Purification, Reconstitution, and Functional Analysis of Connexin Hemichannels

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26.