Human Kir7.1 Channel Mutation (R162W), Associated with Snowflake Vitreoretinal Degeneration (SVD), Renders Non-Functional Channel Due to a Dominant-Negative Effect

Abstract

Purpose: : Mutations in the KCNJ13 gene impair the function of the Retinal Pigment Epithelial Kir7.1 channel and are associated with an autosomal dominant disorder SVD that causes degeneration of multiple ocular tissues. We previously showed that the mutation results in a non-functional channel. In this work we test our hypothesis that the mutant channel alters the function of wild-type protein through a dominant negative effect.

Methods: : Fusion clones of wild-type (eGFP-hKir7.1, WT) and mutant (mCherry-hKir7.1M, M) were generated. K⁺- current due to WT, M or both transfected in CHO cells were studied by whole-cell patch-clamp electrophysiology. Protein localization in transfected cells were detected by live cell imaging. We used recently published molecular structure to determine alterations in M proteins. Total protein from transfected CHO cells was used for western blot analysis of Kir7.1 and βactin antibodies.

Results: : Cells expressing WT channel had a membrane potential of -64 ± 2.4 mV (n=9) compared to -12 ± 2.1 mV (n=18) for the M and -34 ± 4.2 mV for WT+M expressing cells (p<0.005). Current-voltage curves for the WT was typical with a preference for Rb⁺ (WT: 9.2 ± 0.7, M: 1.4 ± 0.4, WT + M: 4 ± 1.5 fold increase). The WT localized to the membrane while the M was visible throughout the cell. Substitution of arginine to tryptophan results in a stiff C-linker domain in the protein structure. Mutant protein expression was less than the WT.

Conclusions: : hKir7.1WT expression in CHO cells results in a highly selective current while the mutant did not exhibit any functional channels. The mutation alters the conserved C-linker domain and perhaps alters electrostatic interactions necessary for channel function and selectivity. The mutant channel makes the WT non -functional due to a dominant negative effect. All-in-all, the non-functional Kir7.1 channel may contribute to the classic phenotype of SVD, by distorting RPE transport function and altering RPE physiology.

Keywords: ion channels • gene/expression • retinal degenerations: hereditary