In vitro assembly of gap junctions

Paul D. Lampe, Joerg Kistler, Andreas Hefti, Jacqui Bond, Shirley Müller, Ross G Johnson, Andreas Engel

Research output: Contribution to journalArticlepeer-review

27 Scopus citations

Abstract

Gap junction structures were assembled in vitro from octyl-β-d-glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = b = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg2+ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.

Original languageEnglish (US)
Pages (from-to)281-290
Number of pages10
JournalJournal of Structural Biology
Volume107
Issue number3
DOIs
StatePublished - Dec 1991

Bibliographical note

Funding Information:
We are grateful to Dr. K. Stauffer for providing a preprint of her work on recombinant connexin 32. We also thank Ms. H. Frefel and Ms. M. Zoller for their expert photographic work. This project was supported by grants from the National Institutes of Health, GM37230 and CA28548 (to P.D.L. and R.G.J.), from the Medical Research Council of New Zealand (to J.K.), from the Swiss National Foundation for Scientific Research, 31-25684.88 (to A.E.), the M. E. Miiller-Foundation of Switzerland, and the Department of Education of the Kanton Basel-Stadt.

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