Remo, al of Sodium Channel Inactivation in Squid Giant Axons by N-Bromoacetamide

The group-specific protein reagents, N-bromoacetamide (NBA) and N-bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an allor-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur-containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel. I N T R O D U C T I O N The widely held view that ionic channels in excitable membranes are composed of protein molecules has been b rough t into clear focus by the demonst ra t ion that certain proteases can modify the inactivation gating behavior o f sodium channels (Rojas and Armst rong , 1971; Arms t rong et al., 1973; Sevcik and Narahashi , 1975; Rojas and Rudy, 1976). In addition, a correlation between the spectral dependence o f selective sodium channel destruction by ultraviolet radiation and absorption spectra o f aromatic amino acids has been demons t ra ted (Fox, 1974; Oxford and Pooler, 1975a). Such data have p rompted more systematic investigations o f the possible role o f specific protein chemical groups in the function o f both sodium and potassium channels in nerve membrane (Shrager, 1974, 1975, 1977; Keana and Stfimpfli, 1974; Marquis and Mautner , 1974; Gainer et al., 1974; Inoue et al., 1976). The application o f group-specific protein reagents to probe the molecular interactions within ionic channels unde r voltage clamp conditions is a potentially T H E J O U R N A L OF G E N E R A L P H Y S I O L O G Y " V O L U M E 71, 1978 • p a g e s 2 2 7 2 4 7 227 on Jne 6, 2017 D ow nladed fom Published March 1, 1978