Connexin26 is a ubiquitous gap junction protein that serves critical homeostatic functions. Four single-site mutations found in the transmembrane helices (M1-M4) cause different types of dysfunctional channels: 1), C×26T135A in M3 produces a closed channel; 2), C×26M34A in M1 severely decreases channel activity; 3), C×26P87L in M2 has been implicated in defective channel gating; and 4), C×26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP3 transfer. These mutations do not affect C×26 trafficking in mammalian cells, and make normal-appearing channels in baculovirus-infected Sf9 membranes when imaged by negative stain electron microscopy. Upon dodecylmaltoside solubilization of the membrane fraction, C×26M34A and C×26V84L are stable as hexamers or dodecamers, but C×26T135A and C×26P87L oligomers are not. This instability is also found in C×26T135A and C×26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type C×26 and C×26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of C×26T135A hemichannels could not be rescued by coexpression with WT. Thus, T135 and P87 residues are in positions that are important for oligomer stability and can affect gap junction gating.
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