Analysis of the dominant mutation N188T of human connexin46 (hCx46) using concatenation and molecular dynamics simulation.

Patrik Schadzek,Matthias Preller,Anaclet Ngezahayo,Yannick Stahl

doi:10.1002/2211-5463.12624

Patrik Schadzek, Matthias Preller + Show 2 more

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https://doi.org/10.1002/2211-5463.12624

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Abstract

Connexins (Cx) are proteins that form cell‐to‐cell gap junction channels. A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract. As it is dominantly inherited, it is possible that the mutant variant affects the co‐expressed wild‐type Cx and/or its interaction with other cellular components. Here, we proposed to use concatenated hCx46wt‐hCx46N188T and hCx46N188T‐hCx46wt to analyze how hCx46N188T affected co‐expressed hCx46wt to achieve a dominant inheritance. Heterodimer hCx46wt‐hCx46N188T formed fewer gap junction plaques compared to homodimer hCx46wt‐hCx46wt, while the hCx46N188T‐hCx46N188T homodimer formed almost no gap junction plaques. Dye uptake experiments showed that hemichannels of concatenated variants were similar to hemichannels of monomers. Molecular dynamics simulations revealed that for docking, the N188 of a protomer was engaged in hydrogen bonds (HBs) with R180, N189, and D191 of the counterpart protomer of the adjacent hemichannel. T188 suppressed the formation of HBs between protomers. Molecular dynamics simulations of an equimolar hCx46wt/hCx46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co‐expressing the variants.

Highlights

Connexins (Cx) are proteins that form cell-to-cell gap junction channels
In agreement with previous results ([13]), here we found that hCx46N188T rarely formed gap junction plaques (Fig. 1)
It is possible that the presence of the hCx46N188T either as homomer or as tandem in any combination strongly reduced the presence of the protein in the membrane

Summary

Introduction

A mutation at position 188 in the second extracellular loop (E2) domain of hCx46 has been linked to an autosomal dominant zonular pulverulent cataract As it is dominantly inherited, it is possible that the mutant variant affects the co-expressed wild-type Cx and/or its interaction with other cellular components. Molecular dynamics simulations of an equimolar hCx46wt/ hCx46N188T gap junction channel revealed a reduced number of HBs between protomers, suggesting reduction of gap junction channels between lens fibers co-expressing the variants. Structural modeling and molecular dynamics (MD) simulations of a human Cx46 (hCx46) model, using the crystallized Cx26 [12] as template, indicate that N188 in a protomer within a connexon of one cell forms hydrogen bonds (HB) with residues R180, T189, and D191 of the counterpart protomer within the connexon in the adjacent cell to stabilize the docked hCx46 gap junction channel. Concatenation was shown to be a good technique that allows to gain insight in the architecture of multimeric membrane proteins such as acetyl choline or c-aminobutyric acid receptors as well as K+ channels or Cx [15,16,17,18,19,20,21]

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