Favre I, Moczydlowski E, Schild L
Institut de Pharmacologie et Toxicologie de l'Université, Lausanne, Switzerland.
J Gen Physiol. 1995 Aug;106(2):203-29. doi: 10.1085/jgp.106.2.203.
bTyrosine 401 of the skeletal muscle isoform (mu 1) of the rat muscle Na channel is an important determinant of high affinity block by tetrodotoxin (TTX) and saxitoxin (STX) in Na-channel isoforms. In mammalian heart Na channels, this residue is substituted by cysteine, which results in low affinity for TTX/STX and enhanced sensitivity to block by Zn2+ and Cd2+. In this study, we investigated the molecular basis for high affinity block of Na channels by STX and divalent cations by measuring inhibition of macroscopic Na+ current for a series of point mutations at residue Tyr401 of the rat mu 1 Na channel expressed in Xenopus oocytes. Substitution of Tyr401 by Gly, Ala, Ser, Cys, Asp, His, Trp, and Phe produced functional Na+ currents without major perturbation of gating or ionic selectivity. High affinity block by STX and neosaxitoxin (NEO) with Ki values in the range of 2.6-18 nM required Tyr, Phe, or Trp, suggestive of an interaction between an aromatic ring and a guanidinium group of the toxin. The Cys mutation resulted in a 7- and 23-fold enhancement of the dissociation rate of STX and NEO, respectively, corresponding to rapid toxin dissociation rates of cardiac Na channels. High affinity block by Zn2+ (Ki = 8-23 microM) required Cys, His, or Asp, three residues commonly found to coordinate directly with Zn2+ in metalloproteins. For the Cys mutant of mu 1 and also for the cardiac isoform Na channel (rh1) expressed in the L6 rat muscle cell line, inhibition of macroscopic Na+ conductance by Zn2+ reached a plateau at 85-90% inhibition, suggesting the presence of a substate current. The Asp mutant also displayed enhanced affinity for inhibition of conductance by Ca2+ (Ki = 0.3 mM vs approximately 40 mM in wild type), but block by Ca2+ was incomplete, saturating at approximately 69% inhibition. In contrast, Cd2+ completely blocked macroscopic current in the Cys mutant and the L6 cell line. These results imply that the magnitude of substate current depends on the particular residue at position 401 and the species of divalent cation. The His mutant also exhibited enhanced sensitivity to block by H+ with a pKa of approximately 7.5 for the His imidazole group. Our findings provide further evidence that residue 401 of mu 1 is located within the outer vestibule of the Na channel but external to the single-filing region for permeant ions.
大鼠肌肉钠通道骨骼肌亚型（μ1）的酪氨酸401是钠通道亚型中河豚毒素（TTX）和石房蛤毒素（STX）高亲和力阻断的重要决定因素。在哺乳动物心脏钠通道中，该残基被半胱氨酸取代，这导致对TTX/STX的亲和力较低，并增强了对Zn2+和Cd2+阻断的敏感性。在本研究中，我们通过测量非洲爪蟾卵母细胞中表达的大鼠μ1钠通道残基Tyr401处一系列点突变对宏观Na+电流的抑制作用，研究了STX和二价阳离子对钠通道高亲和力阻断的分子基础。用甘氨酸、丙氨酸、丝氨酸、半胱氨酸、天冬氨酸、组氨酸、色氨酸和苯丙氨酸取代Tyr401产生了功能性Na+电流，而门控或离子选择性没有受到重大干扰。STX和新石房蛤毒素（NEO）的高亲和力阻断，其Ki值在2.6 - 18 nM范围内，需要酪氨酸、苯丙氨酸或色氨酸，这表明芳香环与毒素的胍基之间存在相互作用。半胱氨酸突变分别导致STX和NEO的解离速率提高了7倍和23倍，这与心脏钠通道快速的毒素解离速率相对应。Zn2+（Ki = 8 - 23 μM）的高亲和力阻断需要半胱氨酸、组氨酸或天冬氨酸，这三个残基在金属蛋白中通常被发现直接与Zn2+配位。对于μ1的半胱氨酸突变体以及在L6大鼠肌肉细胞系中表达的心脏亚型钠通道（rh1），Zn2+对宏观Na+电导的抑制在85 - 90%抑制时达到平台期，这表明存在亚状态电流。天冬氨酸突变体对Ca2+抑制电导也表现出增强的亲和力（Ki = 0.3 mM，而野生型约为40 mM），但Ca2+的阻断不完全，在约69%抑制时达到饱和。相反，Cd2+完全阻断了半胱氨酸突变体和L6细胞系中的宏观电流。这些结果表明亚状态电流的大小取决于401位的特定残基和二价阳离子的种类。组氨酸突变体对H+阻断也表现出增强的敏感性，组氨酸咪唑基团的pKa约为7.5。我们的研究结果进一步证明，μ1的401位残基位于钠通道的外前庭，但在通透离子的单列区域之外。
