Chemical modification of sodium channel surface charges in frog skeletal muscle by trinitrobenzene sulphonic acid

Abstract

1. We have investigated the effects of externally applied trinitrobenzene sulphonic acid (TNBS) on sodium currents of voltage-clamped frog skeletal muscle fibres. TNBS is a membrane-impermeant reagent which reacts specifically with amino groups under physiological conditions. 2. TNBS shifts the voltage dependence of steady-state sodium current inactivation (h infinity) by approximately 18 mV to more hyperpolarized potentials when measured at pH 9. This effect of TNBS is irreversible, suggesting that the reagent permanently modifies some membrane component(s). 3. Time constants for the development of and recovery from inactivation of sodium current are similarly shifted to more hyperpolarized potentials by TNBS treatment. External TNBS did not affect the completeness of sodium current inactivation during depolarizing pulses. 4. The activation of sodium current is less affected by TNBS than is inactivation. The voltage dependence of peak sodium current activation was shifted by only approximately 4 mV by TNBS treatment at pH 9. The kinetics of sodium current activation during depolarization were little affected by TNBS. 5. The TNBS-induced shift in h infinity and sodium current activation are independent of pH when measured at pHs between 7.4 and 11. 6. The results are consistent with the hypothesis that TNBS increases the negative surface charge of the membrane by reacting with membrane amino groups having a high pK.