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C. A. Rubinos, B. Cooper, M. Srinivas; Functional Characterization of Cx50 Mutations Underlying Congenital Cataracts. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4606.
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Mutations in Cx50 have been reported to cause a variety of dominant cataracts in both humans and mice. How these distinct pathologies arise from Cx50 mutations is not known. To determine the underlying mechanism in cataract formation, we characterized the functional properties of gap junctions made by Cx50 mutations.
Human Cx50 cDNA subcloned in pCS2+ was used as a template to produce the D47N, V44E, and V79L mutations by site-directed mutagenesis and expressed in N2A cells, a communication-deficient cell line. The dual whole-cell voltage clamp technique was used to examine macroscopic and unitary properties of junctional currents.
No detectable currents were found in cells expressing the V44E (n= 18) and D47N mutations (n = 7). In contrast, V79L mutation was found to form functional intercellular channels (n = 22), although macroscopic junctional conductance was low compared to wildtype Cx50 (~ 50-60 % reduction in compared to wildtype). Unitary conductance of Cx50 channels was not significantly altered by the V79L mutation (~ 210 ± 5 pS). Single channel open probability of V79L channels was found to be significantly lower than that of wildtype Cx50 channels even at low transjunctional voltages, indicating that mutant subunits are likely to reside largely in closed state at rest. Co-expression of the different mutant subunits with wildtype Cx46 was further studied. Mixing of the V44E mutation with wildtype Cx46 caused a significant inhibition of coupling. In contrast, co-expression of V79L and D47N subunits with wildtype Cx46 resulted in the formation of heteromeric channels with unique channel properties.
These results indicate that Cx50 mutations cause different alterations in channel properties, possibly explaining the variety of cataract phenotypes found in humans with Cx50 variants. Whether these alterations can lead to changes in permeability of essential nutrients and metabolites remains to be determined.
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