Single Hemichannels Recorded in Lipid Bilayers and Artificial Gap Junction Formation with Cells

Abstract

Connexins (Cx) are members of a multigene family of membrane-spanning proteins that form gap junctions, which are composed of two hexameric hemichannels, called connexons. These gap junctions, organized in so-called gap junctional plaques, span the extracellular space/matrix of adjacent cells and thus allow a passive exchange of small molecules up to about 1 kDa. Connexins are widely distributed with various subtypes of connexin and are involved in different biological processes such transmission of information and propagation of action potential for e.g. Recent studies indicates that hemichannels do open under physiological and pathological conditions. In our study, we investigated the biophysical properties of hemichannels Cx26 and Cx43 which were isolated biochemically and reconstituted into synthetic lipid membranes. Both hemichannels are present in different tissues and involved in different pathologies. The results on a study of the Cx26 are presented. Reconstitutions of functional Cx26 and mutant hemichannels were performed. Secondly, Cx43 was purified and reconstituted into bilayers. The hemichannel Cx43 properties were compared to previous studies and showed similarities of conductance on single channel recordings of Cx43 in cells. Our focus was then to form artificial gap junctions, first between two unrelated cells and then between cells and bilayers containing functional hemichannels. This was done using Cx26 or Cx43. The bilayer-cell configuration allows to measure electrophysiological properties of the cells indirectly via gap junctions. Single channel recordings of gap junctions were recorded using a bilayer containing Cx43 and Cardiomyocytes expressing Cx43. Macroscopic currents were as well recorded between bilayers and cell lines expressing Cx26 or Cx43.