G-PROTEIN MODULATION OF N-TYPE CALCIUM-CHANNEL GATING CURRENT IN HUMAN EMBRYONIC KIDNEY-CELLS (HEK-293)

1. Voltage-dependent inhibition of N-type calcium currents by G-protei ns contributes importantly to presynaptic inhibition. To examine the e ffect of G-proteins on key intermediary transitions leading to channel opening, we measured both gating and ionic currents arising from reco mbinant N-type channels (alpha(1B), beta(1b) and alpha(2)) expressed i n transiently transfected human embryonic kidney cells (HEK 293). Reco mbinant expression population of channels provided a favourable system for rigorous examination of the mechanisms underlying G-protein modul ation. 2. During intracellular dialysis with GTP gamma S to activate G -proteins, ionic currents demonstrated classic features of voltage-dep endent inhibition, i.e. strong depolarizing prepulses increased ionic currents and produced hyperpolarizing shifts in the voltage-dependent activation of ionic current. No such effects were observed with GDP be ta S present to minimize G-protein activity. 3. Gating currents were c learly resolved after ionic current blockade with 0.1. mM free La3+, e nabling this first report of gating charge translocation arising exclu sively from N-type channels. G-proteins decreased the amplitude of gat ing currents and produced depolarizing shifts in the voltage-dependent activation of gating charge movement. However, the greatest effect wa s to induce a similar to 20 mV separation between the voltage-dependen t activation of gating charge movement and ionic current. Strong depol arizing prepulses largely reversed these effects. These modulatory fea tures provide telling clues about the kinetic steps affected by G-prot eins because gating currents arise from the movement of voltage sensor s that trigger channel activation. 4. The mechanistic implications of concomitant G-protein-mediated changes in gating and ionic currents ar e discussed. We argue that G-proteins act to inhibit both voltage-sens or movement and the transduction of voltage-sensor activation into cha nnel opening.