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Jean X Jiang, Zhengping Hu, Bin Wang, Robert Brenner, Sumin Gu; Connexin 46 G143R Mutation in Intracellular Loop Domain Alters its Interaction with C-terminal domain and Calmodulin, and Gating of Hemichannels . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4658.
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© ARVO (1962-2015); The Authors (2016-present)
Connexin channels play a critical role in maintaining lens homeostasis and transparency. The G143R missense mutation on connexin (Cx) 46 is associated with congenital Coppock cataracts; however, the underlying molecular mechanism is largely unknown.
Cx46 hemichannel activities were analyzed in Xenopus oocytes and Hela cells. Wild-type (WT) and G143R mutant of Cx46 cRNA were injected into Xenopus oocytes and electrical conductance was measured. The whole cell currents were determined in Hela cells transfected with either WT or mutant Cx46 cDNA. The secondary structures of intracellular loop domains were studied with circular dichroism (CD) technique. The interactions between intracellular loop, C-terminal domains and calmodulin were assessed by protein pull-down and isothermal titration calorimetry (ITC) assays with purified proteins.
G143R mutation diminished the hemichannel conductance in Xenopus oocytes. Moreover, it functioned in a dominant negative manner by inhibiting the conductance by WT Cx46. Whole cell currents of Cx46 hemichannels was only detected in Hela cells expressing WT, but not mutant Cx46. The CD analysis showed that α-helical structural content of the intracellular loop domain of Cx46 was greatly reduced as a result of the mutation. Previous studies have shown that the interaction between the intracellular loop and C-terminal domains or calmodulin affect Cx43 gap junction channel function, but its impact on Cx46 hemichannels remains unexplored. Protein pull-down assay showed that as compared to WT control, the interaction between the intracellular loop and C-terminal domains was decreased. Conversely, the mutation enhanced the interaction between the intracellular domain and calmodulin with a 2-fold increase of the binding affinity by ITC assay. Given that Cx46 G143 residue on intracellular loop domain is located in the potential calmodulin binding pocket and is closely adjacent to Cx46 C-terminus binding motif, it is likely that competitive binding between the C-terminus and calmodulin regulates gating of Cx46 channels.
These results suggest that G143R mutation results in the impairment of electrical conductance mediated by Cx46 hemichannels, which is likely to be caused by structural changes of the intracellular loop domain leading to the altered interaction with C-terminal domain and calmodulin.
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