Snowflake Vitreoretinal Degeneration (SVD) Mutation R162W Provides New Insights into Kir7.1 Ion Channel Structure and Function

<div><p>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 I_Kir7.1 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.</p></div

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

<p>(<b>A</b>) 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>B</b>) R162 interacts with PIP₂ through 3 hydrogen bonds as shown by the green dotted lines. (<b>C</b>) R162W structure showing the tryptophan residue and its side chain orientation with respect to PIP₂. (<b>D</b>) 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). (<b>E</b>) 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. (<b>F)</b> 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.</p

Rb⁺ has no effect on Kir7.1 R162W.

<p>Average I–V plot of pEGFP-hKir7.1 (<b>A</b>) and pmCherry-R162W (<b>B</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. (<b>C</b>) 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.</p

Cellular localization of the Kir7.1 channel.

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

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

<p>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. (<b>A</b>) 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>B</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. (<b>C</b>) Suppression of Kir current by the R162W mutation is illustrated by comparing the relative preference for Rb⁺ over K⁺ current responses at −150 mV (<b>I</b>_Rb⁺/<b>I</b>_K⁺: black bar) and the measure of the conductance of the inward current between −50 to −130 mV (<b>G</b>: gray bar). Mean values ± SEM from at least 5 experiments are represented.</p