Kinetics and Gβγ modulation of Ca_v2.2 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_v2.2 channel subunits (N-type, α₁B and either β_1b or β_2a). These were co-expressed with either Gβ₁γ₂ or the Gβγ-binding domain of β-adrenergic-receptor kinase-1 to sequester endogenous Gβγ. Since G protein modulation of Ca_v channels may affect both inactivation and activation, we examined Gβγ modulation of Ca_v2.2 channels in the presence of two different β-subunits that affect inactivation differently and compared in detail the single-channel characteristics of N-type channels expressed with either of these β-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 β_1b and β_2a-subunits by co-transfection with Gβ₁γ₂. This effect was absent at lower voltages as examined for channels with the β_1b-subunit. The distribution of latency-to-first-opening of Ca_v2.2 channels was similar for both β-subunit isoforms. However, the inclusion of the β_2a subunit resulted in channels with an additional, prominent, slow activation phase. Co-transfection of Gβ₁γ₂ with Ca_v2.2 channels markedly reduced the ensemble current amplitude and slowed the first latency. The inhibition imposed by Gβ₁γ₂ was largely independent of the β-subunit species. Facilitation of Gβγ-modulated currents (the channel response following a large and brief depolarising prepulse) was observed for channels with both β-subunits and involved mainly enhancement of the activation, as assessed by the faster first latency. The inactivation process was strongly dependent on the β-subunit species, with β_1b supporting inactivation and β_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 β_1b-subunit had a fast component of inactivation (time constant ~180 ms, 50%) and a slow phase with time constant of ~1 s, while the β_2a-subunit supported only a very slow inactivation process with time constant of ~5 s. Co-transfection of Gβ₁γ₂ with the Ca_v2.2 channel had no effect on the inactivation properties with either β-subunit. In summary, we show that the inactivation properties of expressed Ca_v2.2 channels depend largely on the β-subunit species and to a minor extent only on the presence or absence of the Gβγ modulator. Furthermore, the activation, amplitude, mean open and closed times and G protein modulation of N-type channels were similar for both β_1b- and β_2a-subunits.