Kinetics and Gβγ modulation of Cav2.2 channels with different auxiliary β subunits

Abstract

Modulation of calcium channels by both auxiliary subunits and G proteins was studied in cell-attached patches from COS-7 cells transfected with Ca(v)2.2 channel subunits (N-type, alpha(1)B and either beta(1b) or beta(2a)). These were co-expressed with either Gbeta(1)gamma(2) or the Gbetagamma-binding domain of beta-adrenergic-receptor kinase-1 to sequester endogenous Gbetagamma. Since G protein modulation of Ca(v) channels may affect both inactivation and activation, we examined Gbetagamma modulation of Ca(v)2.2 channels in the presence of two different beta-subunits that affect inactivation differently and compared in detail the single-channel characteristics of N-type channels expressed with either of these beta-subunit isoforms. The single-channel mean amplitude and mean open time were not influenced by the transfection combination. However, the mean closed time at +40 mV was increased for both beta(1b) and beta(2a)-subunits by co-transfection with Gbeta(1)gamma(2). This effect was absent at lower voltages as examined for channels with the beta(1b)-subunit. The distribution of latency-to-first-opening of Ca(v)2.2 channels was similar for both beta-subunit isoforms. However, the inclusion of the beta(2a) subunit resulted in channels with an additional, prominent, slow activation phase. Co-transfection of Gbeta(1)gamma(2) with Ca(v)2.2 channels markedly reduced the ensemble current amplitude and slowed the first latency. The inhibition imposed by Gbeta(1)gamma(2) was largely independent of the beta-subunit species. Facilitation of Gbetagamma-modulated currents (the channel response following a large and brief depolarising prepulse) was observed for channels with both beta-subunits and involved mainly enhancement of the activation, as assessed by the faster first latency. The inactivation process was strongly dependent on the beta-subunit species, with beta(1b) supporting inactivation and beta(2a) reducing this process. This difference was assessed by estimation of both steady-state inactivation (prepulse influence on test pulse responses) and the inactivation time course during depolarisation. At +40 mV, channels with the beta(1b)-subunit had a fast component of inactivation (time constant ~180 ms, 50%) and a slow phase with time constant of approximately 1 s, while the beta(2a)-subunit supported only a very slow inactivation process with time constant of approximately 5 s. Co-transfection of Gbeta(1)gamma(2) with the Ca(v)2.2 channel had no effect on the inactivation properties with either beta-subunit. In summary, we show that the inactivation properties of expressed Ca(v)2.2 channels depend largely on the beta-subunit species and to a minor extent only on the presence or absence of the Gbetagamma modulator. Furthermore, the activation, amplitude, mean open and closed times and G protein modulation of N-type channels were similar for both beta(1b)- and beta(2a)-subunits.