RESUMO
1. The open time of single Na+ channels in excised (outside-out) patches from cultured late-fetal rat ventricular myocytes was prolonged to several minutes by germitrine (0.5 mM) in order to analyse tetrodotoxin (TTX) blocking kinetics. 2. The germitrine modification appeared during depolarizing pulses that activated normal Na+ channels. Following repolarization to -100 mV, the modified Na+ channel remained activated for 136 +/- 186 s (mean +/- S.D., n = 54) with an open-channel current amplitude of -0.5 pA. The predominant open state with a mean open time of 0.13 s was interrupted by brief closing events lasting for milliseconds. Replacing extracellular Na+ by Cs+ decreased the current amplitude to -0.1 pA. 3. Extracellular superfusion with TTX (3 x 10(-7) M) of a single germitrine-activated Na+ channel induced full channel closures lasting seconds (blocked events) separated by channel reopenings (unblocked events) that were indistinguishable in terms of amplitude and gating kinetics from the germitrine-activated state in the absence of TTX. 4. Cumulative probability histograms of blocked and unblocked events (n greater than 140) collected during long-lasting germitrine modifications at 10(-7) and 3 x 10(-7) M-TTX are well described by single exponentials. The 3-fold increase in [TTX] decreased the time constant of the unblocked state, tau o, from 11.9 to 4.7 s, while the time constant of the blocked state, tau c, was not significantly altered from 8.6 to 9.7 s. A microscopic association rate constant of 7.7 x 10(5) M-1 s-1, dissociation rate constant of 0.11 s-1, and equilibrium dissociation constant of 1.4 x 10(-7) M (at -100 mV) were calculated (20 degrees C). 5. Increasing [TTX] to 10(-5) M decreased tau o to 86 ms. This argues against the existence of a slower conformational step interposed between the binding of TTX to an open channel and the resultant channel closure. 6. Setting the membrane potential to -50 or 0 mV subsequent to a germitrine modification at -100 mV did not significantly alter TTX (3 x 10(-7) M) blocking kinetics: tau o was 6.7 s at -50 mV and 5.2 s at 0 mV; tau c was 8.9 and 8.1 s, respectively. 7. These results suggest that blocked events correspond to the random times that a TTX molecule resides on the Na+ channel before it dissociates, and unblocked events correspond to the random waiting times of an unoccupied channel before it binds another toxin molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
