Cataracts are caused by alterations of a critical N-terminal positive charge in connexin50.

Cataracts are a major cause of impaired vision and blindness throughout the world.1 Congenital cataracts are responsible for 10% of childhood blindness and are a common treatable cause of childhood visual impairment.2 A substantial fraction of isolated (nonsyndromic) childhood cataracts are inherited. Inheritance is most commonly autosomal dominant, although autosomal recessive and X-linked forms have also been reported. Hereditary congenital cataracts have been linked to mutations of several different genes, including the gap junction proteins connexin46 (CX46) and connexin50 (CX50).3-17 Maintenance of a transparent lens depends on gap junction-mediated communication between its cells. Gap junctions are plasma membrane specializations containing clusters of intercellular channels. These channels allow intercellular transfer of ions and molecules of ≤1000 Da. Within the lens, gap junction channels form a network of cell-to-cell channels that facilitate exchange of water, ions, nutrients, and metabolites, especially between fiber cells.18,19 A gap junction channel contains 12 subunit proteins (connexins) distributed among two coaxially aligned hexameric hemichannels located in the plasma membranes of the apposing cells. Lens fiber cells primarily express CX5020 and CX46,21 whereas the lens epithelial cells mainly express connexin43 (CX43).22,23 Large numbers of gap junction channels may cluster together within the appositional membranes to form plaques. Genetic data from rodents have also emphasized the importance of alterations of the lens fiber connexins in cataractogenesis. Several strains of mice or rats that develop cataracts harbor mutations of the Gja3 (CX46) or Gja8 (CX50) genes.24-28 Targeted ablation of the murine Gja3 (CX46)29 or Gja8 (CX50)30,31 genes also causes cataracts. In fact, not only loss of CX50, but also its overexpression, can result in cataracts.32 Investigators in several previous studies have examined the behavior of cataract-associated mutant connexins in expression systems, to elucidate cellular and physiological abnormalities that may lead to cataract formation. Some lens connexin mutants exhibit loss of function-for example, small or no gap junction conductances were detected when they were expressed in paired Xenopus oocytes.17,28,33,34 When coexpressed with wild-type connexins, some mutants exert a dominant-negative effect abolishing conductance,15,35,36 whereas some other mutants do not eliminate coupling, but may affect channel gating properties.37 Several mutants do not form gap junction plaques.15,36 In one of these cases a unique sequence in the mutant protein (generated by a frame shift) impairs trafficking to the plasma membrane.38 The human mutation, CX50R23T (which leads to the substitution of a threonine for arginine-23), was first described in members of an Iranian family with autosomal dominant, bilateral, congenital nuclear cataract that progressed to cause visual impairment by the second decade of life.9 The mechanisms responsible for the development of this cataract are not understood. However, this mutation affects a critical position within the connexin molecule at the predicted interface between the N-terminal cytoplasmic domain and the first transmembrane domain (Fig. 1). Structural models suggest the importance of this residue for maintaining associations of transmembrane domains.39 Substitutions of this amino acid have been identified in the mutant forms of at least three other connexins associated with other diseases.40-44 The present experiments were designed to elucidate the mechanism of cataract formation in patients carrying the mutation CX50R23T by examining the cellular distribution and physiological function of the CX50R23T mutant and of site-directed mutants containing other amino acid substitutions at this position. FIGURE 1 Diagram of the membrane topology of human CX50 showing the position of the R23T substitution.