Cataract-associated D3Y mutation of human connexin46 (hCx46) increases the dye coupling of gap junction channels and suppresses the voltage sensitivity of hemichannels

Abstract

Connexin46 (Cx46), together with Cx50, forms gap junction channels between lens fibers and participates in the lens pump-leak system, which is essential for the homeostasis of this avascular organ. Mutations in Cx50 and Cx46 correlate with cataracts, but the functional relationship between the mutations and cataract formation is not always clear. Recently, it was found that a mutation at the third position of hCx46 that substituted an aspartic acid residue with a tyrosine residue (hCx46D3Y) caused an autosomal dominant zonular pulverulent cataract. We expressed EGFP-labeled hCx46wt and hCx46D3Y in HeLa cells and found that the mutation did not affect the formation of gap junction plaques. Dye transfer experiments using Lucifer Yellow (LY) and ethidium bromide (EthBr) showed an increased degree of dye coupling between the cell pairs expressing hCx46D3Y in comparison to the cell pairs expressing hCx46wt. In Xenopus oocytes, two-electrode voltage-clamp experiments revealed that hCx46wt formed voltage-sensitive hemichannels. This was not observed in the oocytes expressing hCx46D3Y. The replacement of the aspartic acid residue at the third position by another negatively charged residue, glutamic acid, to generate the mutant hCx46D3E, restored the voltage sensitivity of the resultant hemichannels. Moreover, HeLa cell pairs expressing hCx46D3E and hCx46wt showed a similar degree of dye coupling. These results indicate that the negatively charged aspartic acid residue at the third position of the N-terminus of hCx46 could be involved in the determination of the degree of metabolite cell-to-cell coupling and is essential for the voltage sensitivity of the hCx46 hemichannels.

Properties of two cataract-associated mutations located in the NH2 terminus of connexin 46

“…In this study, we show that substitution of a polar tyrosine at residue 3 for the negatively charged aspartic acid present in wild-type Cx46 results in a rightward shift in the activation curve and appears to disrupt hemichannel closure at positive potentials. In another recent report, Schlingmann et al (29) found that this same D3Y substitution in human wild-type Cx46 resulted in the lack of voltage-sensitive hemichannels when expressed in Xenopus oocytes. The macroscopic gating properties of Cx46D3Y hemichannels resemble those previously reported for Cx46D3N.…”

“…Several mechanisms have been proposed to account for the role of these mutations in the development of disease: 1) inability to form functional gap junctions; 2) alterations in the gating and permeability properties of gap junctions; 3) dominant negative effects on other connexins; 4) aberrant hemichannel activity; and 5) mistrafficking leading to cytoplasmic accumulation of protein. With a few exceptions (20,29,34), most of the cataract-associated connexin mutations that have been studied are unable to form gap junctional plaques when expressed by themselves and show mistrafficking (2-4, 7, 19, 24, 25, 32, 42). Some of them also act as dominant negative inhibitors of wild-type connexin activity.…”

Connexin expression patterns in diseased human corneas

“…Also, it is becoming increasingly clear that Cxs have profound effects on gene expression during the process of development. Gap junction-deficient and -mutant mice present a range of debilitating phenotypes, such as hereditary diseases of the lens (zonular pulverulent cataract) (18), nervous system (X-linked Charcot-Marie-Tooth disease) (19), hereditary non-syndromic deafness (20), skin (palmoplantar keratoderma) (21), and bone and teeth (occulodigitoldental dysplasia) (22). These are additional reasons that altered expressions of gap junction proteins in injured human corneas require further investigation.…”

Motifs in the permeation pathway of connexin channels mediate voltage and Ca2+ sensing

“…It is unknown whether these changes in the permeability properties correlate with structural rearrangements of the NT inside the pore. A number of mutations at the NT have been shown to affect selectivity of small molecules, voltage dependence and unitary conductance, expected if the NT is folded within the pore (Xin et al, 2010; Beyer et al, 2012; Schlingmann et al, 2012; Xin and Bai, 2013). …”