TY - JOUR
T1 - Cataract-associated connexin 46 mutation alters its interaction with calmodulin and function of hemichannels
AU - Hu, Zhengping
AU - Riquelme, Manuel A.
AU - Wang, Bin
AU - Bugay, Vladislav
AU - Brenner, Robert
AU - Gu, Sumin
AU - Jiang, Jean X.
N1 - Funding Information:
This work was supported by National Institutes of Health Grant EY012085 (to J. X. J.), Welch Foundation Grant AQ-1507 (to J. X. J.), and National Science Foundation Grant 1456862 (to R. B.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2018/2/16
Y1 - 2018/2/16
N2 - Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the -helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca2-dependent fashion, an observation confirmed by immuno-fluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46 –CaM interaction and attenuated its abolishment by Ca2 depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca2-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca2 sensitivity. The data suggest that the G143R-induced enhancement of the CaM–Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.
AB - Connexin channels help maintain eye lens homeostasis and transparency. The G143R missense substitution in connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown. Here, we report that compared with WT Cx46, the G143R substitution abolishes hemichannel conductance in Xenopus oocytes and in HeLa cells. Moreover, this substitution is dominant-negative and inhibits conductance of WT Cx46. CD analysis indicated that the substitution greatly reduces the -helical structure of the intracellular Cx46 loop domain. Protein pulldown assays and isothermal titration calorimetry revealed that this Cx46 domain directly interacts with calmodulin (CaM) in a Ca2-dependent fashion, an observation confirmed by immuno-fluorescent co-localization of Cx46 with CaM. Interestingly, the G143R substitution enhanced the Cx46 –CaM interaction and attenuated its abolishment by Ca2 depletion. Moreover, Cx46 increased dye influx, and the G143R substitution augmented this effect. Inhibition of Ca2-mediated CaM activation blocked hemichannel permeability. The membrane potential plays a crucial role in Cx46 membrane permeability. We found that the activity of hemichannels is detectable under rest and hyperpolarization conditions but is eliminated with depolarization. These results suggested that the G143R substitution impairs voltage-dependent electrical conductance and alters membrane permeability mediated by Cx46 hemichannels. The latter likely is caused by the substitution-induced structural changes of the intracellular loop domain associated with the increased interaction with CaM and reduced Ca2 sensitivity. The data suggest that the G143R-induced enhancement of the CaM–Cx46 interaction results in altered hemichannel activities and might be related to cataract formation.
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U2 - 10.1074/jbc.RA117.001348
DO - 10.1074/jbc.RA117.001348
M3 - Article
C2 - 29298900
AN - SCOPUS:85042199974
VL - 293
SP - 2573
EP - 2585
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 7
ER -