CALCIUM-DEPENDENT SHIFTS OF NA-BINDING TO THE PORE( CHANNEL ACTIVATION CORRELATED WITH THE STATE DEPENDENCE OF CALCIUM)

Calcium ions block the open configuration and antagonise the tonic bin ding of TTX to the closed state of sodium channels in very different r anges of extracellular concentration, [Ca](0). We measured the open-st ate block in channels expressed in Xenopus oocytes by a-subunits from rat brain (rBIIa) or adult fat skeletal muscle (rSkMI). Recordings of instantaneous tail-currents from cell-attached macro patches show that the binding of Ca2+ to the blocking site has a dissociation constant of about 20 mM at 0 mV and senses about 30% of the membrane potential drop, whereas the concentration of half-inhibition of TTX-binding is l ess than 1 mM and voltage-insensitive. Assuming that both effects invo lve a single binding site, a simple model predicts that the state-depe ndency of the dissociation constant entails positive shifts of activat ion and faster kinetics of deactivation at increasing [Ca](0). The shi fts of activation measured for rBIIA and rSkM1 channels are comparable in size to those predicted by the model, which accounts also for the observed larger shifts of the rBIIA-mutant K226Q as a consequence of i ts reduced voltage-sensitivity. Shifts attributable to surface-charge screening effects seem smaller in the oocyte than in native cell-membr anes. The experimental [Ca](0)-dependence of deactivation kinetics is also consistent with the model and with the idea that Ca2+-binding cha nges to the same extent, but in opposite directions, the activation fr ee-energies of both opening and closing transitions.