Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520, USA.
J Membr Biol. 2012 Aug;245(8):453-63. doi: 10.1007/s00232-012-9457-z. Epub 2012 Jul 24.
Connexins form channels with large aqueous pores that mediate fluxes of inorganic ions and biological signaling molecules. Studies aimed at identifying the connexin pore now include a crystal structure that provides details of putative pore-lining residues that need to be verified using independent biophysical approaches. Here we extended our initial cysteine-scanning studies of the TM1/E1 region of Cx46 hemichannels to include TM2 and TM3 transmembrane segments. No evidence of reactivity was observed in either TM2 or TM3 probed with small or large thiol-modifying reagents. Several identified pore residues in E1 of Cx46 have been verified in different Cx isoforms. Use of variety of thiol reagents indicates that the connexin hemichannel pore is large and flexible enough, at least in the extracellular part of the pore funnel, to accommodate uncommonly large side chains. We also find that that gating characteristics are largely determined by the same domains that constitute the pore. These data indicate that biophysical and structural studies are converging towards a view that the N-terminal half of the Cx protein contains the principal components of the pore and gating elements, with NT, TM1 and E1 forming the pore funnel.
连接蛋白形成具有大亲水性孔道的通道，介导无机离子和生物信号分子的流动。目前旨在鉴定连接子孔的研究包括一个晶体结构，该结构提供了推测的孔衬里残基的详细信息，这些残基需要使用独立的生物物理方法进行验证。在这里，我们将最初对 Cx46 半通道 TM1/E1 区域的半胱氨酸扫描研究扩展到 TM2 和 TM3 跨膜片段。用小或大的巯基修饰试剂探测 TM2 或 TM3 时，均未观察到反应性。在不同的 Cx 异构体中已经验证了 Cx46 的 E1 中几个已鉴定的孔残基。使用各种巯基试剂表明，连接蛋白半通道孔足够大且灵活，至少在孔漏斗的细胞外部分，足以容纳不常见的大侧链。我们还发现门控特性主要由构成孔的相同结构域决定。这些数据表明，生物物理和结构研究正在趋于一致，认为 Cx 蛋白的 N 端包含孔和门控元件的主要成分，NT、TM1 和 E1 形成孔漏斗。
