Erkkila Brian E, Sedelnikova Anna V, Weiss David S
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
Biophys J. 2008 Jun;94(11):4299-306. doi: 10.1529/biophysj.107.118455. Epub 2008 Feb 29.
Picrotoxin (PTX) is a noncompetitive antagonist of many ligand-gated ion channels, with a site of action believed to be within the ion-conducting pore. In the A-type gamma-aminobutyric acid receptor, a threonine residue in the second transmembrane domain is of particular importance for the binding of, and ultimate inhibition by, PTX. To better understand the relationship between this residue and the PTX molecule, we mutated this threonine residue to serine, valine, and tyrosine to change the structural and biochemical characteristics at this location. The known subunit stoichiometry of the A-type gamma-aminobutyric acid receptor allowed us to create receptors with anywhere from zero to five mutations. With an increasing number of mutated subunits, each amino acid substitution revealed a unique pattern of changes in PTX sensitivity, ultimately encompassing sensitivity shifts over several orders of magnitude. The electrophysiological data on PTX-mediated block, and supporting modeling and docking studies, provide evidence that an interaction between the PTX molecule and three adjacent uncharged polar amino acids at this position of the pore are crucial for PTX-mediated inhibition.
印防己毒素（PTX）是许多配体门控离子通道的非竞争性拮抗剂，其作用位点被认为在离子传导孔内。在A型γ-氨基丁酸受体中，第二个跨膜结构域中的一个苏氨酸残基对于PTX的结合及最终抑制作用尤为重要。为了更好地理解该残基与PTX分子之间的关系，我们将这个苏氨酸残基突变为丝氨酸、缬氨酸和酪氨酸，以改变该位置的结构和生化特性。A型γ-氨基丁酸受体已知的亚基化学计量关系使我们能够创建含有从零到五个突变的受体。随着突变亚基数量的增加，每个氨基酸取代都揭示了PTX敏感性变化的独特模式，最终涵盖了几个数量级的敏感性变化。关于PTX介导的阻断的电生理数据以及支持性的建模和对接研究提供了证据，表明PTX分子与孔这个位置的三个相邻不带电极性氨基酸之间的相互作用对于PTX介导的抑制作用至关重要。
