Characterization of the R162W Kir7.1 mutation associated with snowflake vitreoretinopathy

Abstract

KCNJ13 encodes Kir7.1, an inwardly rectifying K(+) channel that is expressed in multiple ion-transporting epithelia. A mutation in KCNJ13 resulting in an arginine-to-tryptophan change at residue 162 (R162W) of Kir7.1 was associated with snowflake vitreoretinal degeneration, an inherited autosomal-dominant disease characterized by vitreous degeneration and mild retinal degeneration. We used the Xenopus laevis oocyte expression system to assess the functional properties of the R162W (mutant) Kir7.1 channel and determine how wild-type (WT) Kir7.1 is affected by the presence of the mutant subunit. Recordings obtained via the two-electrode voltage-clamp technique revealed that injection of oocytes with mutant Kir7.1 cRNA resulted in currents and cation selectivity that were indistinguishable from those in water-injected oocytes, suggesting that the mutant protein does not form functional channels in the plasma membrane. Coinjection of oocytes with equal amounts of mutant and WT Kir7.1 cRNAs resulted in inward K(+) and Rb(+) currents with amplitudes that were ∼17% of those in oocytes injected with WT Kir7.1 cRNA alone, demonstrating a dominant-negative effect of the mutant subunit. Similar to oocytes injected with WT Kir7.1 cRNA alone, coinjected oocytes exhibited inwardly rectifying Rb(+) currents that were more than seven times larger than K(+) currents, indicating that mutant subunits did not alter Kir7.1 channel selectivity. Immunostaining of Xenopus oocytes or Madin-Darby canine kidney cells expressing mutant or WT Kir7.1 demonstrated distribution of both proteins primarily in the plasma membrane. Our data suggest that the R162W mutation suppresses Kir7.1 channel activity, possibly by negatively impacting gating by membrane phosphadidylinositol 4,5-bisphosphate.

Fig. 1.

Wild-type (WT) and R162W mutant Kir7.1 currents. A: families of macroscopic currents recorded in a WT Kir7.1 cRNA-injected Xenopus oocyte bathed in ND96 solution in the absence (control) and presence of 10 mM Ba²⁺ and their algebraic difference (right). Horizontal line to the left of each set of currents represents zero-current level. Voltage-clamp protocol used to evoke the currents is shown below currents in the Ba²⁺ recording (middle). B: families of macroscopic currents recorded in a R162W mutant Kir7.1 cRNA-injected oocyte bathed with ND96 solution in the absence and presence of 10 mM Ba²⁺ and their algebraic difference. Voltage-clamp protocol and scales as described in A. C: steady-state current-voltage (I-V) relationships in Xenopus oocytes injected with WT Kir7.1 cRNA (WT), R162W mutant Kir7.1 cRNA (MT), or water and bathed in ND96 solution. Values are means ± SE for 17–18 oocytes. D: I-V relationships of Ba²⁺-sensitive currents obtained in the same oocytes used for measurements in C.

Fig. 2.

Effect of cation substitution on WT and mutant Kir7.1 currents. A: Ba²⁺-sensitive currents recorded in a representative Xenopus oocyte injected with WT Kir7.1 cRNA and bathed with 98 mM K⁺ (left) or 98 mM Rb⁺ (right) solution. B: Ba²⁺-sensitive currents recorded in a representative Xenopus oocyte injected with mutant Kir7.1 cRNA and bathed with 98 mM K⁺ (left) or 98 mM Rb⁺ (right) solution. Voltage-clamp protocol as depicted in A. C: I-V relationships of Ba²⁺-sensitive currents obtained in Xenopus oocytes injected with WT Kir7.1 cRNA, R162W mutant Kir7.1 cRNA, or water and bathed in 98 mM K⁺ solution. Values are means ± SE for 12–18 oocytes. D: I-V relationships of Ba²⁺-sensitive currents in Xenopus oocytes injected with WT Kir7.1 cRNA, mutant Kir7.1 cRNA, or water and bathed in 98 mM Rb⁺ solution. Values are means ± SE for 12–18 oocytes.

Fig. 3.

Coinjection of WT and mutant Kir7.1 cRNAs decreases Kir7.1 current amplitude but does not alter cation selectivity. A: Ba²⁺-sensitive currents in a representative Xenopus oocyte injected with a 1:1 mixture of WT and mutant Kir7.1 cRNAs bathed with 98 mM K⁺ (left) or 98 mM Rb⁺ (right) solution. B: I-V relationships of Ba²⁺-sensitive currents in Xenopus oocytes injected with WT + mutant Kir7.1 cRNAs and bathed with 98 mM K⁺ or 98 mM Rb⁺ solution. Values are means ± SE for 15 oocytes. C: comparison of inward K⁺ and inward Rb⁺ current amplitudes in oocytes injected with WT Kir7.1 cRNA alone or WT + mutant Kir7.1 cRNAs (WT + MT). Values are means ± SE for 16–18 oocytes. D: superimposition of normalized I-V curves of Ba²⁺-sensitive Rb⁺ currents in Xenopus oocytes injected with WT cRNA or WT + mutant Kir7.1 cRNAs. For each group, mean current at each voltage was normalized by mean current measured at −150 mV.

Fig. 4.

Immunolocalization of WT and mutant Kir7.1 proteins expressed in Xenopus oocytes. A and B: representative confocal images obtained from animal poles of cryosectioned Xenopus oocytes injected with WT Kir7.1 or mutant Kir7.1 cRNA and incubated first with rabbit anti-Kir7.1 antibody and then with goat anti-rabbit secondary antibody labeled with Alexa Fluor 488. C and D: confocal images obtained from cryosections of animal poles of oocytes from the same batch of oocytes and processed identically, except primary antibody was preabsorbed with excess antigenic peptide. Laser power and photomultiplier tube gain were the same for all images. Scale bar, 50 μm.

Fig. 5.

Immunofluorescence localization of WT and mutant human Kir7.1 proteins in Madin-Darby canine kidney (MDCK) cells. A: confocal immunofluorescence image of WT Kir7.1-transfected MDCK cells labeled with anti-Kir7.1 antibodies (a1 and a2). Basolateral membrane proteins were selectively biotinylated and labeled with fluorescently tagged streptavidin (b1 and b2). a1 and b1, Projections generated from 4 consecutive 0.29-μm z sections obtained midway through the monolayer; a2 and b2, orthogonal z-y projections from the same cells; c1 and c2, overlays of a1 and b1 and a2 and b2, respectively. B: confocal immunofluorescence images of mutant Kir7.1-transfected MDCK cells labeled with anti-Kir7.1 antibodies (d1 and d2). Basolateral membrane was selectively biotinylated and labeled with fluorescently tagged streptavidin (e1 and e2). d1 and e1, Projections generated from 4 consecutive 0.29-μm z sections obtained midway through the monolayer; d2 and e2, orthogonal z-y projections from the same cells; f1 and f2, overlays of d1 and e1 and d2 and e2, respectively. ap, apical membrane; ba, basolateral membrane. Scale bars, 10 μm.

Fig. 6.

Immunofluorescence localization of Kir7.1 in human retina. A human central retinal section was double-labeled first with rabbit anti-Kir7.1 antibodies and mouse antibodies against glutamine synthetase (GS), a marker of Müller cells, and then with fluorescence-conjugated goat anti-rabbit IgG (green) and goat anti-mouse IgG (red) and 4′,6-diaminido-2-phenylindole (DAPI, blue). Kir7.1 antibodies strongly labeled the retinal pigment epithelium (RPE) apical microvilli (left and right) but not the neural retina. Arrow, RPE apical processes; arrowhead, RPE basal border. ILM, internal limiting membrane; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptor inner and outer segments; Ch, choroid. Scale bar, 50 μm.