Interaction between calcium channel ligands and guanine nucleotides in cultured rat sensory and sympathetic neurones.

Abstract

Voltage-activated Ca2+ channel currents were recorded from cultured rat dorsal root ganglion (DRG) neurones using the whole-cell clamp technique with Ba2+ as the charge carrier. Inclusion of the GTP analogue guanosine 5''-O-3-thiotriphopshate (GTP-.gamma.-S, 500 .mu.M) or guanylylimidodiphosphate (GMP-PNP, 500 .mu.m) or GTP itself (1 mM) in the patch pipette solution resulted in a smaller, slowly activating Ca2+ channel current which did not inactivate during a 100 ms voltage step. This current was inhibited by CdCl2 (10-110 .mu.M) and .omega.-conotoxin (1 .mu.M). Nifedipine (5 .mu.M), (-)-(R)-201-791 (5 .mu.M), D600 (10 .mu.M), and diltiazem (30 .mu.M) inhibited Ca2+ channel currents recorded from control neurones, although in some cells a biphasic response was observed, with an initial increase preceding the inhibition of the currents. In the presence of internal GTP-.gamma.-S, at a holding potential (VH) of -80 mV, only potentiation of the Ca2+ channel current was observed in the presence of all three Ca2+ channel ligands. Internal GMP-PNP, while less effective than GTP-.gamma.-S, also resulted in D600 showing an agonist response. Similarly, in the presence of internal GTP (1 mM), (-)-(R)-202-791 gave a prolonged agonist response. Nifedipine, whether acting as an anatogonist in control cells or as an agonist in GTP-.gamma.-S-containing cells, induced a shift to more hyperpolarized potentials of the steady-state inactivation curves. Potentiation of Ca2+ channel currents induced by D600 in GTP-.gamma.-S-containing cells, was not observed when the neurones were pre-treated with pertussis toxin. The presence of internal GDP-.beta.-S (500 .mu.M) did not significantly alter the maximum inhibitory action of D600 compared with controls. However, 1 mM-GDP-.beta.-S increased the rate of onset of inhibition by (-)-(R)-202-791. Depolarizing VH to -30 mV accelerated the onset of inhibition induced by the Ca2+ channel ligands in control cells. In the presence of internal GTP-.gamma.-S at VH -30 mV, biphasic responses were produced by all the Ca2+ channel antagonist ligands with initial stimulation for 1-2 min being followed by inhibition of the Ca2+ channel currents. The agonist actions of (+)-(S)-202-791 were potentiated by the presence of internal GTP-.gamma.-S. The expression of an agonist response to (-)-(R)-202-791 induced by internal GTP-.gamma.-S was also present in sympathetic neurones cultured from adult rat superior cervical ganglion (SCG). Internal GTP-.gamma.-S markedly increased .tau.act of the Ca2+ channel current and .tau.deact of the tail current at both VH - 80 mV and VH -30 mV. (-)-(R)-202-791 acting as an agonist increased the rate of activation of the current in GTP-.gamma.-S-containing cells, and decreased the rate of tail current decay. The slower gating kinetics of Ca2+ channels in the presence of GTP-.gamma.-S may underlie the altered response to Ca2+ channel ligands. The potentiation of Ca2+ channel currents induced by Ca2+ channel ligands and the prevention of ligand-mediated inhibition in the presence of internal GTP-.gamma.-S suggests a functional link between binding sites for the Ca2+ channel ligands and the site at which the activated pertussis toxin-sensitive-G-protein (directly or indirectly) has its action.