Purified Functional Human Connexin 26 Hemichannels Expressed in E. Coli

Abstract

Gap junctions channels communicate the cytoplasms of adjacent cells, and are formed by head-to-head association of two hemichannels, one from each of the adjacent cells. Gap-junction channels and hemichannels mediate electrical and chemical communication between cells due to their permeability to ions and hydrophilic molecules with molecular weights of up to 1,000 Da. Mutations of Cx26 are the most frequent cause of genetic deafness, and it is therefore important to understand the structure-function relationship of wild-type and deafness-associated mutants. Currently the preferred system for connexin overexpression is the insect cell/baculovirus system. This system has many drawbacks, including complexity and cost, which severely limit high-throughput screening of mutants. Here, we present an E. coli-based expression system for Cx26 that yields functional protein. The functional and biochemical properties of Cx26 hemichannels purified from insect cells and E. coli were indistinguishable. We also show that a poly-His tag fused to the C-terminal end affects hemichannel gating. We conclude that the E. coli-based expression system is suitable to produce milligram amounts of purified and functional Cx26 hemichannels. Supported by National Institutes of Health grants R01GM79629 and 3R01GM079629-03S1, Fondecyt #1120214, and Anillo ATC1104.