Eaholtz G, Colvin A, Leonard D, Taylor C, Catterall W A
Graduate Program in Neurobiology, University of Washington, Seattle, Washington 98195-7280, USA.
J Gen Physiol. 1999 Feb;113(2):279-94. doi: 10.1085/jgp.113.2.279.
Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH2, measured on sodium channels whose inactivation was slowed by the alpha-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH2 with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH2 did not cause appreciable fast block, but the divalent cation IFM-NH(CH2)2NH2 was as effective as the pentapeptide ac-KIFMK-NH2. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH2 showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.
钠通道的失活被认为是由连接结构域III和IV的细胞内环形成的失活门介导的。失活过程需要一个包含异亮氨酸、苯丙氨酸和甲硫氨酸（IFM）氨基酸序列的疏水基序。含有IFM基序的肽，当应用于这些通道的细胞质侧时，会产生两种类型的阻断：快速阻断，类似于失活过程；以及由强去极化脉冲刺激产生的缓慢、使用依赖性阻断。在被来自金蝎（Leiurus quinquestriatus, LqTx）的α-蝎毒素减慢失活的钠通道上测量，肽ac-KIFMK-NH2的快速阻断在复极化时以0.9毫秒的时间常数逆转。相比之下，对照和LqTx修饰的钠通道从使用依赖性阻断中恢复较慢。对于快速阻断，含有IFM基序和两个正电荷的三到六个氨基酸残基的线性肽比带有一个正电荷的肽更有效，而不带电荷的IFM肽则无效。用较小、疏水性较低的残基取代肽ac-KIFMK-NH2中的IFM残基可防止快速阻断。带正电荷的三肽IFM-NH2不会引起明显的快速阻断，但二价阳离子IFM-NH(CH2)2NH2与五肽ac-KIFMK-NH2一样有效。含有两个正电荷的受限肽环KIFMK不会引起快速阻断。这些结果表明，正电荷的位置并不重要，但含IFM肽的灵活性或构象对于实现快速阻断很重要。观察到含有三到六个残基、带有一个或两个正电荷的含IFM肽会产生缓慢、使用依赖性阻断，但二肽或苯丙氨酸酰胺则不会。与缺乏快速阻断相反，环KIFMK是一种有效的使用依赖性阻断剂。三肽IFM-NH2中氨基酸残基的取代表明，在所有三个位置上，大的疏水残基对于缓慢、使用依赖性阻断是优选的。然而，用大的疏水残基二苯丙氨酸或受限残基苯甘氨酸或四氢异喹啉取代苯丙氨酸会降低效力，这表明该苯丙氨酸残基在IFM肽结合期间必须能够进入一个受限的疏水口袋。总之，关于快速阻断和缓慢、使用依赖性阻断的结果表明，IFM肽与钠通道上的受体位点形成两种具有不同稳定性和结构特异性的不同复合物。有人提出，快速阻断代表这些肽与失活门受体的结合，而缓慢、使用依赖性阻断代表IFM肽在孔中的更深层次结合。
