Calmodulin Directly Interacts with the Cx43 Carboxyl-Terminus and Cytoplasmic Loop Containing Three ODDD-Linked Mutants (M147T, R148Q, and T154A) that Retain α-Helical Structure, but Exhibit Loss-of-Function and Cellular Trafficking Defects.

Li Zheng,Paul L Sorgen,Andrew Trease,Asokan Anbanandam,Sylvie Chenavas,Fabien Kieken,Gaelle Spagnol,Sarah Brownell

doi:10.3390/biom10101452

Li Zheng, Paul L Sorgen + Show 6 more

Open Access

https://doi.org/10.3390/biom10101452

Copy DOI

Abstract

The autosomal-dominant pleiotropic disorder called oculodentodigital dysplasia (ODDD) is caused by mutations in the gap junction protein Cx43. Of the 73 mutations identified to date, over one-third are localized in the cytoplasmic loop (Cx43CL) domain. Here, we determined the mechanism by which three ODDD mutations (M147T, R148Q, and T154A), all of which localize within the predicted 1-5-10 calmodulin-binding motif of the Cx43CL, manifest the disease. Nuclear magnetic resonance (NMR) and circular dichroism revealed that the three ODDD mutations had little-to-no effect on the ability of the Cx43CL to form α-helical structure as well as bind calmodulin. Combination of microscopy and a dye-transfer assay uncovered these mutations increased the intracellular level of Cx43 and those that trafficked to the plasma membrane did not form functional channels. NMR also identify that CaM can directly interact with the Cx43CT domain. The Cx43CT residues involved in the CaM interaction overlap with tyrosines phosphorylated by Pyk2 and Src. In vitro and in cyto data provide evidence that the importance of the CaM interaction with the Cx43CT may lie in restricting Pyk2 and Src phosphorylation, and their subsequent downstream effects.

Highlights

Gap junctions are integral membrane proteins that enable direct cytoplasmic exchange of ions and low molecular-mass molecules between adjacent cells [1]
Cx43CL100-158 oculodentodigital dysplasia (ODDD) mutants M147T, R148Q, T154A polypeptides, and Cx43CT236-382 S255,279,282D and Y265,313D were obtain by introducing the specific point mutation in the Cx43CL or Cx43CT plasmids, respectively, using the same kit
A well-accepted regulation mechanism for Cx43 relies on the pH-dependent interaction between the Cx43CT and Cx43CL [9,14,54]

Summary

Introduction

Gap junctions are integral membrane proteins that enable direct cytoplasmic exchange of ions and low molecular-mass molecules between adjacent cells [1]. They provide a pathway for propagating and amplifying intercellular signal transduction cascades triggered by cytokines, growth factors, and other molecules involved in development, growth, and differentiation. Dysfunctional gap junction intercellular communication causes a number of human pathologies and genetic diseases [2]. Gap junctions are formed by the apposition of connexons from adjacent cells. Each connexon is a hexamer of connexin proteins. Though the 21 human connexin isoforms share significant sequence homology, there is major divergence in the primary sequence of the cytoplasmic domains, the cytoplasmic loop (CL) and carboxyl-terminus (CT)

Objectives

Methods

Results

Discussion

Conclusion