Characterization of gap junctional channels of lens connexin 50 and its mutations in heterologous expression systems.

摘要

These studies have been designed to characterize the properties of gap junctional channels of lens Cx50 and investigate the consequence of its mutations on gap junctional coupling. (1) A mouse Cx50 mutation D47A, which has been associated with congenital cataracts, lost its ability to form functional gap junctional channels, but did not inhibit the coupling of Cx46 or Cx50 wild-type when expressed in Xenopus oocyte pairs. (2) Human Cx50 was stably expressed in communication-deficient N2A cells. Human Cx50 formed functional gap junctional channels, which were sensitive to transjunctional voltage with V0 = 43.8 mV, and its single channel conductance was 212 pS. (3) Two human Cx50 C-terminal truncation mutants, hCx50V284stop and hCx50A294stop, were constructed and transiently expressed in N2A cells. V284stop failed to form functional gap junctional channels. A294stop formed functional gap junctional channels, which showed voltage sensitivity with V0 = 39.3 mV, and its single channel conductance was 185 pS. These were not significantly different from those of wild-type hCx50 channels. However, gap junctional channels made of A294stop failed to close at intracellular acidification as low as pH 5.8, while gap junctional coupling of wild-type hCx50 was reversibly blocked when intracellular pH was reduced to 6.3. (4) When stably expressed in HeLa cells, human Cx50 mutation P88S, which is associated with congenital cataracts, rarely formed gap junctional plaques at appositional membranes, but instead exhibited large intracellular protein accumulation. The expression of P88S inhibited endogenous gap junctional coupling in HeLa cells, while the expression of wild-type hCx50 induced a very high level of gap junctional coupling.