Snowflake vitreoretinal degeneration (SVD) mutation R162W provides new insights into Kir7.1 ion channel structure and function

Abstract

Snowflake Vitreoretinal Degeneration (SVD) is associated with the R162W mutation of the Kir7.1 inwardly-rectifying potassium channel. Kir7.1 is found at the apical membrane of Retinal Pigment Epithelial (RPE) cells, adjacent to the photoreceptor neurons. The SVD phenotype ranges from RPE degeneration to an abnormal b-wave to a liquid vitreous. We sought to determine how this mutation alters the structure and function of the human Kir7.1 channel. In this study, we expressed a Kir7.1 construct with the R162W mutation in CHO cells to evaluate function of the ion channel. Compared to the wild-type protein, the mutant protein exhibited a non-functional Kir channel that resulted in depolarization of the resting membrane potential. Upon co-expression with wild-type Kir7.1, R162W mutant showed a reduction of IKir7.1 and positive shift in '0' current potential. Homology modeling based on the structure of a bacterial Kir channel protein suggested that the effect of R162W mutation is a result of loss of hydrogen bonding by the regulatory lipid binding domain of the cytoplasmic structure.

Figure 1. Kir7.1 channels are localized to the apical processes of adult rhesus RPE cells.

(A) Low-magnification (10X, NA 0.30) view of a frozen section showing Kir7.1 immunopositive staining of the RPE cell layer. Kir7.1 (red) was detected in the RPE cell layer but was not detected in the neural retina. Cone photoreceptors (PR) are immunopositive for cone arrestin (green). Nuclear staining (blue) shows an intact retinal structure. (B) Images acquired using a 60X objective (60X water, NA 1.0) show the magnified outer nuclear layer (ONL) and the RPE cell layer. Z-stack images of the retina were obtained to generate a three-dimensional reconstructed image. The image above the top white line (B) is the optical cross section at the horizontal cursor, and the image on the right side of vertical white line is the optical cross section of the vertical cursor. Note the apical localization of the red fluorescence signal (arrow) which is present anterior to the RPE nuclear staining. (C) Higher magnification image (100X Oil, NA 1.4) showing 4 adjacent RPE cells illustrating the distribution of Kir7.1 immunostaining at the apical processes (upper panel). The middle panel shows the position of the RPE cell nuclei (DAPI) and the lower panel is a superimposed image of Kir7.1 and DAPI nuclear staining. Abbreviations used are: choroid (Ch), retina pigment epithelium (RPE), outer nuclear layer (ONL), inner nuclear layer (INL), ganglion cell layer (GCL), photoreceptors (PR), basal side (ba), and apical side (ap).

Figure 2. Kir7.1 R162W channels display altered function.

(A) Representative recordings showing current responses to voltage pulses of 1.5 seconds. Hyperpolarizing potentials yield large inward currents and small outward currents due to the hKir7.1 channel (upper panel) whereas amplitudes of the outward and inward current were of similar magnitude for R162W channels (lower panel). The horizontal dashed line represents the zero current line. The voltage pulse protocol is indicated in the inset. (B) Average current amplitude vs. applied voltage (I–V) plot from non-transfected (NT-: closed triangles), pmCherry-R162W (R162W: open circles) and pEGFP-hKir7.1 (hKir7.1: open squares) transfected cells. The responses were averaged from at least 5 experiments. (C) Normalized currents (I/I_{-150 mV}) reflecting a clear positive shift in the zero current potential (square-hKir7.1 and circle-R162W). (D) I–V plot from cells transfected with hKir7.1 channels showing whole cell current (open squares), and current in the presence of 20 mM Cs⁺ (open circle). The Kir current (closed triangles) is the Cs⁺ sensitive component that is obtained by mathematical subtraction (square minus circle). (E) I–V plot from cells transfected with pmCherry-R162W with symbols as in D. Error bars represent ± SEM.

Figure 3. Rb⁺ has no effect on Kir7.1 R162W.

Average I–V plot of pEGFP-hKir7.1 (A) and pmCherry-R162W (B) transfected cells. The recordings were obtained in HEPES Ringer’s (Ctrl: open square), 135 mM extracellular K⁺ (closed triangle), or 135 mM extracellular Rb⁺ (open circle). Each data point is the mean ± the SEM of at least 5 experiments. (C) Comparison of the mean fold-increase in the current amplitude due to the exposure of cells to either 135 mM external Rb⁺ (gray bar) or 135 mM external K⁺ (white bar) measured at −140 mV. Error bars are ± SEM.

Figure 4. Mutation R162W affects the function of the Kir7.1 channel.

Currents were elicited in cells co-transfected with equal amounts of pEGFP-hKir7.1 and pmCherry-R162W DNA during 500 msec 20 mV step voltage pulses from −160 to +40 mV. (A) Raw data of current recordings showing that responses were of similar amplitude in both ‘–ve’ and ‘+ve’ voltages. The dashed line represents zero current. (B) I–V relationship from seven co-transfected cells (solid triangle) shows a linear response. For comparison, the responses of hKir7.1 and R162W channel are shown as a solid line and a dashed line, respectively. Note that the current responses lacked rectification and that the zero current potential was intermediate between that of the wild-type and the mutant channel response. (C) Suppression of Kir current by the R162W mutation is illustrated by comparing the relative preference for Rb⁺ over K⁺ current responses at −150 mV (I _Rb ⁺/I _K ⁺: black bar) and the measure of the conductance of the inward current between −50 to −130 mV (G: gray bar). Mean values ± SEM from at least 5 experiments are represented.

Figure 5. hKir7.1 and R162W protein expression in transfected CHO cells.

(A) Western blot of transfected cell extracts with anti-Kir7.1 antibody showing Kir7.1 protein intensities at 54 kDa. The β-actin control confirms that consistent amounts of protein were loaded in each lane. The plasmids used for each transfection are indicated. (B) Total Kir7.1 levels were normalized against β-actin. The average total soluble Kir7.1 protein expression from the three different transfections used for the electrophysiology studies are presented as a ratio of protein expression to wild-type hKir7.1 protein levels.

Figure 6. Cellular localization of the Kir7.1 channel.

CHO cells expressing the pEGFP-hKir7.1, pEGFP-R162W (A) or both pEGFP-hKir7.1+ pmCherry-R162W (C, D, E) were studied by live cell fluorescence microscopy using a 60X water immersion objective. Kir7.1 localized mainly to the plasma membrane (A. upper panel green: hKir7.1, red: ER and blue: nucleus) in the pEGFP-hKir7.1 transfected cells. pEGFP-R162W expression co-localized with ER labeling (A. middle panel). Control pEGFP expressing cells are shown in the lower panel (A). (B) Line scans (white arrow) of fluorescence intensity distribution of pEGFP-hKir7.1 (A. black trace upper panel), pEGFP-R162W (A. green trace middle panel), and pEGFP (A. dark green trace lower panel) transfected cells. Red and blue traces (B. upper panel and middle panel) represent ER labeling and Hoechst nucleus staining, respectively. In co-transfection experiments, the GFP fluorescence localized to the cellular membrane (C), whereas mCherry fluorescence shows an intracellular aggregated localization (D). Superposition of both red and green fluorescence (E) further illustrates that there is very little co-localization of the wild-type and mutant channel signals. (F) Fluorescence quantification of membrane vs. cytoplasmic expression from five independent co-transfections with pEGFP-hKir7.1 and pmCherry-R162W plasmids is shown in F, p<0.01.

Figure 7. Human Kir7.1 model and Kir channel family homology within the C-linker domain.

(A) Tetrameric structural model of Kir7.1 protein and four interacting PIP₂ molecules. The highlighted structure is enlarged for clarity of the interactions between the C-terminal hotspot and the PIP₂ head group. (B) R162 interacts with PIP₂ through 3 hydrogen bonds as shown by the green dotted lines. (C) R162W structure showing the tryptophan residue and its side chain orientation with respect to PIP₂. (D) Comparison of the interaction of both R and W at position 162 with PIP₂ (green dotted line), along with the adjacent K-sharing hydrogen bond (purple dotted line). (E) Topology of the Kir7.1 subunit showing the relative position of the C-linker and Arg (R) 162 residue located adjacent to 2nd trans-membrane domain. (F) The conserved basic residues amongst Kir channels are indicated by upper-case letters. Disease mutations are highlighted by bold-face letters. Residues in the C-linker region are shaded. Numbers represent the first and last residues in the corresponding sequence. The species, name and accession numbers for proteins used for this comparison were as follows: hKir1.1 NM_000220, hKir2.1 NM_010603, hKir2.2 GI: 23110982, hKir3.1 NM_002239, hKir4.1 NM_002241, hKir5.1 NM_018658, hKir7.1 NM_002242, and cKir2.2 GI: 118097849.