The Connexin50D47A Mutant Causes Cataracts by Calcium Precipitation

Abstract

Purpose Mutations in connexin50 (Cx50) and connexin46 (Cx46) cause cataracts. Because the expression of Cx46fs380 leads to decreased gap junctional coupling and formation of calcium precipitates, we studied Cx50D47A lenses to test whether Cx50 mutants also cause cataracts due to calcium precipitation. Methods Connexin levels were determined by immunoblotting. Gap junctional coupling conductance was calculated from intracellular impedance studies of intact lenses. Intracellular hydrostatic pressure was measured using a microelectrode/manometer system. Intracellular free calcium ion concentrations ([Ca 2+ ] i ) were measured using Fura-2 and fluorescence imaging. Calcium precipitation was assessed by Alizarin red staining and compared to the distribution of opacities in darkfield images. Results In Cx50D47A lenses, Cx50 levels were 11% (heterozygotes) and 1.2% (homozygotes), and Cx46 levels were 52% (heterozygotes) and 30% (homozygotes) when compared to wild-type at 2.5 months. Gap junctional coupling in differentiating fibers of Cx50D47A lenses was 49% (heterozygotes) and 29% (homozygotes), and in mature fibers, it was 24% (heterozygotes) and 4% (homozygotes) compared to wild-type lenses. Hydrostatic pressure was significantly increased in Cx50D47A lenses. [Ca 2+ ] i was significantly increased in Cx50D47A lenses. Alizarin red-stained calcium precipitates were present in homozygous Cx50D47A lenses with a similar distribution to the cataracts. Conclusions Cx50D47A expression altered the lens internal circulation by decreasing connexin levels and gap junctional coupling. Reduced water and ion outflow through gap junctions increased the gradients of intracellular hydrostatic pressure and concentrations of free calcium ions. In these lenses, calcium ions accumulated, precipitated, and formed cataracts. These results suggest that mutant lens fiber connexins lead to calcium precipitates, which may cause cataracts.

Connexin Mutants Compromise the Lens Circulation and Cause Cataracts through Biomineralization

“…Mice with mutations in the Cx46 or Cx50 genes develop cataracts that are inherited with a dominant or semi-dominant pattern [12][13][14][15][16][17]. The lenses of mice with targeted deletion of Cx46 or Cx50, or with dominant mutations in either of these genes, have very reduced levels of Cx46 and/or Cx50 [10,11,[16][17][18] and of intercellular communication between lens fiber cells (measured as an increase in intracellular resistivity) [18][19][20][21]. Transgenic mice with lens over-expression of a mutant ubiquitin (K6W-Ub) that is conjugation competent but proteolytically incompetent also have decreased gap junctional coupling between fiber cells in the core of the lens, likely because these mice have lower levels of Cx46, as detected by immunoblotting [22].…”

“…Other parameters related to the lens circulation have been determined in these lenses. The hydrostatic pressure is increased in lenses from heterozygous Cx46 knockout mice and lenses from mice expressing Cx46 or Cx50 dominant mutants [3,21,23]. The intracellular concentration of Na + is increased in lenses from Cx46 mutant mice [3].…”

Connexin 50-R205G Mutation Perturbs Lens Epithelial Cell Proliferation and Differentiation

“…A recent study reports that Cx50-D47A, a point mutation occurring on extracellular loop 1 (E1) of the protein, decreases both Cx50 and Cx46 gap junction coupling in lens fibers and increases the gradients of intracellular hydrostatic pressure and calcium level, which leads to calcium precipitates and cataracts. 17 Mouse Cx50-R205G mutation, on extracellular loop 2, displays semidominant ocular phenotypes that differed from Cx50 knockout and other Cx50 point mutations reported previously. [17][18][19] The Cx50-R205G heterozygous mice show reduced lens size and mild cataracts while homozygous mice develop much more severe phenotypes, including a much smaller lens with vacuole-like structures in lens cortical fibers and a dense nuclear cataract.…”

“…The Cx50-D47A, Cx50-G22R, and Cx50-S50P mutations have improper and reduced gap junction communication in the lens and have been implicated in inducing stress pathways or promoting calcium elevation and deposits. 17,25,26 Increased ER stress is also involved in the cell death in skin disease-associated Cx31 mutations. 32,33 However, our data indicate that the BiP expression is not significantly increased, and there is no obvious accumulation of Cx50-R205 mutant proteins in cultured Cx50(R205G/R205G) LECs (Fig.…”

Do Connexin Mutants Cause Cataracts by Perturbing Glutathione Levels and Redox Metabolism in the Lens?

“…The lenses of both lines develop cataracts. In both mouse lines, lenses have severely reduced levels of Cx46 and Cx50, drastically impaired lens cell–cell communication, increased hydrostatic pressure, and accumulation of Ca 2+ [ 12 , 13 , 14 , 15 , 16 ]. However, there are also several differences between these two models (summarized in Table 1 ).…”