Application of SCAM (Substituted Cysteine Accessibility Method) to gap junction intercellular channels

I. M. Skerrett, E. Kasperek, F. L. Cao, J. H. Shin, J. Aronowitz, S. Ahmed, B. J. Nicholson

Research output: Contribution to journalArticlepeer-review

Abstract

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 sub-stitution 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.

Original languageEnglish (US)
Pages (from-to)179-185
Number of pages7
JournalCell Adhesion and Communication
Volume8
Issue number4-6
StatePublished - Dec 1 2000
Externally publishedYes

Keywords

  • Accessibility
  • Gap junction
  • Pore-lining
  • SCAM

ASJC Scopus subject areas

  • Cell Biology

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