Naren A P, Quick M W, Collawn J F, Nelson D J, Kirk K L
Department of Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL 35294-0005, USA.
Proc Natl Acad Sci U S A. 1998 Sep 1;95(18):10972-7. doi: 10.1073/pnas.95.18.10972.
Previously we showed that the functional activity of the epithelial chloride channel that is encoded by the cystic fibrosis gene (CFTR) is reciprocally modulated by two components of the vesicle fusion machinery, syntaxin 1A and Munc-18. Here we report that syntaxin 1A inhibits CFTR chloride channels by means of direct and domain-specific protein-protein interactions. Syntaxin 1A stoichiometrically binds to the N-terminal cytoplasmic tail of CFTR, and this binding is blocked by Munc-18. The modulation of CFTR currents by syntaxin 1A is eliminated either by deletion of this tail or by injecting this tail as a blocking peptide into coexpressing Xenopus oocytes. The CFTR binding site on syntaxin 1A maps to the third predicted helical domain (H3) of this membrane protein. Moreover, CFTR Cl- currents are effectively inhibited by a minimal syntaxin 1A construct (i.e., the membrane-anchored H3 domain) that cannot fully substitute for wild-type syntaxin 1A in membrane fusion reactions. We also show that syntaxin 1A binds to and inhibits the activities of disease-associated mutants of CFTR, and that the chloride current activity of recombinant DeltaF508 CFTR (i.e., the most common cystic fibrosis mutant) can be potentiated by disrupting its interaction with syntaxin 1A in cultured epithelial cells. Our results provide evidence for a direct physical interaction between CFTR and syntaxin 1A that limits the functional activities of normal and disease-associated forms of this chloride channel.
先前我们发现，由囊性纤维化基因（CFTR）编码的上皮氯离子通道的功能活性受到囊泡融合机制的两个组分，即 syntaxin 1A 和 Munc - 18 的相互调节。在此我们报告，syntaxin 1A 通过直接且具有结构域特异性的蛋白质 - 蛋白质相互作用抑制 CFTR 氯离子通道。Syntaxin 1A 以化学计量方式结合到 CFTR 的 N 端胞质尾巴，并且这种结合被 Munc - 18 阻断。通过缺失该尾巴或将该尾巴作为阻断肽注射到共表达的非洲爪蟾卵母细胞中，均可消除 syntaxin 1A 对 CFTR 电流的调节作用。Syntaxin 1A 上的 CFTR 结合位点定位于该膜蛋白的第三个预测螺旋结构域（H3）。此外，CFTR Cl - 电流可被一种最小的 syntaxin 1A 构建体（即膜锚定的 H3 结构域）有效抑制，该构建体在膜融合反应中不能完全替代野生型 syntaxin 1A。我们还表明，syntaxin 1A 结合并抑制 CFTR 的疾病相关突变体的活性，并且在培养的上皮细胞中，通过破坏重组 DeltaF508 CFTR（即最常见的囊性纤维化突变体）与 syntaxin 1A 的相互作用，可增强其氯离子电流活性。我们的结果为 CFTR 与 syntaxin 1A 之间的直接物理相互作用提供了证据，这种相互作用限制了该氯离子通道正常形式和疾病相关形式的功能活性。
