Skerrett M, Kasperek E, Cao F L, Shin J H, Aronowitz J, Ahmed S, Nicholson B J
Department of Biological Sciences, SUNY at Buffalo, NY 14260, USA.
Cell Commun Adhes. 2001;8(4-6):179-85. doi: 10.3109/15419060109080720.
The pore-lining residues of gap junction channels determine their permeability to ions and small cellular metabolites. These residues can be identified through systematic cysteine substitution and accessibility analysis, commonly known as SCAM (Substituted Cysteine Accessibility Method). However, application of this technique to intercellular channels is more complicated than for their transmembrane counterparts. We have utilized a novel dual-oocyte perfusion device to apply cysteine reagents to the cytoplasmic face of paired, voltage-clamped Xenopus oocytes. In this configuration, a large and irreversible cysteine reagent MBB (maliemidobutyryl biocytin, mw 537) was shown to readily traverse the gap junction pore and induce conductance changes upon reaction of accessible sites. Of the 11 reactive sites identified, 6 were located in M3, where they span the bilayer. They display a periodicity characteristic of the tilted helix that lines the pore in the gap junction structure of Unger et al. (1999). Access to several of the other sites was attributed to aqueous crevices between transmembrane helices. Reactive sites were slightly different than those identified for gap junction hemichannels (Zhou et al. 1997), suggesting that conformational changes occur upon docking.
间隙连接通道的孔衬残基决定了其对离子和小细胞代谢物的通透性。这些残基可通过系统的半胱氨酸取代和可及性分析来确定，即通常所说的SCAM（取代半胱氨酸可及性方法）。然而，将该技术应用于细胞间通道比应用于跨膜通道更为复杂。我们利用一种新型的双卵母细胞灌注装置，将半胱氨酸试剂应用于成对的、电压钳制的非洲爪蟾卵母细胞的细胞质面。在这种配置下，一种大的、不可逆的半胱氨酸试剂MBB（马来酰亚胺丁酰生物素，分子量537）被证明能够轻易穿过间隙连接孔，并在可及位点发生反应时诱导电导变化。在鉴定出的11个反应位点中，有6个位于M3，它们跨越双层膜。它们呈现出倾斜螺旋的周期性特征，该螺旋排列在Unger等人（1999年）间隙连接结构的孔中。其他几个位点的可及性归因于跨膜螺旋之间的水性缝隙。反应位点与间隙连接半通道鉴定出的位点略有不同（Zhou等人，1997年），这表明对接时会发生构象变化。
