Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification.
Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.
Wang
G K
GK
Department of Anesthesia Research Laboratories, Harvard Medical School, Boston, Massachusetts.
Wang
S Y
SY
D001496:Batrachotoxins / Q000494:pharmacology*; D001566:Benzocaine / Q000494:pharmacology; D002460:Cell Line; D002700:Chloramines / Q000494:pharmacology; D004347:Drug Interactions; D004357:Drug Synergism; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D012604:Scorpion Venoms / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism*; D014105:Tosyl Compounds
D001496:Batrachotoxins; D002700:Chloramines; D012604:Scorpion Venoms; D015222:Sodium Channels; D014105:Tosyl Compounds; C016300:chloramine-T; D012964:Sodium; D001566:Benzocaine