Functional reconstitution of connexons in artificial membranes: expression, purification and characterization of connexin 43

Connexin 43 (Cx43) is the most prominent member of the connexin family in mammalian tissues. In biological lipid membranes, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, forming gap junction channels. Membrane channel proteins are pivotal for communication between cells. Dysfunctional ion channels are associated to diseases like osteoporosis, epilepsy, ataxia, myotonia and cardiac arrhythmia. Therefore, there is an interest in studying these channel proteins to elucidate their functional properties and determine how to manipulate their functionality. This is commonly carried out by ion channel recordings, which require invasive contacting by voltage clamp methods in cells. The alternative approach taken here was to utilize connexon-doped artificial membranes for electrical coupling of connexon-expressing cells via gap junction channels. In this work, an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (Cx4