The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8) have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating) or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death) and formation of cytoplasmic accumulations (that may act as light scattering particles). These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.

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.

Purpose.: To characterize the properties of connexin 46 hemichannels in differentiating fiber cells isolated from mouse lenses. Methods.: Differentiating fiber cells were isolated from mouse lenses using collagenase. Cellular localization of connexin 50 (Cx50) and connexin 46 (Cx46) was assessed by immunofluorescence. Membrane currents were recorded using whole cell patch clamping. Dye uptake was measured using time-lapse imaging. Results.: In freshly dissociated fiber cells isolated from knockout Cx50 (KOCx50) mouse lenses, removal of external divalent cations induced a macroscopic current composed of large conductance channels. This current was reduced at a holding potential of −60 mV, activated on depolarization, and had a reversal potential near 0 mV. These properties were very similar to those of Cx46 hemichannel currents recorded in single Xenopus oocytes. If the currents observed in divalent cation-free Ringer's solution were due to Cx46 hemichannel opening, then dye influx by gap junctional/hemichannel permeable dyes should be measurable in the fiber cells. To measure dye influx, the authors used the positively charged dyes, propidium iodide (PrI) and 4′-6-diamidino-2-phenylindole (DAPI). In the absence of external calcium, fiber cells took up both dyes. Furthermore, dye influx could be inhibited by hemichannel blockers. To confirm that this current was due to Cx46 hemichannels, the authors studied fiber cells isolated from the lenses of double knockout (Cx46^−/−; Cx50^−/−) mice and demonstrated that both the calcium-sensitive conductance and dye influx were absent. Conclusions.: These results show that Cx46 can form functional hemichannels in the nonjunctional membrane of fiber cells.