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, 7 (1), 10651

Abnormal Electroretinogram After Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

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Abnormal Electroretinogram After Kir7.1 Channel Suppression Suggests Role in Retinal Electrophysiology

Pawan K Shahi et al. Sci Rep.

Abstract

The KCNJ13 gene encodes the inwardly rectifying potassium channel, Kir7.1. Mutations in this gene cause childhood blindness, in which the a- and b-wave responses of electroretinogram (ERG) are abolished. The ERG a-wave is the light-induced hyperpolarization of retinal photoreceptors, and the b-wave is the depolarization of ON-bipolar cells. The Kir7.1 channel is localized to the apical aspects of retinal pigment epithelium (RPE) cells and contributes to a delayed c-wave response. We sought to understand why a defect in an RPE ion-channel result in abnormal electrophysiology at the level of the retinal neurons. We have established the expression of Kir7.1 channels in the mouse RPE. ERGs recorded after mice Kir7.1 suppression by shRNA, or by blocking with VU590, showed reduced a-, b- and c-wave amplitudes. In contrast, the Kir7.1 blocker had no effect on the ex-vivo isolated mouse retina ERG where the RPE is not attached to the isolated retina preparation. Finally, we confirmed the specificity of VU590 action by inhibition of native mouse RPE Kir7.1 current in patch-clamp experiment. We propose that mutant RPE Kir7.1 channels contribute directly to the abnormal ERG associated with blindness via alterations in sub-retinal space K+ homeostasis in the vicinity of the photoreceptor outer segment.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
mRNA expression of Kir7.1 in RPE and retina. (A) Gel electrophoresis image illustrates the expression of Kir7.1 mRNA in both RPE and retinal tissue, as well as in pools of 10 isolated cells. Complete gel image is included as Supplemental Fig. 1A. (B) Images of representative single RPE, bipolar, and Müller glial cell used for single-cell RNA extraction. (C) mRNA expression for Kir7.1 and GAPDH in RPE cells (R1–R3), bipolar cells (B1–B3), and Müller glial cells (M1-M2) along with negative control water and perfusion bath solution. A Supplemental Fig. 1C is included showing full gel image.
Figure 2
Figure 2
Kir7.1 protein expression. (A) Protein expression of Kir7.1 in RPE and retinal tissue detected by Western blot analysis. Complete Western blot image is included as Supplemental Fig. 2A. (B) Bar graph showing the relative expression of Kir7.1 protein in the RPE and the retina by densitometry expressed as a Kir7.1/b-actin ratio. (C) Immunohistochemistry against Kir7.1 and Ezrin; ezrin (red) labels the microvilli of the RPE cells and Kir7.1 (green) co-localizes with ezrin confirming its presence in the apical processes of the RPE cell. Outer nuclear layer (ONL) is stained with DAPI (blue). Scale bar is indicated. A video is included as a Supplemental data. (D) Higher magnification image of the RPE layer shows ezrin expression (red) in apical processes extending towards the retina. Kir7.1 (green) staining also appeared in apical membrane extensions with co-localization of both proteins (yellow) in long apical processes (arrow). Scale bar is included. (E) A quantitative distribution plot representation of signals acquired in green (488) vs. red (594).
Figure 3
Figure 3
Reduced RPE/retina function after sub-retinal administration of lentivirus containing shRNA for Kir7.1. (A) Gel electrophoresis showing that Kir7.1 mRNA expression is reduced after Kir7.1 shRNA injection when compared to the un-injected contralateral eye. Full image of the gel is included as Supplemental Fig. 3A. (B) Expression of Kir7.1/b-actin ratio in shRNA injected and un-injected eye (part A) represented in a bar graph. (C) Representative scotopic traces comparing the shRNA-injected mice 14 d post injection, as well as control and PBS-injected mice. (D) Fluorescent image of live RPE sheet of cells displaying the expression of GFP that is fused with the shRNA Kir7.1. (E) Normalized a-wave and (F) normalized b-wave comparing the shRNA injected eye with the PBS injected eye at 30 cd.s/m2 at 0, 2, 4, 7, and 14 d post-injection. (G) Comparison of c-wave for eyes injected with Kir7.1 shRNA and PBS at 0, 2, 4, 7 and 14 d post injection. Dotted line represents the control-normalized response from non-injected eyes.
Figure 4
Figure 4
Kir7.1 channel blocker VU590 inhibits c-wave originating from RPE cell. (A) Representation of the scotopic traces at different intensities from control mice and mice injected with VU590 or VU-608, an inactive analogue of VU-590. (B) Amplitude of a-wave and (C) b-wave after VU590 injection (red) compared with the VU608 injected eye (blue) and the control (black) saline injected eye. (D) Scotopic ERG trace representing the reduction of c-wave after the injection of VU590 but not with VU608 when compare with control. (E) Graphical representation of the c-wave reduction after Kir7.1 channel inhibition. Data is mean ± SEM and *P < 0.005.
Figure 5
Figure 5
Optical Coherence Tomography imaging of saline (A) and shRNA (B) injected eyes to visualize the structural integrity of the RPE and retina. In both panels A and B, the fundus photo shown in the left panel indicates the orientation (arrow) of the cross-sectional OCT. The scanned images with the outer nuclear (ONL) and inner nuclear (INL) layers is highlighted in the panel on the right. In image B, GFP fluorescence is evident in the white patches. (C) Ex-vivo ERG allows recording the response from the retina, only. ERG traces from the retina before (black) and after the treatment with 50 µM VU590 (red) are shown. The proper functioning of the ERG and the retina is confirmed by the treatment with 100 µM Ba2+ (blue). (D) Bar graph representing the amplitude of a- and b-waves in ex-vivo ERG recording after treatment with VU590 and Ba2+ are shown as compared to control.
Figure 6
Figure 6
Inhibition of Kir7.1 current by VU590. Representative traces of whole cell currents recorded from single isolated RPE cells from mouse (A) and CHO-K1 cells (B) overexpressing the Kir7.1 channel, respectively. Whole cell currents were recorded by voltage ramping from +40 mV to −160 mV. The traces represent current density, which is the whole cell current normalized for the cell capacitance. In both cell types, the normal baseline Kir7.1 current is recorded prior to cells being treated with 50 µM VU590. The current is reduced after treatment with VU590, but is not affected by the treatment with 50 µM VU608. (C) and (D) The normalized Kir7.1 current at −160 mV is compared before, and after, treatment with VU590 or VU608.
Figure 7
Figure 7
Control of sub-retinal space K+ homeostasis by Kir7.1 is crucial for photoreception. On the left is a representation of a normally functioning Kir7.1 channel which is able to maintain normal K+ levels. On the right, a reduction in K+ in the sub-retinal space due to disease that alters the ERG. The model: 1- Depolarization of RPE, 2- Changes in the sub-retinal space volume and K+, and 3- PR ionic conductance. RPE: retinal pigment epithelium, PR: photoreceptor, RSRS: sub-retinal space resistance.

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