Regulation of recombinant CaV3.1 protein membrane expression by Cdk5.
A) Representative macroscopic current traces recorded from HEK-293 cells transiently transfected with a plasmid encoding CaV3.1 channels alone or in conjunction with plasmids encoding Cdk5 and p35. Currents were elicited by depolarizing steps from a Vh of −80|mV to-30|mV. Ba2+ (2 mM) was used as the charge carrier. B) Conductance-voltage (G-V) curves were calculated for each cell. An increase in conductance was observed in Cdk5/p35-coexpressing cells. C) Cdk5/p35 overexpression in HEK-293 cells stably expressing CaV3.1 channels did not affect the voltage dependence of current activation and steady-state inactivation. D) Comparison of the time constant of current activation (τact) and inactivation (τinact) at different membrane voltages in untransfected (control) and Cdk5/p35-coexpressing cells. E) Representative cell surface protein extraction assay followed by Western blot using an anti-CaV3.1 specific antibody. F) Densitometric quantification of three repetitions of the experiment shown in E. Asterisk denotes significant differences (P>0.05) between cells expressing the CaV3.1 channels only, and cells transfected with the channels plus the Cdk5/p35 complex.