Role of domain I of neuronal Ca2+ channel alpha1 subunits in G protein modulation

Abstract

1. We studied the G protein inhibition of heteromultimeric neuronal Ca2+ channels by constructing a series of chimeric channels between the strongly modulated alpha1B subunit and the alpha1E(rbEII) subunit, which showed no modulation. 2. In parallel studies, alpha1 subunit constructs were co-expressed together with the accessory Ca2+ channel alpha2-delta and beta2a subunits in mammalian (COS-7) cells and Xenopus oocytes. G protein inhibition of expressed Ca2+ channel currents was induced by co-transfection of Gbeta1 and Ggamma2 subunits in COS-7 cells or activation of co-expressed dopamine (D2) receptors by quinpirole (100 nM) in oocytes. 3. The data indicate that transfer of the alpha1B region containing the N-terminal, domain I and the I-II loop (i.e. the alpha1B1-483 sequence), conferred G protein modulation on alpha1E(rbEII), both in terms of a slowing of activation kinetics and a reduction in current amplitude. 4. In contrast, the data are not consistent with the I-II loop and/or the C-terminal forming a unique site for G protein modulation.

Figure 1. Effect of Gβ1γ2 on Ca²⁺ channel constructs in COS-7 cells

Ca²⁺ channel constructs between α1B and α1E(rbEII) subunits (as shown in A) were transfected together with cDNA coding for α2-δ and β2a subunits. B, example current density-voltage profiles for control cells (in the presence of GDPβS) and in the presence of Gβ1γ2. The initial test potential (V_t) shown was always -50 mV and was increased in 10 mV increments; holding potential V_H= -100 mV. Values for scale bars on the left apply also to scale bars on the right. C, time constant of activation (τ_act) at -10 mV for Ca²⁺ channel constructs coexpressed with Gβ1γ2 (black columns) or in control conditions in the presence of GDPβS (open columns); number of experiments, n, is given in parentheses. Only currents resulting from constructs containing the α1B_1-483 sequence showed a clear slowing of activation kinetics.

Figure 2. Effect of activation of D2 receptors on Ca²⁺ channel constructs in Xenopus oocytes

Ca²⁺ channel constructs were injected together with cDNA coding for α2-δ and β2a subunits and dopamine (D2) receptors in all cases. A, activation of D2 receptors by quinpirole caused an inhibition of currents formed by constructs containing the α1B_1-483 sequence; V_t= 0 mV and V_H= -100 mV; number of experiments, n, is given in parentheses. B, time course of barium current (I_Ba) inhibition by quinpirole (Q, 100 nm) in selected constructs (V_t= 0 mV, left panel) and corresponding effects of quinpirole on the current-voltage relationship (right panel). Current-voltage data were fitted with the equation: where G_max is maximum slope conductance, V_½ is the voltage at which 50 % of the current is activated, V_rev is the null potential and k is the slope factor. Quinpirole inhibition (•) was accompanied by a reduction in current amplitude, particularly at hyperpolarized potentials, with a corresponding shift in V_½ values.

Figure 3. Reversal of Gβγ inhibition of Ca²⁺ channel constructs by large depolarizing prepulses in COS-7 cells

Application of depolarizing prepulses reversed Gβ1γ2-induced inhibition, as shown for selected constructs. A, I_Ba (V_t= -40 to 0 mV) was examined immediately before (P1) and 10 ms after (P2) application of a depolarizing prepulse to +120 mV; V_H= -100 mV. B, in constructs containing the α1B_1-483 sequence, prepulses (pp) reversed Gβ1γ2-induced slowing of activation kinetics to control levels (recorded separately at P1 in control cells). C, in constructs containing the α1B_1-483 sequence, prepulses reversed Gβ1γ2-induced inhibition of current amplitude (P2 : P1 measured at 50 ms). In all instances, note the lack of effects on α1E(rbEII). Number of experiments, n, is given in parentheses.

Figure 4. Reversal of D2 receptor-induced inhibition of Ca²⁺ channel constructs by large depolarizing prepulses in Xenopus oocytes

Application of depolarizing prepulses caused a reversal of quinpirole-induced inhibition of I_Ba for constructs containing the α1B_1-483 sequence. A, percentage facilitation of control (open columns) and quinpirole-inhibited I_Ba (black columns) induced by a prepulse to +100 mV. B, in constructs containing the α1B_1-483 sequence, inhibition of control I_Ba (1) by quinpirole (2) was partially reversed by prepulse to +100 mV (3). In all cases, V_t= 0 mV and V_H= -100 mV. Number of experiments, n, is given in parentheses.