Green W N, Weiss L B, Andersen O S
J Gen Physiol. 1987 Jun;89(6):873-903. doi: 10.1085/jgp.89.6.873.
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.
研究了胍盐毒素对通过电压依赖性钠通道的电流的抑制作用，该通道是整合到不带净电荷的平面脂质双分子层中的经蟾毒素修饰的通道。为了确定毒素结合位点附近是否存在净负电荷，我们研究了在广泛的[Na⁺]范围内由河豚毒素（TTX）和石房蛤毒素（STX）诱导的通道关闭情况。这些毒素分别带有+1和+2的电荷。毒素诱导的通道关闭的频率和持续时间取决于电压。STX和TTX的电压依赖性相似，与[Na⁺]无关，这表明结合位点位于钠通道细胞外表面的浅表位置。毒素解离常数KD和毒素诱导的通道关闭的速率常数kc随[Na⁺]而变化。STX的Na⁺依赖性比TTX大。同样，添加四乙铵（TEA⁺）或Zn²⁺对STX的KD增加和kc降低的影响比对TTX的影响更大。这些差异效应被解释为是由毒素结合位点附近的静电势变化引起的。产生这种电势的电荷一定位于通道上，因为双分子层没有净电荷。KDSTX/KDTTX和kcSTX/kcTTX比值的Na⁺依赖性被用于估计毒素结合位点附近的表观电荷密度约为-0.33 e×nm⁻²。Zn²⁺引起单通道电流的电压依赖性阻断，就好像Zn²⁺结合在渗透路径内的一个位点上，从而阻断Na⁺的移动。在其阻断位点的Zn²⁺与在其结合位点的STX或TTX之间没有可测量的相互作用，这表明毒素结合位点与通道入口是分开的。毒素结合位点与Zn²⁺阻断位点之间的距离估计至少为1.5 nm。基于毒素结合后通道的构象变化，提出了一个毒素诱导的通道关闭模型。
