FACILITATION OF RABBIT ALPHA(1B) CALCIUM CHANNELS - INVOLVEMENT OF ENDOGENOUS G-BETA-GAMMA SUBUNITS

1. The alpha(1B) (N-type) calcium channel shows strong G protein modul ation in the presence of G protein activators or G beta gamma subunits . Using transient expression in COS-7 cells of a,, together with the a ccessory subunits alpha(2)-delta and beta(2a), we have examined the ro le of endogenous G beta gamma subunits in the tonic modulation of alph a(1B) and compared this with modulation by exogenously expressed G bet a gamma subunits. 2. Prepulse facilitation of control alpha(1B)/alpha( 2)-delta/beta(2a) currents was always observed. This suggests the exis tence of tonic modulation of alpha(1B) subunits. To determine whether endogenous G beta gamma is involved in the facilitation observed in co ntrol conditions, the beta ARK1 G beta gamma-binding domain (amino aci ds 495-689) was overexpressed, in order to bind free G beta gamma subu nits. The extent of control prepulse-induced facilitation was signific antly reduced, both in terms of current amplitude and the rate of curr ent activation. In agreement with this, GDP beta S also reduced the co ntrol facilitation. 3. Go-expression of the G beta(1) gamma(2) subunit , together with the alpha(1B)/alpha(2)-delta/beta(2a) calcium channel combination, resulted in a marked degree of depolarizing prepulse-reve rsible inhibition of the whole-cell I-Ca or I-Ba. Both slowing of curr ent activation and inhibition of the maximum current amplitude were ob served, accompanied by a depolarizing shift in the mid-point of the vo ltage dependence of activation. Activation of endogenous G beta gamma subunits by dialysis with GTP gamma S produced a smaller degree of pre pulse-reversible inhibition. 4. The rate of reinhibition of alpha(1B) currents by activated G protein, following a depolarizing prepulse, wa s much faster with G beta(1) gamma(2) than for the decay of facilitati on in control cells. Furthermore, beta ARK1 (495-689) co-expression ma rkedly slowed the control rate of reinhibition, suggesting that the ki netics of reinhibition depend on the concentration of free endogenous or exogenously expressed G beta gamma in the cells. In contrast, the r ate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined. 5. These find ings indicate that, in this system, the voltage-dependent facilitation of alpha(1B) that is observed under control conditions occurs as a re sult of endogenous free G beta gamma binding to alpha(1B).