1. Voltage-dependent inhibition of N-type calcium currents by a-proteins contributes importantly to presynaptic inhibition. To examine the effect of G-proteins on key intermediary transitions leading to channel opening, we measured both gating and ionic currents arising from recombinant N-type channels (α(1B), β(1b) and α2) expressed in transiently transfected human embryonic kidney cells (HEK 293). Recombinant expression of a homogeneous population of channels provided a favourable system for rigorous examination of the mechanisms underlying G-protein modulation. 2. During intracellular dialysis with GTPγS to activate G-proteins, ionic currents demonstrated classic features of voltage-dependent 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 GDPβS present to minimize G-protein activity. 3. Gating currents were clearly resolved after ionic current blockade with 0.1 mM free La3+, enabling this first report of gating charge translocation arising exclusively from N-type channels. G-proteins decreased the amplitude of gating currents and produced depolarizing shifts in the voltage-dependent activation of gating charge movement. However, the greatest effect was to induce a ~ 20 mV separation between the voltage-dependent activation of gating charge movement and ionic current. Strong depolarizing prepulses largely reversed these effects. These modulatory features provide telling clues about the kinetic steps affected by G-proteins because gating currents arise from the movement of voltage sensors that trigger channel activation. 4. The mechanistic implications of concomitant G-protein-mediated changes in gating and ionic currents are discussed. We argue that G-proteins act to inhibit both voltage-sensor movement and the transduction of voltage-sensor activation into channel opening.
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