Focus on Kir7.1: physiology and channelopathyMohit Kumar1,* and Bikash R Pattnaik2,3,4,*

1Departments of Biotechnology and Bioinformatics; NIIT University; Neemrana, Rajasthan, India; 2Department of Pediatrics; University of Wisconsin; Madison, WI USA;3Department of Ophthalmology and Visual Sciences; University of Wisconsin; Madison, WI USA; 4McPherson Eye Research Institute; University of Wisconsin; Madison, WI USA

Keywords: channelopathy, LCA, potassium channel, retinal degeneration, retinal pigment epithelium, SVD

Genetic studies have linked alterations in Kir7.1 channelto diverse pathologies. We summarize functional relevance ofKir7.1 channel in retinal pigment epithelium (RPE), regulationof channel function by various cytoplasmic metabolites, andmutations that cause channelopathies. At the apicalmembrane of RPE, KC channels contribute to subretinal KC

homeostasis and support NaC/KC pump and NaC-KC-2Cl¡

cotransporter function by providing a pathway for KC

secretion. Electrophysiological studies have established thatbarium- and cesium-sensitive inwardly rectifying KC (Kir)channels make up a major component of the RPE apicalmembrane KC conductance. Native human RPE expressestranscripts for Kir1.1, Kir2.1, Kir2.2, Kir3.1, Kir3.4, Kir4.2, andKir6.1, albeit at levels at least 50-fold lower than Kir7.1. Kir7.1 isstructurally similar to other Kir channels, consisting of 2 trans-membrane domains, a pore-forming loop that contains theselectivity filter, and 2 cytoplasmic polar tails. Within thecytoplasmic structure, clusters of amino acid sequences formregulatory domains that interact with cellular metabolites andcontrol the opening and closing of the channel. Recentevidence indicated that intrinsic sequence motifs present inKir7.1 control surface expression. Mutant Kir7.1 channels areassociated with inherited eye pathologies such as SnowflakeVitreoretinal Degeneration (SVD) and Lebers CongenitalAmaurosis (LCA16). Based on the current evidence, mutationsimplicated in channelopathies have the potential to be usedfor genetic testing to diagnose blindness due to Kir7.1.

Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of pig-mented cells of neuro-ectodermal origin. These cells are situatedbetween the neuroretina and the choroids. The apical membraneof the polarized RPE cells faces the photoreceptor (PR) outer seg-ments. The basolateral membrane faces Bruch’s membrane,which separates the RPE from the fenestrated endothelium of thechoriocapillaris (Figs. 1 and 2a).1-4 RPE is a hexagonally packed,tight-junction connected, single sheet of post-mitotic cells con-taining pigment granules. Interactions between both the RPEand the PR are essential for visual function.4,5 Without anydirect vascular supply to the PR, RPE cell must manage the

bi-directional flow of molecules and ions in and out of the retina.This includes removing wastes from the retina, and maintainingionic gradients necessary for phototransduction to occur.5-8 Theprimary roles of the RPE are highlighted in Fig. 1: (1) Transportof nutrients, ions, and water, (2) absorption of light and protec-tion against photo oxidation of proteins and phospholipids of theouter segments,9 (3) re-isomerization of all-trans-retinal into 11-cis-retinal, which is a central component of the visual cycle,10-12

(4) phagocytosis of shed photoreceptor membranes,13 and (5)secretion of essential elements for the morphological integrity ofthe retina.14,15 The involvement of various ion channels and trans-porters in these functions (Fig. 1) emphasizes the importance offunctional membrane molecules. Furthermore, analyses of heredi-tary retinal degeneration reveal a strong dependence of RPE onthe photoreceptors and vice versa.16

KC Ions and Channels in RPE Function

In the dark, cGMP-gated cation channels are open in the PRouter segments. The influx of NaC and Ca2C through these chan-nels is counterbalanced by an outflow of KC at the inner segment(Fig. 2C).16 When the retina is illuminated, the cGMP-dependentcation channels in the PR are closed and the KC outflow at theinner segment is severely reduced (Fig. 2D). Simultaneously,NaC/KC-ATPase at the inner segment takes up KC into the PR.This reduces the KC concentration in the subretinal space from5 mM to 2 mM.17 Decrease in the subretinal KC concentrationresults in the left shift of the KC electrochemical equilibriumpotential reflecting an increase in KC current, in addition to theslowing of the apical Na/KC-ATPase.18,19 The combined effect isthus a net efflux of KC into the subretinal space (Fig. 2B), and anincrease of the subretinal KC concentration back to normal val-ues.20 Several different subtypes of inwardly rectifying KC channelsubunits have been identified in the RPE.21-25 Reverse transcrip-tion-polymerase chain reaction (RT-PCR) analysis indicated thatin addition to Kir7.1, 7 other Kir channel subunits (Kir1.1, Kir2.1,Kir2.2, Kir3.1, Kir3.4, Kir4.2 and Kir6.1) are expressed in nativehuman RPE. However, transcripts of these channels are signifi-cantly less abundant than Kir7.1 transcripts.25 While Kir channelsubtypes Kir4.1

22,23 and Kir6.2,21 are detected in rat RPE, they

were not detected in human RPE.25 The functional significanceof the Kir subtypes beyond Kir7.1 detected in human RPE isunclear. Determining the subcellular location of the other identi-fied Kir channel subunits may be an important key to understandtheir physiological function in the RPE. In a blocker sensitivitystudy on intact toad RPE-choroid, millimolar concentrations of

*Correspondence to: Mohit Kumar; Email: kumarmohit@yahoo.com; Bikash RPattnaik; Email: bikashp@pediatrics.wisc.eduSubmitted: 08/18/2014; Revised: 08/25/2014; Accepted: 08/27/2014http://dx.doi.org/10.4161/19336950.2014.959809

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REVIEW

barium were necessary to block the RPE apical membrane KC

conductance,26 consistent with Kir7.1 properties.24 Moreover,Snowflake Vitreoretinal Degeneration (SVD) and Leber Congeni-tal Amaurosis (LCA) arise from mutations in Kir7.1 channel sug-gesting this ion channel plays an important role in RPE healthand disease (Fig. 4). This underscores the importance of evaluat-ing the basic structure, synthesis, transport signals, function, regu-lation, and channelopathies associated with Kir7.1 channel.

Kir7.1

Kir7.1 ion channel is the most recently identified member ofthe inwardly rectifying KC channel family. It was independentlyidentified by 3 groups in the secretory epithelial cells of the cho-roid plexus,27 central neuron cells28 and in the small intestine.29

The primary structure of Kir channels consists a common motifof 2 membrane spanning domains (TM1 and TM2) linked by anextracellular pore forming region (H5) and cytoplasmic amino(NH2-) and carboxy (COOH-) terminal domains. This topologyis common to all Kir channels.

27 The H5 region serves as the ‘ionselectivity filter’ that shows the signature sequence T-X-G-Y-G.Unlike the Kv channels, lack of S4 helix in the Kir channels (apart of the voltage sensor region S1-S4 helices), makes theminsensitive to the membrane voltage changes. Inward rectifica-tion, a defining feature of the Kir channels, is not an intrinsicfunction of the channel protein, but is arises when intracellularcations like polyamines and Mg2C block outward KC flow.30

The primary structure of 2 transmembrane strands is insufficientto form a complete ion channel, and functional Kir channels aremade up of 4 such subunits in a tetrameric complex.31,32 Thisstoichiometry was confirmed by velocity sedimentation withsucrose density gradients, size exclusion column chromatography,and chemical cross-linking.33

A reference gene sequence (NG016742) spanning 111327–129090 of the original human BAC library clone RP11-174L18,constructed from chromosome 2 of a male blood DNA(AC064852) harbored the Kir7.1 DNA sequence.34 Analysis of thehuman Kir7.1 gene (KCNJ13) organization revealed 3 exons, 2introns, and a novel alternative 50 splice site in exon 2. Kir7.1cDNA is 1080 bp long consisting of a single ORF (open readingframe) encoding a protein of 360 amino acid residues. Kir7.1 is themost divergent in amino acid sequence among the Kir members. Itssequence shares »50% homology at the protein level with theclosely related Kir4.1 and Kir4.2 ion channels. Human RPE showedthat the alternative usage of 2 competing 50 splice sites in exon 2gives rise to transcripts encoding either full-length Kir7.1 orKir7.1S, which is predicted to encode a truncated protein.35

Kir7.1S transcript lacks 236 bp of the 476 bp exon 2 leading to atranslation frame shift and introduction of stop codon.27 As aresult, the predicted protein (94 aa) is truncated with a shorter C-terminus and has a stretch of 19 aa residues that are not present inKir7.1. Naturally occurring transcript variants of Kir7.1 form trun-cated proteins which may co-assemble with Kir7.1 channel subunitsand alter Kir7.1 channel properties or surface expression. However,electrophysiology studies in Xenopus oocytes indicated that Kir7.1Sdoes not interact functionally with full length Kir7.1 protein.

One of the key features of Kir7.1 sequence is the presence ofphophorylation sites (discussed below) in the cytoplasmic regionsthat may play a role in the intracellular trafficking of the protein.Another noteworthy sequence feature is the presence of an N-glycosylation site at position 95 in the extracellular M1-H5region, which has been shown to control the open probability ofanother inward rectifier Kir1.1.

36 Kir7.1 channel functions as atetramer; however there is no report of heteromeric assembly ofKir7.1. Immunoreactivity studies demonstrated the presence ofNaC, KC-ATPase on the apical membrane of RPE and choroidplexus32 and suggested the possible functional coupling withKir7.1 in KC recycling (Fig. 2).

Biophysical Properties of Kir7.1

Kir7.1 exhibits unusual current-voltage dependence in the slopeof the I-V curve as current increases with negative potential com-pared to other inward rectifier channels.28 Heterologous expressionof Kir7.1 shows extremely small»50fS single channel conductanceand an increase in outward current. Negative shift of the mem-brane potential due mainly to the left shift of electrochemical equi-librium potential upon reduction of extracellular KC from 5 mMto 2 mM results in an increase in net potassium current (Fig. 2B).An unusual feature of Kir7.1 channel is the large RbC-to-KC con-ductance ratio of the inward current.37 The sensitivity of the chan-nel to Ba2C and CsC is very low with IC50 values of »1 mM and»10 mM respectively, which are >10 times greater than other Kir

channels.38 Inward rectification of Kir7.1 is weak when extracellu-lar [KC] is low and when extracellular [KC] is high the channelbecomes strongly inwardly rectifying (Fig. 2B). This is alsoreflected by the crossover of the 2 I-V curves at positive potential.The resting membrane potential of human RPE cells is ¡74 mV

Figure 1. RPE cell functions crucial for vision. In combination with NaC/ATPase/KC pump, Kir7.1 channels regulate the direction of fluid transportacross the RPE, subretinal space volume, and help to maintain KC

homeostasis around the photoreceptor outer-segment.

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whereas the zero-current potential ofinward rectifier channel is ¡71 §2.1 for 5 mM [KC]o and ¡104 §3.2 for 1 mM [KC]o.39

There is no evidence of Kir7.1channel interaction with intracellularmolecules like Mg2C and polyaminesat the lining of the channel pore;such interactions generally block KC

permeation. The competitive block-age of Kir channels by Mg2C andpolyamines is in general crucial forthe control of the magnitude of out-ward current. However, the Kir7.1conductance was not affected bychanges in either external or internaldivalent cation concentrations. Thisis attributed to the presence of M125(Arg in all other Kir channels) in thepore region. When M125 is replacedwith R125, channel conductance andBa2C sensitivity increases »20 foldand»10 fold respectively.20

Identification of Signalsin Kir7.1 Protein forMembrane Targeting

As noted by Pattnaik et al.40

mutation of arginine at position 162to bulky tryptophan in Kir7.1 doesnot traffic to the plasma membraneduring heterologous expression inCHO-K1 cells. The authors hypoth-esized that mutant R162W preventsPIP2 binding, which may then pre-vent translation of the cytoplasmicdomain toward the membrane. Thisstudy further confirms that phos-phoinositides like PIP2 may play acritical role in trafficking of the ionchannels to the membrane. Con-versely, Zhang et al.41 reported thatR162W mutant Kir7.1 channel suc-cessfully localized to both the oocyteand Madin Darby canine kidney (MDCK) cell membrane. Oocytesystem is a robust system employed for expression of membraneproteins so merely having transmembrane domain may drive theprotein to the membrane. On the other hand MDCK cells arewidely used for the study of epithelial transport with caution.Given these conflicting data, directed trafficking of Kir7.1 channelsto the membrane is poorly studied, and the molecular interactionsby R162 residue within the C-terminal remains to be established.42

In eukaryotic cells, the machinery dedicated to protein foldingand secretion is highly conserved. Recent evidence indicated thatintrinsic sequence motifs are present in the ion channel proteins

that control surface expression by regulating distinct intracellulartrafficking steps.43 Since there are few to no reports regarding sig-nals responsible for transport of Kir7.1 protein from the endo-plasmic reticulum (ER) to plasma membrane either directly orthrough the Golgi complex, we will consider potential signals forKir7.1 protein localization based on the data available for otherKir ion channels.

Phophorylation sitesYoo et al.38 reported that phosphorylation is essential for cell

surface expression of Kir1.1. Six phosphorylation sites are present

Figure 2. (A) Normal retinal architecture in showing the photoreceptors (PR), subretinal space (SRS), the ret-inal pigmented epithelium (RPE), the Bruch membrane (BM), and the choroidal vascular network. (B) Repre-sentative Kir7.1 current recording showing hyperpolarizing shift in membrane potential on switchingextracellular KC concentration from 5 mM to 2 mM (Modified from Doring et al. 1998). Gray rectangle rep-resents current activation around the resting membrane potential of RPE cells in response to change inextracellular KC. (C) Schematic of rod PR: The dark current circulates between the inner and outer segments.The cytoplasmic concentration of cGMP is high, and maintains the cGMP-gated channels in an open stateand allows a steady inward current (dark current) to depolarize the photoreceptor. In dark, the KC concen-tration of the subretinal space is approximately 5 mM. Adjacent RPE cell mechanisms active during dark areshown on the right with simplified representation of ion transport. (D) On light exposure, fewer KC ionsleave the photoreceptors and the KC concentration in the subretinal space decreases from 5 to 2 mM.Kir7.1 channels in the apical membrane of RPE recycle efflux KC. Additionally, the movement of water andCl- is shown. Abbreviations are: Ap – apical, Ba – Basolateral, AQP – aquaporine, KCNQ – M-type KC channel,NKCC – NaC/KC/2Cl transporter, and ATPase – NaC/KC/ATPase pump. The thickness and length of thearrows depicts the relative magnitude of the driving forces.

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in Kir7.1 protein, Casein Kinase II (T321 and T337), PKA(S287) and PKC (S14, S169 and S201) sensitive sites are shownas red filled squares in Fig. 4. Substitution of Casein Kinase II(T321 and T337) and PKA (S287) sites with alanine did notaffect the plasma membrane localization of Kir7.1 protein,43

however the role of other phosphorylation sites remains elusive.

Length of proteinTateno et al.43 demonstrated that the C-terminus length is a

critical determinant for the plasma membrane localization ofKir7.1 protein. They showed that 1–54 aa residues of N-terminushad no effect on the protein transport to the plasma membrane,but reducing the length of the C-terminus to 166 aa residuesfrom the original 204 aa residues, eliminated membrane targeting.Kir7.1 protein truncated at the C-terminus (323–360aa) co-local-ized with calreticulin, an ER marker, suggested that loss of surfaceexpression of deletion mutants is due to a defect in ER exit.

From ER to golgi complexExport from ER to the Golgi complex is a key event in intra-

cellular traffic of proteins. N- and C- terminal diacidic motifsconsisting of 2 acidic amino acid residues separated by any otheramino acid such as DXE44 or DXD45 render selective ER export,most likely binding to COPII (Coat Protein II) complexes

(Fig. 3).44,45 Based on these findings, we identified 3 suchdiacidic motifs in Kir7.1 protein. The first 7 aa residue longstretch (84–90 aa) is located post TM1, and another 2 (211–213aa and 338–340 aa) are present in the C-terminus. These areshown as purple diamonds in Fig. 4. The export signals found inKir7.1 protein share the presence of one or more diacidic motifwith the Kir1 (VLSEVDETD), Kir2 (FCYENE), Kir3.2(DQDVESPV and ELETEEEE), and Kir3.4 (NQDMEIG) sub-families. However, Tateno et al. showed that deletion of 37 C-terminus residues that included the diacidic motif (338–340 aa)did not affect localization to the cell surface. In addition, as thereare many membrane proteins without diacidic motif that areexported from the ER efficiently, the diacidic motif cannot bethe only signal for export from the ER.

RXR, a known ER retention signal, ensures that incorrectlyassembled and improperly regulated channel proteins do notreach the cell surface. The RXR motif is present in the N-termi-nal of Kir7.1 protein at position 16–19 shown in green boxes(Fig. 4). Scaffold proteins like PDZ domain-containing proteinsare important for protein complex assembly and may associatewith the ion channel proteins early in the secretory pathway,which can facilitate or inhibit ER to Golgi transport.46 Kir ionchannels contain the PDZ binding motif (-S/T-X-V/I/M-COOH) for sorting and cluster formation of membrane proteins,but this motif is absent from the C-terminal of Kir7.1 protein.

43

Post-golgi traffickingThe presence of YXXF (X is any amino acid and F being a

bulky and hydrophobic residue) or di-leucine motifs favor cla-thrin coated vesicles.47 Di-lysine motifs in the C-terminal bindto COPI (Fig. 4).48 Sequence analysis of Kir7.1 protein revealedthe presence of YSHI (244–247 aa) in the C-terminal that resem-bles the consensus sequence YXXF signal shown as blue dia-monds in Fig. 4. The second sorting motif, the di-leucine motif(LL) was also detected in Kir7.1 protein sequence shown as bluedimonds in Fig. 4. One di-leucine motif is present in the N-ter-minal (12–13 aa), 2 are present in transmembrane domain 2(137–138aa and 143–144 aa) and 2 are present in the C-terminal(256–257 aa and 302–303 aa).

It is intriguing that in spite of both tyrosine-based and di-leucine-based peptide sequences classified as basolateral sortingsignals49 (Fig. 3), Kir7.1 is trafficked to the RPE apical processes?Two possible scenarios may provide an explanation. In the firstscenario, RPE cannot recognize basolateral information in Kir7.1as it does in N-CAM or EMMPRIN. In the second scenario,transcytosis may solve this puzzle as it does in case of Influenzahemagglutinin (HA) (Fig. 3). HA is delivered to the apical sur-face via an indirect pathway, first being delivered to the basolat-eral surface, and then to the apical plasma membrane viatranscytosis in RPE.3 Alternately, Kir7.1 harbor possible uniquesignals for apical sorting. Schuck and Simons42 reported that api-cal sorting motifs are localized in the extracellular or transmem-brane domains of proteins, in contrast to the cytoplasm-orientedbasolateral sorting motif. Apical sorting signals are much morediverse than basolateral signals and consist of post-translationalmodifications, rather than distinct peptide sequences. Perhaps

Figure 3. Proposed model for targeted protein secretion from the ER-Golgi secretory pathway. Proteins containing apical sorting signals(orange) and basolateral sorting signals (black) are delivered to Golgi.Upon entering the Golgi apparatus, proteins progress to the TGN andsorting occurs. In pathway A, apical signals (glycans, GPI linkages) inter-act with specific cellular machinery, e.g., apical targeting receptors, lipidrafts, resulting in packaging into apical vesicles. These vesicles can thentraffic directly to the apical plasma membrane (PM). Alternatively, tar-geted protein may first traffic to the basolateral PM (Pathway B). Basallytrafficked proteins are subsequently redirected to the apical PM viatranscytosis, either directly (pathway C), or via endosomes (pathway D).Proteins in the endosomal pathway are subsequently trafficked to theapical PM. Abbreviations are: ER – Endoplasmic reticulum, COP – CoatProtein, AP – Associate protein, CL – Clathrin, TGN – Trans golgi network.

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the most extensively studied of suchmodifications are N- and O-linked gly-cans (Fig. 3). D€oring et al.27 reportedan N-glycosylation site at position 95 inthe extracellular M1-H5 region inKir7.1 within a consensus sequence ofN-X-T/S. We have predicted 2 morepotential N-glycosylation sites at posi-tion 261 and 335 in Kir7.1.

Taken together, the trafficking ofKir7.1 to apical membrane of RPE mayemploy any of the speculated signals dis-cussed above or an altogether differentmechanism. Clearly, this provides bothan opportunity to understand biology ofdisease and a challenge to study the traf-fic route of Kir7.1 and other like pro-teins in RPE.

Functional Relevance of Kir7.1and RPE

Properly folded Kir7.1 proteinreaches the apical membrane of RPEwhere it maintains the ionic homeostasisof the subretinal space by epithelialtransport of ions, metabolites, and fluidbetween the subretinal space and thechoroid.35 Illumination of the retina,resulting in a reduction of the KC con-centration in the subretinal space from5 mM to 2 mM,50 accompanied with an increase in the volumeof the subretinal space. The decrease in the subretinal KC concen-tration hyperpolarizes the apical RPE membrane and contributesto tight regulation of subretinal space KC homeostasis coupledwith NaC-KC-ATPase in active state.51 The weak inwardly recti-fying property of Kir7.1 provides a compensatory pathway tosupport the RPE response to either rapidly decrease or increasethe subretinal KC concentration. La Cour51 reported thatdecreases in subretinal KC concentration increase the KC perme-ability of RPE apical membrane, promoting KC efflux. Similarly,Shimura et al.24 reported that decreases in extracellular KC con-centration increases the slope conductance of Kir7.1. NaC gradi-ent also causes the active transport of Cl¡ into the cell across theapical membrane via of NaC-KC-Cl¡ co-transporter activity.Nevertheless, the decrease in the subretinal KC concentrationdecreases uptake of Cl¡ and results in the hyper polarization ofthe basolateral membrane to decrease Cl¡ transport from thecell.52 Similarly, light-dependent hyper polarization of the apicalmembrane reduces the transport rate of apical NaC-HCO3

¡ co-transporter and thus causes an intracellular acidification by»0.35 pH units that facilitates Cl¡ transport through the baso-lateral membrane from subretinal space to the choroid direction.This Cl¡ transport across the cell also supports movement ofwater in the same direction which holds the retina in the

proximity of RPE via suction and prevents retinal detachment.Intracellular acidification may also increase KC efflux andenhance the buffering capacity of subretinal space.53

Functional Regulation of Kir7.1

Many cytoplasmic metabolites that interact with the intrinsicamino acid residues have been reported to modulate the Kir7.1channel. Zhang et al.54 showed that renal Kir7.1 ion channel isregulated by cAMP dependent protein kinase A (PKA) and pro-tein kinase C (PKC). They showed that an increase in Kir7.1 cur-rent is observed with increasing intracellular cAMP concentration,and is suppressed by the mutating the sole PKA site (S287). Thesame study also demonstrated that mutation of PKC site S201inhibits Kir7.1 channel, yet 2 other PKC sites (S14 and S169) donot affect the sensitivity of Kir7.1 channel to PKC.

Both extracellular pH (pH)o and intracellular pH (pH)i regu-late the function of the Kir7.1 channel. While Kir7.1 conductanceis relatively independent of pHo in the range from 6.5 to 9.0, it isstrongly inhibited by further lowering the pHo below 6.0.53-55

On the other hand, Kir7.1 conductance showed a biphasicresponse to the pHi.

55 Acidification of the cytoplasm from 7.2 to6.8 activates the Kir7.1 current and further lowering the pHi to6.0 or 5.5 results in a transient activation followed by inhibition.

Figure 4. Kir7.1 membrane topology. Localization of consensus sites for phophorylation by CaseinKinase II (T321 and T337), PKA (S287) and PKC (S14, S169 and S201) shown in red filled squares, as wellas various protein trafficking signals (green boxes: ER retention signal; purple diamonds: diacidicmotif; blue diamonds: dileucine motif). LCA and SVD mutation locations are also shown as filled circlesindicated by arrows. Localization of mutations on the topological image is based on the TOPO2 pro-gram. TM – transmembrane.

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In contrast, alkalization of the cytoplasm causes a reversible andrapid inhibition.53,55 Histidine at position 26 appears to act asthe pH sensor of Kir7.1 since mutation of H26 to alanine or argi-nine affects both proton-induced activation, as well as proton-induced inhibition.55

One of the best characterized lipid modulators of Kir channelactivity is membrane PIP2 (phosphatidylinositol 4, 5-bisphos-phate).56-60 PIP2 is found principally in the cytoplasmic leaflet ofthe plasma membrane that comprises »1 % of plasma membranephospholipids.57 Membrane PIP2 abundance is a dynamic entityand is precisely controlled by lipid kinases, phospholipases, andphosphatases. Kir7.1 channels are inhibited upon depletion ofmembrane (PIP2).58 The apical membrane of the RPE cell is alsothe site of many G-protein coupled receptors (GPCR), includinga1-adrenergic, muscarinic, and P2Y2. These receptors are linkedto Phospholipase C signaling which serves to hydrolyze mem-brane-bound PIP2. Numerous studies of electrophysiological, bio-chemical and molecular simulation collectively suggest that PIP2activates Kir channels directly with variable sensitivity by bindingto positively charged residues in the C-terminal cytoplasmic “hotspot.”61 This hot spot in Kir7.1 is located at aa 151–170 residuesat the beginning of the C-terminal cytoplasmic domain and con-tains a cluster of basic residues (R or K). The R162W mutationwithin the ‘hotspot’ in the KCNJ13 gene converts a basic arginineresidue to a bulky tryptophan and is associated with SnowflakeVitreoretinal Degeneration (SVD). Recently, it was inferred thatR162W alters the Kir7.1 channel-PIP2 interaction.40,41

Channelopathies Associated with Kir7.1

Genetic alterations in Kir7.1 underlie hereditary ion channeldiseases known as channelopathies, and are primarily associatedwith congenital blindness.

Snowflake Vitreoretinal Degeneration (SVD, MIM 193230)is a developmental and progressive hereditary eye disorder thataffects the retina and vitreous. The prevalence of SVD is low(<1/1000000), however the disorder has been described in sev-eral families.62 SVD is one of the vitreoretinal degenerationscharacterized by early onset cataract, congenital liquefaction ofthe vitreous humor, and abnormalities of the interface betweenthe vitreous and retina leading to increased risk of retinal detach-ment and the presence of small yellow-white crystalline depositsin the peripheral retina.63 Electrophysiological studies reveal anelevated dark adaptation and reduced scotopic waves. Jiao et al.64

identified the chromosomal location of the gene linked tomarkers in a region of chromosome 2q36 defined by D2S2158and D2S2202. Hejtmancik et al.65 reported that affected mem-bers were heterozygous for a missense mutation in the Kir7.1(KCNJ13), an arg162-to-trp substitution (R162W) that arosefrom c.484 C-T transition. This mutation resulted in a non-functional channel in heterologous expression studies and ren-dered wild-type channel non-functional through a dominantnegative mechanism. Some of the variable phenotypes in theseheterologous expression studies may be due to the specific expres-sion system.66,67 Functionally, R162W mutant Kir7.1 channel

may result in the premature depolarization of the RPE cells,resulting in Ca2C overload and cell death.40,41

The mechanism involved in SVD pathology is still unknown.In principle, the lack of channel activity could be caused byimpaired surface expression, which can result from defects in anumber of processes, including protein folding, posttranslationalmodification, assembly, and membrane trafficking, or ER reten-tion and degradation. Zhang et al.41 showed that mutant Kir7.1protein is localized to the plasma membrane when over expressedin Xenopus oocytes or MDCK cells. Thus the loss of function inmutant Kir7.1 channels is not due to impaired processing or traf-ficking to the plasma membrane, but, rather, some other mecha-nism, such as suppressed channel activity. We previouslysuggested that there may have been a significant loss of the wildtype channel function due to oligomerization with mutant subu-nits which may affect the assembly of functional tetrameric com-plexes able to pass the ER quality control machinery.40 Structuralmodeling of wild type and mutant Kir7.1 based on the homolo-gous structure of mammalian Kir channel suggested that thismutation causes major structural changes in the vicinity of “hotspot” in KCNJ13 which may be involved in channel activation byPIP2.58 The cytoplasmic domain can influence channel selectivityeither due to a non-selective channel or due to the lack of regula-tion by PIP2 that will result in non-functional Kir7.1 channel.

Leber Congenital Amaurosis (LCA, MIM 204000) is anotherloss-of-function homozygous mutation in the KCNJ13. LCA israre, hereditary disorder that leads to retinal dysfunction andvisual impairment from the first year of life.68 This suggests bothimpaired retinal development and severe retinal degeneration,which involves both rod and cone photoreceptor pathways. LCAis inherited in an autosomal recessive manner and is caused by 16different gene mutations, including KCNJ13.67,68 LCA accountsfor the blindness of more than 20% of those blind from birth.

Sergouniotis et al.67 recently identified a single loss-of-func-tion homozygous variant c.496C>T (p.R166X) in KCNJ13.R166X results in an early stop codon and produces a truncatedKir7.1 protein lacking most of the cytoplasmic C-terminalsequence. The absence of C-terminal sequence in the mutantwould likely lead to mislocalization as discussed above. Anotherchange c.380A>G (p.Q117R) was observed in a patient of whiteEuropean ancestry with early onset retinal dystrophy and heavilypigmented fundus.67 Q117 is located within the conserved P-loop domain or selectivity loop. Thus, this mutation is predictedto give rise to either altered KC selectivity or complete loss of KC

permeability by the channel. Another variation c.485G>A (p.R162Q) was identified in the heterozygous state in male patientsof Turkish ancestry. While R162 is the site of defect in SVDpatients, R162Q had no phenotype as reported for LCA screen-ing. There are also several other genetic variations withinKCNJ13 gene reported by Sergouniotis et al.67 but their func-tional characteristics are still to be explored. Further studies arenecessary to investigate the potential loss or gain-of-functionunderlying pathophysiology of LCA due to Kir7.1 channelop-athy. Moreover, it would be interesting to study the electrophysi-ological outcome of various genetic variations on RPE cellfunction.

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Open Questions

Despite the progress attained in our understanding of theKir7.1 channelopathy, several questions remain unanswered. Whydoes Kir7.1 channelopathy have only a retinal phenotype?69

Within the eye Kir7.1 is also localized to other cell types, yet thephenotype is specific to RPE. How does an RPE apical membraneion-channel defect generate ERG phenotype, signature of retinaelectrical output in response to light exposure? Kir7.1 channels arealso regulated by membrane cholesterol so future studies shouldaddress the role of Kir7.1 in age-related blindness. To addressKir7.1 channel biology, future studies could test the effects of chan-nel knock down (by RNA interference) or channel mutation. Per-taining to Kir7.1 molecule, mutations might alter ion channelfunction either due to defective trafficking to the membrane, defec-tive channel opening and closing, and/or loss of selectivity for thespecific ion. Molecular understanding of the functional abnormali-ties associated with a specific mutation need to be established forour understanding of the disease pathology. As an example, amutation of residue 162 to tryptophan results in SVD, whereas inscreening for LCA genotype, a mutation to glutamine at the sameposition is tolerated. Severity in the outcome of the mutation isperhaps a direct measure of severe pathological consequence.Although ion-channel research mostly builds on studies using het-erologous expression, stem cell approaches have a remarkable

potential, as well. Successful reprogramming of patient fibroblaststo retina iPSCs for disease modeling and drug testing is promis-ing.70-73 iPS cells will be a key for both LCA and SVD modeling,drug discovery and cell therapies using clinically-relevant patientdonor samples. When successful, there is a chance to circumventKir7.1 channelopathy through drugs or gene or cell therapy.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

Our gratitude goes to Laura Hogan for her editorial assistanceand Aparna Lakkaraju for her comments.

Funding

Rebecca Meyer Brown Professorship of the McPherson EyeResearch Institute (Retina Research Foundation), UW-Depart-ment of Pediatrics, School of Medicine and Public Healthresearch grant, UW-Foundation. The project described was sup-ported by the Clinical and Translational Science Award (CTSA)program, through the NIH National Center for AdvancingTranslational Sciences (NCATS), grant UL1TR000427. Thecontent is solely the responsibility of the authors and does notnecessarily represent the official views of the NIH.

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