromaffin cells (MCCs) with the aim to determine the isoform of L-type Ca(2+) channel (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 the BK channel blocker paxilline altered the shape of action potentials (APs), suggesting close coupling of LTCCs to BK channels. WT-MCCs expressed 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 Ca(2+) 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-inactivating BK currents 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 Ca(2+) current and the corresponding Ca(2+)-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.
Marcantoni
Andrea
A
Department of Neuroscience, Nanostructured Interfaces and Surfaces Centre of Excellence, Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia, 10125 Torino, Italy.
Vandael
David H F
DH
Mahapatra
Satyajit
S
Carabelli
Valentina
V
Sinnegger-Brauns
Martina J
MJ
Striessnig
Joerg
J
Carbone
Emilio
E
D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester / Q000494:pharmacology; D000302:Adrenal Cortex / Q000378:metabolism; D000313:Adrenal Medulla / Q000166:cytology*; D000818:Animals; D055592:Biophysical Phenomena / Q000187:drug effects / Q000235:genetics; D001703:Biophysics; D002120:Calcium Channel Agonists / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D019439:Chromaffin Cells / Q000187:drug effects / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004558:Electric Stimulation / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics; D007211:Indoles / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D018408:Patch-Clamp Techniques; D026902:Potassium Channel Blockers / Q000494:pharmacology; D013111:Spider Venoms / Q000494:pharmacology; D013997:Time Factors
C514350:CACNA1C protein, mouse; C485995:Cacna1d protein, mouse; D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D020746:Calcium Channels, L-Type; D002614:Chelating Agents; D007211:Indoles; D007476:Ionophores; C419385:L-type calcium channel alpha(1C); D051036:Large-Conductance Calcium-Activated Potassium Channels; D026902:Potassium Channel Blockers; C115521:SNX 482; D013111:Spider Venoms; C048220:paxilline; D004533:Egtazic Acid; D001498:3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid