We studied the effects of modification of native cysteines present in squid giant axon Na channels with methanethiosulfonates. We find that intracellular, but not extracellular, perfusion of axons with positively charged [(2-trimethylammonium)-ethyl]methanethiosulfonate (MTSET), or 3(triethylammonium)propyl]methanethiosulfonate (MTS-PTrEA) irreversibly reduces sodium ionic (I(Na)) and gating (I(g)) currents. The rate of modification of Na channels was dependent on the concentration of the modifying agent and the transmembrane voltage. Hyperpolarized membrane potentials (e.g., -110 mV) protected the channels from modification by MTS- PTrEA. In addition to reducing the amplitudes of I(Na) and I(g), MTS-PTrEA also altered their kinetics such that the remaining/(Na) did not appear to inactivate, whereas I(g) was made sharper and declined to baseline more quickly. The shape and amplitude of I(g) after modification of channels with MTS-PTrEA appeared to be 'charge-immobilized,' as if the modified channels were inactivated. MTS-PTrEA did not affect/(Na) or I(g) when inactivation was removed by internal perfusion of the axon with pronase. In addition, we find that the steady-state inactivation curve of modified Na channels is made much shallower and is markedly shifted to hyperpolarized potentials. The rates of activation, deactivation, or open-state inactivation were not altered in MTS- PTrEA-modified channels. The uncharged sulfhydryl reagent methymethanethiosulfonate (MMTS) did not affect either I(Na) or I(g), but prevented the irreversible effects of MTS-PTrEA or MTSET on Na channels. It is proposed that the positively charged methanethiosulfonates MTS-PTrEA and MTSET modify a native internal cysteine(s) in squid Na channels, and by doing so promote inactivation from closed states, resulting in charge immobilization and reduction of I(Na).
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