Abstract
Some dyes of the methylene blue family serve as artificial inactivators of the sodium channels when present inside squid axons at a concentration of approximately 0.1 mM. The dyes restore a semblance of inactivation after normal inactivation has been destroyed by pronase. In fibers that inactivate normally, the dyes hasten the decay of sodium current. Many dye-blocked channels conduct transiently on exit of the dye molecule after repolarization to the holding potential. In contrast, normally inactivated channels do not conduct during recovery from inactivation. Kinetic evidence shows that inactivation of a dye-blocked channel is unlikely or impossible, which suggests that dye molecules compete with inactivation "particles" for the same site. In the absence of tetrodotoxin, the dyes do not affect the ON gating current unless the interpulse interval is very short. If sufficient equilibration time is allowed during a pulse, the initial amplitude of the OFF gating current is reduced to near zero. This suggests that a dye molecule is a Na channel completely blocks that channel's gating current, even the fraction that is resistant to normal inactivation. Dyes block INa and Ig with the same time course. This provides the strongest evidence to date that virtually all of recorded "gating current" is associated with Na channels. Tetrodotoxin greatly slows dissociation of dye molecules from Na channels and reduced gating current during both opening and closing of the channels.