Channel-forming activity of immunoaffinity-purified connexin32 in single phospholipid membranes.
Connexin32, a member of the family of proteins that forms gap junction channels between cells, was immunoaffinity-purified from rat liver using a monoclonal antibody, under nondenaturing conditions and reconstituted into unilamellar phospholipid liposomes and bilayers. Gel-filtration studies indicate that the connexin32 is purified predominantly in structures of a size consistent with that of single hemichannels and too small to be junctional channels (dimers of hemichannels). Purified connexin formed channels permeable to sucrose and to Lucifer Yellow. The permeability was reversibly reduced by acidic pH and unaffected by several agents that modulate coupling between cells. Modeling of the distribution of the permeability in the liposomes indicates that it is mediated by connexin structures that distribute among the liposomes as single hemichannels. Bilayer recordings of the purified connexin show high conductance channels with asymmetric voltage sensitivity. The results show that immunopurified connexin32 can form channels, in single phospholipid membranes, that have permeability similar to that of gap junction channels and thus can be utilized in studies of permeability and its regulation to investigate its role in normal physiological function, development, and disease.
Rhee
S K
SK
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Bevans
C G
CG
Harris
A L
AL
D000818:Animals; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D017630:Connexins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D005960:Glucosides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007546:Isoquinolines / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008099:Liver; D010539:Permeability; D010743:Phospholipids / Q000378:metabolism; D051381:Rats; D012235:Rhodamines / Q000378:metabolism; D013395:Sucrose / Q000378:metabolism; D000097002:Gap Junction beta-1 Protein
D017630:Connexins; D005456:Fluorescent Dyes; D005960:Glucosides; D007546:Isoquinolines; D008081:Liposomes; D010743:Phospholipids; D012235:Rhodamines; D013395:Sucrose; D000097002:Gap Junction beta-1 Protein; C018619:octyl-beta-D-glucoside; C017475:lucifer yellow