SUMMARY1. Single skeletal muscle fibres from the frog Rana pipiens were treated with the carboxyl group modifying reagent trimethyloxonium ion (TMO) and voltage clamped by the method of .2. TMO treatment reduced current through sodium channels to 0 33 + 0 03 that before treatment, but only 45 + 3 % of this remaining current was blocked by 1 #/Mtetrodotoxin (TTX) and only 37 + 5 % by 100 nm-saxitoxin (STX).3. This toxin resistance persisted in 90 sM-TTX, was not due to inactivation of toxin nor to components of the reaction solution other than TMO, but was prevented by the presence of 100 nM-STX during treatment with TMO. TMO-modified sodium channels can be blocked by the local anaesthetic lidocaine. 4. The permeabilities of TMO-modified channels to hydroxylammonium, ammonium, guanidinium, aminoguanidinium, methylammonium and tetramethylammonium ions relative to sodium were not significantly different from the permeabilities of untreated sodium channels.5. Hydrogen ions blocked TMO-modified sodium channels, but the apparent pKa for block at + 38 mV of 5 07 was significantly less than the corresponding value of 5*32 in untreated sodium channels.6. It is suggested that TMO produces toxin resistance by esterifying an ionized carboxyl group which is an essential part of the toxin binding site. Such esterification would electrostatically reduce the local cation concentration, thus reducing the apparent pKa of hydrogen ion block and the single-channel conductance (Sigworth &

Tetrodotoxin (TTX) and saxitoxin (STX) are extremely potent poisons that prevent nerve and muscle cells from producing action potentials by blocking sodium channels. If the channels are modified by reagents that act on carboxyl groups, however, both the binding of these toxins and their effect on the action potential are reduced. One such reagent, trimethyloxonium ion (TMO) converts channels into a form that is not blocked by TTX concentrations 10(5) times greater than its normal Kd (ref. 6). Most such chemical modifications of sodium channels also reduce the measured membrane sodium current, but it has not been known whether such reductions were due to a change in the number of channels, in permeability properties, or in gating properties. We now report that TMO-modified, TTX-resistant sodium channels have a smaller single-channel conductance (gamma) with a more linear instantaneous current-voltage relationship than that of normal channels, and that the measured reduction in gamma accounts for all of the decrease in sodium current after TMO treatment. This change in sodium channel permeability properties can be explained by the removal of a fixed negative charge near the outside of the channel.

Excitation of nerve or muscle requires an orderly opening and closing of molecular pores, the ionic channels, in the plasma membrane. During the action potential, Na channels are opened (activated) by the advancing wave of depolarisation, contributing a pulse of inward sodium current, and then are closed again (inactivated) by the continued depolarisation. As one approach both to obtaining molecular information on the Na channel and towards further defining the recently discovered kinetic interactions of the inactivation and activation gating steps, we have surveyed here the effects of chemical agents reported to slow or prevent Na channel inactivation. We find that many of the agents studied by others on invertebrate giant axons or vertebrate nerve act on our frog skeletal muscle preparation. In addition, we have discovered that simply lowering the intracellular pH nearly eliminates inactivation. The activation mechanism seems to resist modification.