Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because t hey have similar actions, sizes, and functional groups, they have been thou ght to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by Kirsch et al. (1994. Bio phys. J. 67:2305-2315) have suggested that the toxins bind differently to t he isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal mu scle Na+ channel isoform (mu l), we compared the effects on both TTX and ST X affinities of mutations in eight positions known to influence toxin bindi ng. The results permitted the assignment of energies contributed by each am ino acid to the binding reaction. For neutralizing mutations of Asp(400), G lu(755), and Lys(1237), all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule resid ues considered to be more superficial. Specifically, STX affinity was reduc ed much more by neutralizations of Glu(758) and Asp(1532). On the other han d, mutation of Tyr(401) to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer ve stibule residues tested interacted with both toxins early in the binding re action (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We p ropose a revised model of TTX and STX binding in the Na+ channel outer vest ibule in which the toxins have similar interactions at the selectivity filt er, TTX has a stronger interaction with Tyr(401), and STX interacts more st rongly with the more extracellular residues.