Disease processes that result in cell injury and proliferation, such as hypertension, atherosclerosis and ischemia (30C33) alter expression levels. with unique gating and conductance properties. Gap junction channels connect the cytoplasms of adjacent cells and provide a pathway for intercellular diffusion of ions, second messenger molecules, and small metabolites. Functional gap junction channels are formed by connexins (Cxs), a gene family with at least 14 mammalian members that are distinguished from one another by their predicted molecular weights expressed in kilodaltons (e.g., Cx43, the 43-kDa connexin). Cxs oligomerize to form connexons (hemichannels), which are defined as homomeric when the six comprising Cxs are identical or heteromeric when two or more Cxs comprise the connexon. Connexons in adjacent cells join in the extracellular space to form the functional intercellular L-Lysine thioctate channel, which is usually defined as homotypic when the Cx composition of the contributing connexons is usually identical or heterotypic when different. The ability of Cxs to form homomeric/heterotypic channels has been examined in TF the oocyte and HeLa cell expression systems as well as in other settings (for review, see ref. 1). Homomeric Cx43 connexons successfully dock with homomeric connexons comprised of Cxs 30.3, 37, and 45 but not with Cxs 50, 40, 33, 32, 31.3, 31, or 26. Homomeric Cx40 connexons L-Lysine thioctate successfully dock with and form functional homomeric/heterotypic channels with Cxs 37 and 45 but not with Cxs 50, 46, 43, 32, 31.1, 31, or 26. Clearly, there are far more incompatible than compatible combinations. The capacity of L-Lysine thioctate Cxs to form functional heteromeric connexons and channels has only recently received attention. Biochemical and structural tools have exhibited the presence of heteromeric connexons comprised of Cxs 46 and 50 (2) and Cxs 32 and 26 (3). That these Cx pairs form functional heteromeric connexons recently has been exhibited (4, 5), but formation of heteromeric channels by these Cx pairs has not been demonstrated. Based solely on functional data, it appears that Cxs 37 and 43 form heteromeric channels (6). The functional studies of heteromeric connexons and channels demonstrate that these structures display unique L-Lysine thioctate conductances, gating behaviors, or selectivities relative to the homomeric forms. There are no studies in which biochemical (or structural) and functional data are provided in support of formation of functional heteromeric channels by any pair of Cxs, certainly not Cx-pairs incapable of forming homomeric/heterotypic channels. Coexpression of Cxs that fail to form homomeric/heterotypic channels occurs in many tissues and cell types including (tissue or cell type, expressed Cxs): keratinocytes, 43, 37, 31, 31.1, 26; testis, 43, 33; endothelial cells, 43, 40, 37; corneal epithelium, 43, 50; atrioventricular valves, 43, 50; atrial myocytes, 40, 43, 45; vascular easy muscle, 40, 43; and thyroid follicular cells, 43, 32 (1, 7). In view of the changes in permeation and gating apparently induced by heteromerization, the implications of functional heteromers between Cxs incapable of forming functional homomeric/heterotypic channels could be L-Lysine thioctate profound. Thus, the goals of the present study are to determine whether Cxs 40 and 43 form heteromeric channels and, if so, to characterize the functional properties of those channels. We demonstrate that these Cxs form heteromeric channels with functional properties not well predicted by homomeric/homotypic Cx40 or Cx43 channels. Our results represent a demonstration of heteromeric channel formation and function in a mammalian cell line that naturally coexpresses the Cxs. MATERIALS AND METHODS Reagents. A7r5 cells were obtained from American Type Culture Collection. Tissue culture reagents were from GIBCO. Fetal bovine serum was obtained from HyClone or Intergen (Purchase, NY). [35S]methionine (cell-labeling grade) was from New England Nuclear. Anti-Cx40 polyclonal antibody was from Chemicon. All other chemicals were obtained from either Sigma or Fisher Scientific. Preparation and Immunoaffinity Purification of Anti-Cx43 Antibody. Bacterial fusion proteins made up of glutathione for 10 min. The pellet.