Stimulation of recombinant CaV3.2, T-type, Ca2+ channel currents by CaMKIIγC

Abstract

Molecular cloning of low-voltage activated (LVA) T-type calcium channels has enabled the study of their regulation in heterologous expression systems. Here we investigate the regulation of Cav3.2 α1 -subunits (α1H) by calcium- and/or calmodulin-dependent protein kinase II (CaMKII). 293 cells stably expressing α1H were transiently transfected with CaMKIIγc. Using the whole-cell recording configuration, we observed that elevation of pipette free Ca2+ (1 μM) in the presence of CaM (2 μM) increases T-type channel activity selectively at negative potentials, evoking an 11 mV hyperpolarizing shift in the half-maximal potential (V1/2) for activation. The V1/2 of channel inactivation is not altered by Ca2+/CaM. These effects reproduced modulation observed in adrenal zona glomerulosa cells. The potentiation by Ca2+/CaM was dependent on the co-expression of CaMKIIγc and required Ca2+/CaM-dependent kinase activity. Peptide (AIP) and lipophilic (KN-62) protein kinase inhibitors prevented the Ca2+/CaM-induced changes in channel gating without altering basal Cav3.2 channel activity (27 nM free Ca2+) as did replacing pipette ATP with adenylyl imidodiphosphate (AMP-PNP), a non-hydrolysable analogue. CaMKII-dependent potentiation of channel opening resulted in significant increases in apparent steady-state open probability (Po) and sustained channel current at negative voltages. Under identical conditions, CaMKII activation did not regulate the activity of Cav3.1 channels, the first cloned member (α1G) of the T-type Ca2+ channel family. Our results provide the first evidence for the differential regulation of two members of the Cav3 family by protein kinase activation and the first report reconstituting CaMKII-dependent regulation of any cloned Ca2+ channel.