RESUMEN
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), Cx26T135A in M3 produces a closed channel; 2), Cx26M34A in M1 severely decreases channel activity; 3), Cx26P87L in M2 has been implicated in defective channel gating; and 4), Cx26V84L in M2, a nonsyndromic deafness mutant, retains normal dye coupling and electrophysiological properties but is deficient in IP(3) transfer. These mutations do not affect Cx26 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, Cx26M34A and Cx26V84L are stable as hexamers or dodecamers, but Cx26T135A and Cx26P87L oligomers are not. This instability is also found in Cx26T135A and Cx26P87L hemichannels isolated from mammalian cells. In this work, coexpression of both wild-type Cx26 and Cx26P87L in Sf9 cells rescued P87L hexamer stability. Similarly, in paired Xenopus oocytes, coexpression with wild-type restored function. In contrast, the stability of Cx26T135A 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.
Asunto(s)
Conexinas/química , Conexinas/metabolismo , Uniones Comunicantes/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación , Multimerización de Proteína , Secuencia de Aminoácidos , Animales , Conexina 26 , Conexinas/genética , Conexinas/aislamiento & purificación , Detergentes/química , Células HeLa , Humanos , Hidróxidos/metabolismo , Datos de Secuencia Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/aislamiento & purificación , Octoxinol/química , Oocitos/metabolismo , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Ratas , Solubilidad , Treonina/metabolismo , XenopusRESUMEN
Immuno-electron microscopy (immuno-EM) is a technique that has been used widely to determine subcellular localization of proteins. Different approaches are available for immuno-EM: pre-embedding method, post-embedding, and cryosectioning (Tokuyasu "style"). Here we describe a pre-embedding technique that allows the labeling of a target protein in situ, retention of fluorescence signal in plastic, and its localization at the EM level in a given cellular context. The procedure can be technically challenging and labor intensive: it requires optimization of fixation protocols to better preserve the cellular morphology and screening of compatible antibodies. Nevertheless, immuno-EM can be a powerful localization tool.