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X.-M. Zhang, H. Wang, A. K. Sharma, A. O. Edwards, B. A. Hughes; The R162W Mutation in Kir7.1 Causes Dominant-Negative Dysfunction of Wild-Type Kir7.1 Channels. Invest. Ophthalmol. Vis. Sci. 2010;51(13):485.
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© ARVO (1962-2015); The Authors (2016-present)
Kir7.1 channels, which are tetramers formed by subunits encoded by KCNJ13, comprise the apical membrane K+ conductance of the RPE. A mutation in KCNJ13 resulting in a R162W change in the Kir7.1 amino acid sequence was recently found to be associated with Snowflake vitreoretinal degeneration (SVD), an inherited autosomal dominant disease with vitreous degeneration, increased risk of retinal detachment, Fuchs corneal dystrophy, and mild retinal degeneration. The objective of this study was to investigate the mechanism by which the R162W mutation causes Kir7.1 channel dysfunction.
cRNA encoding wild-type (WT) or R162W mutant human Kir7.1 was injected alone or together into Xenopus oocytes. Protein expression was confirmed by Western blot analysis using Kir7.1 antibodies. The two-electrode voltage clamp technique was used to assess channel function.
Western blots of membrane proteins from oocytes injected with WT or mutant Kir7.1 cRNA revealed appropriately sized bands, confirming the expression of both WT and mutant proteins. Oocytes injected with WT Kir7.1 and bathed in 98 mM Rb+ exhibited large inwardly rectifying currents(-22.1 ± 4.4 µA at -150 mV, n = 6), consistent with Kir7.1 channels. In oocytes injected with mutant cRNA, Rb+ currents were small and likely mediated by endogenous channels (-3.0 ± 0.9 µA at -150 mV, n = 9). Co-injection of WT and mutant Kir7.1 cRNAs resulted in Rb+ currents that were of intermediate amplitude (-7.6 ± 1.9 µA at -150 mV, n = 8), indicating that mutant subunits are capable of forming heteromeric channels with WT Kir7.1 subunits and suppressing channel activity.
We conclude that the Kir7.1 R162W mutation results in non-functional subunits that have a dominant negative effect on WT Kir7.1 channels. Our results provide new mechanistic insight into the R162W mutation and may lead to a better understanding of the multiple ocular phenotypes seen in SVD.
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