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B. R. Pattnaik, A. Sharma, S. Tokarz, T. Schroeder, M. Assuma, A. O. Edwards, D.-A. M. Pillers; Kir7.1 Channel Mutation (R162W), Associated With Snowflake Vitreoretinal Degeneration (SVD), Results in a Non-Functional Channel. Invest. Ophthalmol. Vis. Sci. 2010;51(13):682.
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Hereditary snowflake vitreoretinal degeneration (SVD) causes degeneration of multiple ocular tissues due to a mutation in Kir7.1 channel. Kir7.1 channel is predominantly present in the apical membrane of retinal pigment epithelium (RPE) that faces photoreceptor outer segment. This channel is highly conserved amongst mammals and rat Kir7.1 mutant channel produced non-selective cation current. As a first step to understand the underlying biology, we characterized the human (Kir7.1) mutant expressed in mammalian cells.
Wildtype (hKir7.1WT) and 484 C>T (R162W) mutant (hKir7.1M) constructs in pCMV6-XL5 were co-transfected into CHO-M1 cells along with pcDNA3.1/NTGFP using Fugene-6. Kir channel currents were studied by whole-cell configuration of patch clamp electrophysiology. Bath solution: (in mM) 135 NaCl, 5 KCl, 10 HEPES, 10 glucose, 1.8 CaCl2 and 1 MgCl2 pH 7.4 using NaOH. We used 20 mM Cs+ in place of equimolar Na+ to block Kir7.1 current. For selectivity bath Na+ was replaced with either K+ or Rb+. Recording electrodes contained (in mM): 30 KCl, 83 K-gluconate, 10 HEPES, 5.5 EGTA, 0.5 CaCl2, 4 Mg-ATP, and 0.5 GTP adjusted to pH 7.2 using KOH.
GFP positive cells expressing hKir7.1WT channels had a resting membrane potential of -45 ± 4.6 mV (n=10) compared to -3.2 ± 1.8 mV (n=15) for the hKir7.1M channel transfected cells (p < 0.005). Current-voltage curves for the hKir7.1WT channel showed typical inward rectification. This current was inhibited by 20 mM Cs+. The inward current of hKir7.1WT was at least 6-fold larger in the presence of Rb+ and showed higher preference for Rb+ over K+. The hKir7.1M channel on the otherhand exhibited no current over the whole voltage range in which hKir7.1WT is activated. Further application of 20 mM Cs+ or 135 mM Rb+ solution had no effect on the hKir7.1M channel current.
We provide evidence that the hKir7.1M subunit does not form functional channels. Kir7.1 is a mildly inwardly rectifying potassium channel that facilitates potassium homeostasis around the photoreceptor outer segment and also facilitates directional fluid flow by RPE. This non-functional Kir7.1 channel therefore by altering RPE physiology perhaps recapitulates SVD etiology like the minute crystalline deposits and retinal detachments.
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