Batrachotoxin-modified sodium channels in planar lipid bilayers. Characterization of saxitoxin- and tetrodotoxin-induced channel closures.
The guanidinium toxin-induced inhibition of the current through voltage-dependent sodium channels was examined for batrachotoxin-modified channels incorporated into planar lipid bilayers that carry no net charge. To ascertain whether a net negative charge exists in the vicinity of the toxin-binding site, we studied the channel closures induced by tetrodotoxin (TTX) and saxitoxin (STX) over a wide range of [Na+]. These toxins carry charges of +1 and +2, respectively. The frequency and duration of the toxin-induced closures are voltage dependent. The voltage dependence was similar for STX and TTX, independent of [Na+], which indicates that the binding site is located superficially at the extracellular surface of the sodium channel. The toxin dissociation constant, KD, and the rate constant for the toxin-induced closures, kc, varied as a function of [Na+]. The Na+ dependence was larger for STX than for TTX. Similarly, the addition of tetraethylammonium (TEA+) or Zn++ increased KD and decreased kc more for STX than for TTX. These differential effects are interpreted to arise from changes in the electrostatic potential near the toxin-binding site. The charges giving rise to this potential must reside on the channel since the bilayers had no net charge. The Na+ dependence of the ratios KDSTX/KDTTX and kcSTX/kcTTX was used to estimate an apparent charge density near the toxin-binding site of about -0.33 e X nm-2. Zn++ causes a voltage-dependent block of the single-channel current, as if Zn++ bound at a site within the permeation path, thereby blocking Na+ movement. There was no measurable interaction between Zn++ at its blocking site and STX or TTX at their binding site, which suggests that the toxin-binding site is separate from the channel entrance. The separation between the toxin-binding site and the Zn++ blocking site was estimated to be at least 1.5 nm. A model for toxin-induced channel closures is proposed, based on conformational changes in the channel subsequent to toxin binding.
D000818:Animals; D001496:Batrachotoxins / Q000494:pharmacology*; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D004285:Dogs; D004553:Electric Conductivity; D007473:Ion Channels / Q000187:drug effects* / Q000378:metabolism; D007700:Kinetics; D008051:Lipid Bilayers; D008433:Mathematics; D008564:Membrane Potentials; D012530:Saxitoxin / Q000378:metabolism / Q000494:pharmacology*; D012964:Sodium / Q000378:metabolism*; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism; D013779:Tetrodotoxin / Q000378:metabolism / Q000494:pharmacology*; D015032:Zinc / Q000378:metabolism
D001496:Batrachotoxins; D007473:Ion Channels; D008051:Lipid Bilayers; D012530:Saxitoxin; D013779:Tetrodotoxin; D012964:Sodium; D015032:Zinc