Gamma1 Subunit Renders Cav1.2 Channels Dependent on Cell Cycle

Abstract

Auxiliary γ subunits are known to enhance inactivation of L-type calcium channels. When co-expressed in tsA-201 cells with α1C and β2a subunits, the γ1 subunit shifts the voltage-dependence of inactivation by about −20 mV. The voltage of half-maximal inactivation, V1/2, was determined with 5 s long conditioning pre-pulses. On average, addition of γ1 to α1C/β2a channels changed V1/2 from −24±5 mV (n=30) to −44±12 mV (n=92). We noticed that V1/2, but not the steepness of the voltage-dependence of inactivation, varies greatly in cells with γ1 and set up to find the cause of the cell-to-cell variability. Serum starvation and “ER shock” by the N-glycosylation blocker tunicamicin further shifted inactivation to negative voltages. The average V1/2 was −59±12 mV (n=12) in serum-free and −69±13 mV(n=32) in tunicamicin treated cells. These treatments altered inactivation only when γ1 was present and the effects were similar when β3 substituted for β2a. Mutations of γ1 that remove consensus N-glycosylation sites had only partial effect (V1/2=-60±18 mV, n=29) and did not reduce the cell-to-cell variability, indicating that N-glycosylation of γ1 was not its primary cause.Serum starvation and tunicamicin are known to produce cell-cycle arrest in the G0/G1 phase and, therefore, could act on γ1 indirectly by interfering with a cell-cycle dependent pathway. We characterized inactivation in cells expressing fluorescent probes visualizing cell-cycle activity. In support of our hypothesis, V1/2 was −55±16 mV, n=32, in G1 and −36±7 mV, n=20, in S/G2/M cells.Therefore, we propose that a novel cell-cycle dependent regulatory pathway controls voltage-dependent inactivation and functional availability of L-type calcium channels in the presence of γ1 subunit.The project was supported by MH079406 from NIMH.