THE SAXITOXIN TETRODOTOXIN BINDING-SITE ON CLONED RAT-BRAIN IIA NA CHANNELS IS IN THE TRANSMEMBRANE ELECTRIC-FIELD

The rat brain IIa (BrIIa) Na channel alpha-subunit and the brain beta 1 subunit were coexpressed in Xenopus oocytes, and peak whole-oocyte N a current (I-Na) was measured at a test potential of -10 mV. Hyperpola rization of the holding potential resulted in an increased affinity of STX and TTX rested-state block of BrIIa Na channels. The apparent hal f-block concentration (ED(50)) for STX of BrIIa current decreased with hyperpolarizing holding potentials (V-hold). At V-hold of -100 mV, th e ED(50) was 2.1 +/- 0.4 nM, and the affinity increased to a ED(50) of 1.2 +/- 0.2 nM with V-hold of -140 mV. In the absence of toxin, the p eak current amplitude was the same for all potentials negative to -90 mV, demonstrating that all of the channels were in a closed conformati on and maximally available to open in this range of holding potentials . The Woodhull model (1973) was used to describe the increase of the S TX ED(50) as a function of holding potential. The equivalent electrica l distance of block (delta) by STX was 0.18 from the extracellular mil ieu when the valence of STX was fixed to +2. Analysis of the holding p otential dependence of TTX block yielded a similar delta when the vale nce of TTX was fixed to +1. We conclude that the guanidinium toxin sit e is located partially within the transmembrane electric field. Previo us site-directed mutagenesis studies demonstrated that an isoform-spec ific phenylalanine in the BrIIa channel is critical for high affinity toxin block. Therefore, we propose that amino acids at positions corre sponding to this Phe in the BrIIa channel, which lie in the outer vest ibule of the channel adjacent to the pore entrance, are partially in t he transmembrane potential drop.