Search

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

gap junctions contain channels that allow the passage of ions and small molecules between adjacent cells. This intercellular communication has been implicated in the coordination of cellular responses to intracellular signaling molecules. Calcium and inositol phosphates are among the second

Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [35S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like “loss of function” rather than “dominant negative” mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50.

Mutations in gap junctional channels have been linked to certain forms of inherited congenital cataract (D. Mackay, A. Ionides, V. Berry, A. Moore, S. Bhattacharya, and A. Shiels. Am. J. Hum. Genet. 60: 1474-1478, 1997; A. Shiels, D. Mackay, A. Ionides, V. Berry, A. Moore, and S. Bhattacharya. Am. J. Hum. Genet. 62: 526-532, 1998). We used the Xenopus oocyte pair system to investigate the functional properties of a missense mutation in the human connexin 50 gene (P88S) associated with zonular pulverulent cataract. The associated phenotype for the mutation is transmitted in an autosomal dominant fashion. Xenopus oocytes injected with wild-type connexin 50 cRNA developed gap junctional conductances of approximately 5 microS 4-7 h after pairing. In contrast, the P88S mutant connexin failed to form functional gap junctional channels when paired homotypically. Moreover, the P88S mutant functioned in a dominant negative manner as an inhibitor of human connexin 50 gap junctional channels when coinjected with wild-type connexin 50 cRNA. Cells injected with 1:5 and 1:11 ratios of P88S mutant to wild-type cRNA exhibited gap junctional coupling of approximately 8% and 39% of wild-type coupling, respectively. Based on these findings, we conclude that only one P88S mutant subunit is necessary per gap junctional channel to abolish channel function.

Levels (by immunoblotting) and cell distribution (by immunofluorescence) of connexins (Cxs) and glucagon receptors (GRs) were studied during the ontogeny of the rat liver. Cx32, the main rat liver gap junction protein, was present in fetal hepatocytes, and its abundance increased between the 19th and 21st gestational days. The major increase in Cx32 levels occurred between the 7th and 21st postnatal days, after which adult levels were reached. The adult pattern of distribution of Cx26 and Cx32 was established by the 21st postnatal day. Cx43 was present in the liver capsule and perisinusoidal cells, and its levels did not show significant variations throughout development. Levels of GRs were much lower in fetal than in adult livers. After birth, GRs increased progressively, reaching adult levels at about the 28th postnatal day. In adults, GR immunoreactivity on the plasma membrane of hepatocytes was localized to the region facing the sinusoid and showed a density gradient of distribution along the hemiacinus: high in pericentral regions and decreasing toward more glucogenically active cells located in periportal regions. Because cells of the hepatic acinus communicate via gap junctions, which are permeable to second messengers, we propose that signals transduced by GRs in cells expressing high levels of receptor might spread to cells expressing low levels of receptor, thus maximizing metabolic activation.