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Polymorphism of the 86th amino acid in CX26 protein and hereditary deafness.

ABSTRACT: Objective:To investigate the membrane localization function of the CX26 protein when its 86th amino acid is Thr, Ser or Arg, and its relations to deafness. Methods:CX26-GFP protein with either Thr, Ser or Arg as the 86th amino acid was expressed in mouse SGN cells via the GFP fusion type lenti-virus expression system. The membrane localization of the fusion protein was observed under a fluorescence microscope. Results:The mutated protein of CX26 T86S was localized to cell membrane and form gap conjunction structures, showing no difference to the wild type CX26 protein (with Thr as the 86th amino acid). However, the gap conjunction structure disappeared when the mutation was CX26 T86A. Conclusion:These results indicate that the CX26 T86R mutation may be a cause of hearing loss, but CX26 T86S as a non-pathogenic polymorphism mutation does not affect functions of the CX26 protein. The results are in accordance with the results of clinical screening.

SUBMITTER: Shi X

PROVIDER: S-EPMC6002600 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Polymorphism of the 86th amino acid in CX26 protein and hereditary deafness.

Shi Xi X Qiu Shiwei S Yan Fendong F Shi Lizhang L Xuan Yili Y Zhuang Wei W Bei Yingli Y Yao Hanli H Yuan Na N Yang Shiming S Qiao Yuehua Y

Journal of otology 20160610 2

<h4>Objective</h4>To investigate the membrane localization function of the CX26 protein when its 86th amino acid is Thr, Ser or Arg, and its relations to deafness.<h4>Methods</h4>CX26-GFP protein with either Thr, Ser or Arg as the 86th amino acid was expressed in mouse SGN cells via the GFP fusion type lenti-virus expression system. The membrane localization of the fusion protein was observed under a fluorescence microscope.<h4>Results</h4>The mutated protein of CX26 T86S was localized to cell m ...[more]

PMID: 29937815

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Project description:Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca(2+) show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.

Dataset Information

Polymorphism of the 86th amino acid in CX26 protein and hereditary deafness.

Publications

Polymorphism of the 86th amino acid in CX26 protein and hereditary deafness.

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