Cloning and functional expression of human retinal Kir2.4, a pH-sensitive inwardly rectifying K+ channel

Bret A. Hughes1,2, Gyanendra Kumar1, Yukun Yuan1, Anuradha Swaminathan1, Denise Yan1, Ashish Sharma1, Lisa Plumley4, Teresa L. Yang-Feng4, and Anand Swaroop1,3

Departments of 1 Ophthalmology and Visual Sciences, 2 Physiology, and 3 Human Genetics, University of Michigan, Ann Arbor, Michigan 48105; and 4 Department of Genetics, Yale University, New Haven, Connecticut 06520


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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To identify novel potassium channel genes expressed in the retina, we screened a human retina cDNA library with an EST sequence showing partial homology to inwardly rectifying potassium (Kir) channel genes. The isolated cDNA yielded a 2,961-base pair sequence with the predicted open reading frame showing strong homology to the rat Kir2.4 (rKir2.4). Northern analysis of mRNA from human and bovine tissues showed preferential expression of Kir2.4 in the neural retina. In situ hybridization to sections of monkey retina detected Kir2.4 transcript in most retinal neurons. Somatic hybridization analysis and dual-color in situ hybridization to metaphase chromosomes mapped Kir2.4 to human chromosome 19 q13.1-q13.3. Expression of human Kir2.4 cRNA in Xenopus oocytes generated strong, inwardly rectifying K+ currents that were enhanced by extracellular alkalinization. We conclude that human Kir2.4 encodes an inwardly rectifying K+ channel that is preferentially expressed in the neural retina and that is sensitive to physiological changes in extracellular pH.

potassium channels; nucleotide sequence; chromosomal localization


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INWARDLY RECTIFYING K+ channels (Kir) comprise a diverse subset of K+-selective channels that preferentially conduct K+ movement in the inward direction. This widely expressed group of plasma membrane proteins serves both homeostatic and specialized functions. In neurons, Kir channels are important in establishing membrane excitability and shaping action potentials, whereas in other cell types, such as epithelial and glial cells, they help set the membrane potential and play an integral role in ion transport processes.

To date, more than a dozen Kir subunits have been cloned and characterized (for review, see Ref. 22). Although these Kir subunits exhibit structural and functional differences, they share a highly conserved motif consisting of two membrane-spanning domains (M1 and M2) separated by a putative pore region (H5). Kir channels are formed by the assembly of four subunits in the plasma membrane (16) and can comprise either identical or two different types of Kir subunit (12). The ability of different subunits to form heterotetramers may be part of the explanation for the great diversity that exists in the kinetic and conductive properties of native Kir channels found in various cells and tissues.

Electrophysiological studies in cells isolated from the vertebrate retina have identified inwardly rectifying K+ currents in horizontal cells (44, 50), Müller cells (21, 30), and the retinal pigment epithelium (RPE) (20). Relatively little is known, however, about the molecular identity of the Kir subunits that make up the underlying conductances. To date, the only Kir subunit for which expression in the neural retina has been demonstrated is Kir4.1, a subunit originally cloned from rat brain (45) and later localized by in situ hybridization and immunohistochemistry to Müller cells (21) and the RPE (27). Functional differences between the cloned Kir4.1 channel and the inwardly rectifying K+ conductance present in horizontal cells suggest that other Kir subunits may be expressed in the retina.

Here, we report the cloning of the cDNA for an inwardly rectifying K+ channel subunit, human Kir2.4 (hKir2.4), from a human retinal cDNA library. We have demonstrated that transcripts for hKir2.4 are preferentially expressed in the neural retina and are present in most retinal neurons, and that its gene is localized on human chromosome 19 at q13.1-q13.3. We also have shown that expression of hKir2.4 cRNA in Xenopus oocytes generates a K+ conductance exhibiting strong inward rectification and sensitivity to extracellular pH. Preliminary accounts of this work have been presented in abstract form (25, 26).


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Isolation of the cDNAs and Sequence Analysis

Methods used for routine recombinant DNA analysis were essentially as described in laboratory manuals (3, 35). The oligonucleotide primers used for sequencing and PCR experiments were synthesized by Genosys (Woodlands, TX). The Expressed Sequence Tag (EST) database was searched using BLAST 2.0 software (2) for sequences derived from human retinal cDNA libraries that showed homology to Kir channel subunits. The cDNA clone (I.M.A.G.E. clone no. 586857) for one of the identified ESTs (AA504908) was obtained from the repository of American Type Culture Collection (Bethesda, MD). A 1-kb fragment (EcoR I-EcoR I) representing the 3'-untranslated region of the EST was used as a probe to screen a human adult retina directional cDNA library (42) in Charon BS(-) phage vector in LE392 Escherichia coli cells. Filter lifts were hybridized overnight at 65°C in Church's buffer (0.25 M Na-phosphate buffer, pH 7.2, and 5% SDS) and washed at 55°C in 0.1× saline sodium citrate (SSC) and 0.1% SDS. Phage DNA was prepared from tertiary screened positive clones and then converted to plasmid cDNA by digestion with Not I, recircularization by T4 DNA ligase, and amplification in E. coli DH5alpha cells. cDNA clones were characterized by restriction analysis and sequencing of both strands using a cycle sequencing kit (Amersham, Arlington Heights, IL) in the presence of T3, M13, and other nested primers. The complete sequencing of the EST clone was also accomplished using a similar approach. The cDNA sequences of the clones, as well as their predicted protein sequences, were analyzed for homology to other Kir channel sequences using Lasergene software (DNASTAR, Madison, WI).

Northern Analysis

For Northern analysis, we obtained a multiple tissue RNA blot from Clontech (Palo Alto, CA) containing 2 µg of poly(A+) human RNA per lane and prepared a second blot by loading 4 µg of poly(A+) RNA from adult human retina in one lane and 50 µg of total RNA from human fetal brain, placenta, fetus plus placenta, lung, thymus, and HeLa cells in the others. A third blot was prepared by loading 4 µg of poly(A+) RNA from bovine retina and RPE sheets. Poly(A+) RNA was obtained by extracting total RNA from neural retina or washed, dispase-dissociated RPE sheets with TRIzol (Life Technologies, Rockville, MD) and then applying it to an oligo(dT) column (Pharmacia, Piscataway, New Jersey). The blots were sequentially hybridized with a 1-kb probe (EcoR I-EcoR I fragment of EST AA504908) containing the 3'-untranslated region (nucleotides 1,962-2,961) of hKir2.4 cDNA and a beta -actin control probe at 65° in Express-Hyb hybridizing solution (Clontech). The hybridized blots were sequentially washed with 1× SSC solution with 0.1% SDS and 0.1× SSC with 0.1% SDS.

In Situ Hybridization

Adult rhesus monkey and bovine eyes were obtained within 1 h of death and transported to the laboratory on ice. After the anterior segment was dissected away, the vitreous was carefully removed by blunt dissection. Eyecups were sectioned into quarters and placed in 4% paraformaldehyde at 4°C overnight. Tissue pieces were then rinsed in 0.1 M phosphate buffer and cryoprotected as described elsewhere (4). Sections (13 µm) were cut at -20°C, transferred to 3-aminopropyltriethoxysilane-subbed slides (Sigma Chemical, St. Louis, MO), and stored at -70°C until use.

In situ hybridization to monkey and bovine retinal sections was carried out using digoxigenin-labeled cRNA probes and a commercially available DIG Nucleic Acid Detection Kit (Boehringer Mannheim, Indianapolis, IN). For riboprobes, 700-bp Xho I-EcoR I and 1-kb EcoR I-EcoR I fragments of the EST plasmid (AA504908), representing the COOH-terminal coding region (nucleotides 1,321-1,962) and the 3'-untranslated region of hKir2.4 (nucleotides 1,962-2,961), respectively, were cloned into Bluescript vectors (pKS+ or pSK+). Sense and antisense riboprobes were generated from these plasmids in the presence of 11-digoxigenin UTP (Boehringer Mannheim) using either T3 or T7 RNA polymerase.

Chromosomal Localization

For somatic cell hybrid panel analysis, mapping panel no. 2, consisting of 24 human-rodent somatic cell hybrids, was obtained from the National Institute of General Medical Sciences (NIGMS) cell repository. Characterization and human chromosome content of these hybrids are described in detail in the NIGMS catalog. DNA samples were digested with Pst I, separated in 1% agarose gels by electrophoresis, transferred to nylon filters, and hybridized to a 32P-labeled hKir2.4 probe (the entire p17B clone sequence) as previously described (56) to identify human-specific genomic fragments that were then scored in the human-rodent hybrid panel. Regional assignment of the hKir2.4 gene was carried out by the fluorescence in situ hybridization (FISH) of metaphase chromosomes procedure, as previously described (37). Briefly, plasmid containing p17B cDNA was labeled by nick translation with digoxigenin-dUTP and hybridized to human metaphase chromosomes at a concentration of 50 ng/µl. Hybridization signal was detected by rhodamine-conjugated anti-digoxigenin antibody, and chromosomes were counterstained with 4,6-diamino-2-phenylindole. Localization was confirmed by dual-color FISH using p17B and P1 clone RMC19P009 (obtained from Joe W. Gray, University of California, San Francisco), which maps to the distal short arm of chromosome 19. The P1 clone was labeled with biotin-dUTP and detected by fluorescein avidin, whereas the plasmid was labeled with digoxigenin-dUTP and detected by rhodamine-anti-digoxigenin antibody.

Expression in Oocytes

For the construction of a transcription plasmid, the coding region of hKir2.4 cDNA (nucleotides 395-1,760) was PCR amplified with the Expand High-Fidelity PCR system (Boehringer Mannheim) and subcloned into the polyadenylating transcription vector pBSTA (17) at the Bgl II site using a blunt-end ligation procedure. The resulting plasmid contained the cloned cDNA insert flanked by the 3'- and 5'-untranslated regions of the beta -globin gene and allowed sense-strand cDNA to be transcribed by T7 RNA polymerase. Capped poly(A+) cRNA was synthesized from plasmid cDNA linearized at the Sac I site using a commercially available cRNA capping kit (Ambion, Austin, Texas). cRNA was precipitated in ethanol and redissolved in diethyl pyrocarbonate (DEPC)-treated water.

Electrophysiology

Xenopus laevis oocytes were surgically removed from adult female frogs and defolliculated by incubating clusters of oocytes in 0.2% type IV collagenase (Sigma Chemical) in calcium-free ND96 solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 10 mM HEPES, pH 7.4, 200 µg/ml gentamicin, and 550 µg/ml sodium pyruvate). Healthy-looking stage V-VI oocytes were collected and stored overnight at 18°C in ND96 solution plus 1 mM CaCl2.

Defolliculated oocytes were injected with 0.1-5 ng of capped hKir2.4 cRNA in 50 nl and incubated at 18°C for 1-3 days before electrophysiological experiments. Oocytes injected with the same volume of DEPC-treated water served as controls. Whole cell currents were recorded using the two-electrode voltage-clamp technique (39). Thick-wall borosilicate glass microelectrodes having impedance of 0.5-1.5 MOmega when filled with 3 M KCl were used as voltage-sensing and current-passing electrodes. Signals from the current-passing electrode were amplified with a GeneClamp 500 amplifier (Axon Instruments, Burlingame, CA) and stored on a computer hard disk for later analysis with pCLAMP 6.0 software (Axon Instruments). The standard bath solutions for recording comprised modified ND96 solution containing 96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM 2-(N-morpholino)ethanesulfonic acid (pH 5.5, 6.0, and 6.5), 10 mM HEPES (pH 7.0, 7.4, and 8.0), or 10 mM 3-([1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (pH 8.5 and 9.0). In some experiments, 300 µM niflumic acid was added to block endogenous Ca2+-activated Cl- currents. In experiments investigating the relationship between Kir2.4 current and extracellular K+ concentration, NaCl was replaced with equimolar amounts of KCl. For determination of blocker sensitivity, various amounts of Ba2+ or Cs+ were directly added to 98 mM KCl Ringer.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Identification and Sequence Analysis of hKir2.4

A search of the EST database led to the identification of two overlapping human retinal EST clones (W25800 and AA504908) that showed partial homology with known Kir channel subunits. EST AA504908 was then used as a probe to screen >500,000 plaques of an adult human retina cDNA library, resulting in 13 positive phage clones after tertiary screening. Plasmid clones obtained from five of the positive phage clones were characterized by restriction analysis and nucleotide sequencing. All were found to contain the EST AA504908 sequence in their cDNA inserts, which ranged in size from 1.5 to 2.3 kb. One of the cDNA clones, p17B, contained the largest cDNA insert (2.3 kb) and was completely sequenced. Analysis of the sequencing data indicated that p17B contained the entire coding sequence for a Kir2.4 channel subunit as well as a partial 3' end sequence (Fig. 1A). Partial sequencing of the remaining four clones at the 3' and 5' ends revealed that they were smaller fragments of the same transcript from which p17B was derived. Sequencing of EST AA504908 cDNA revealed that it contained a partial coding sequence that overlapped completely with the 3' end of p17B as well as the complete 3' end sequence that included a large poly(A+) tail (Fig. 1A). The composite sequence of the hKir2.4 cDNA was obtained by assembling the p17B and EST AA504908 sequences using Lasergene software (DNASTAR). The full-length hKir2.4 cDNA was 2,961 bp long, with a 1,308-bp coding region flanked at the 5' and 3' ends by 406- and 1,247-bp untranslated regions, respectively (Fig. 1B).


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Fig. 1.   Structure of human Kir2.4 (hKir2.4) cDNA. A: restriction map of hKir2.4, p17B, and Expressed Sequence Tag (EST) AA504908 cDNA. Boxed region indicates the open reading frame. The restriction enzyme abbreviations are as follows: B, BamH I; Bc, Bcl I; D, Dra I; H, Hind III; N, Nco I; P, Pst I; E, EcoR I; S, Sal I; Sc, Sac II; X, Xho I; and Xm, Xma I. The cloning sites for the cDNA are shown in parentheses. B: complete sequence of hKir2.4 cDNA. The GenBank accession number of this sequence is AF081466. The 436-amino acid open reading frame is shown below the nucleotide sequence in single-letter codes. C: hydropathy plot for hKir2.4 protein. M1 and M2, membrane-spanning domains; H5, pore-forming region.

Analysis of hKir2.4 cDNA indicated that it codes for a 436-amino acid-long protein (Fig. 1B) containing major hydrophobic regions that correspond to two membrane-spanning domains, M1 and M2, and an intervening H5 pore-forming region (Fig. 1C), which are the hallmarks of Kir subunit proteins (22). A computer-assisted search for protein motifs in hKir2.4 revealed that it contains one putative Asn glycosylation site at N193, one cAMP- and cGMP-dependent phosphorylation site at S11, four protein kinase C (PKC) phosphorylation sites at T263, S362, S376, and S422, and one tyrosine kinase phosphorylation site at R240. Comparison of the predicted protein sequence of the clone with that of other Kir channel subunits indicated that it is most closely related to members of the Kir2 subfamily of K+ channels (Fig. 2). Amino acids D175 and E227, which have been shown to be critical for conferring strong inward rectification in all homotetrameric Kir2 channels via the interactions with Mg2+ and polyamines (13), are also conserved in hKir2.4. In addition, hKir2.4 contains amino acids C127, H130, and C159, which correspond to residues in Kir2.3 that confer sensitivity to extracellular pH (8). The presence of these sequence motifs suggests that the assembly of Kir2.4 subunits should give rise to a strong inwardly rectifying potassium channel that is sensitive to changes in extracellular pH (see pH sensitivity).


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Fig. 2.   Comparison of the hKir2.4 protein sequence with that of other Kir2 subunits. M1, M2, and the intervening H5 pore-forming regions are indicated. Open and closed circles denote putative cAMP-/cGMP-dependent and protein kinase C phosphorylation sites, respectively. The asterisk marks a tyrosine kinase phosphorylation site.

In addition to an extracellular pH sensor, Kir2.3 has three amino acids in the NH2 terminus (T53, Y57, M60) that have been shown to be responsible for intracellular pH sensitivity (34). This motif, however, is absent from hKir2.4. Unlike all other Kir2 subunits, the COOH terminus of hKir2.4 lacks an RRESXI domain, which contains both a protein kinase A (PKA) phosphorylation site that is involved in channel gating and a PDZ-binding domain that allows channel clustering (7, 51). Although hKir2.4 has a unique PKA phosphorylation consensus site at S11 that could potentially play a role in channel regulation, the lack of an RRESXI sequence seems to suggest that hKir2.4 might have a subcellular distribution different from that of other members of the Kir2 subfamily.

While these studies were in progress, the rat homolog of hKir2.4, rKir2.4, was identified from a rat brain cDNA library (46). Alignment of hKir2.4 and rKir2.4 protein sequences demonstrated a 92% identity (Fig. 2). The pore-forming region H5 and membrane-spanning domains M1 and M2 of these homologs are identical except for a conserved substitution of A105 in the M1 domain of hKir2.4 with T103 in rKir2.4. There is also a substitution of Q138 in rKir2.4 for H130 in hKir2.4, but it can be inferred from the results of site-directed mutagenesis studies on Kir2.3 that this amino acid difference would not affect pH sensitivity (8). Except for an extra PKC phosphorylation site at S422 and five nonconserved amino acid substitutions in nonfunctional regions, all of the other substitutions are either conserved or similar. Alignment of cDNA sequences reveals that whereas the coding regions of hKir2.4 cDNA and rKir2.4 cDNA are 86.9% identical, their 5'- and 3'-untranslated regions share only 29.1% and 39% sequence identities (data not shown).

Expression Pattern of hKir2.4

The expression of hKir2.4 mRNA was evaluated by Northern blot analysis. RNA blots of several human tissues/cell lines, including adult retina, were hybridized with a 32P-labeled probe, which was generated from a 1-kb fragment representing the untranslated region of hKir2.4 (nucleotides 1,962-2,961) (see Fig. 1). The hKir2.4 probe hybridized to a major transcript at ~3 kb in the retina RNA lane only (Fig. 3, A and B, top), which corresponds to the size of the full-length cDNA clone (2.9 kb). A second signal at ~5 kb was also detected, suggesting the possibility of alternative polyadenylation or splicing. The integrity and the relative amounts of RNA in the total RNA samples were established by the hybridization of the same blot with a 32P-labeled beta -actin probe (Fig. 3, A and B, bottom). To confirm the expression of hKir2.4 in the retina and to examine its expression in the RPE, Northern blots of bovine retinal and RPE poly(A+) RNA were hybridized with the same 1-kb probe for the untranslated region of hKir2.4. A single transcript of ~3.5 kb was detected in retinal but not RPE RNA (Fig. 3C, top). Hybridization of the same blot with the actin probe reflected similar loading of the RNA in both lanes (Fig. 3C, bottom). Together, these results indicate that hKir2.4 is preferentially expressed in the neural retina.


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Fig. 3.   Northern blot analysis showing the hybridization of mRNA from various tissues with a 32P-labeled probe specific for the noncoding region of hKir2.4. A: human RNA blot (top) with lanes containing 4 µg of poly(A+) RNA from human retina or 30-50 µg of total RNA from the human tissues indicated. The arrow indicates the major hybridization band at ~3 kb, and the asterisk marks a second band at ~5 kb. B: Clontech human RNA blot (top), with each lane containing 2 µg of poly(A+) RNA isolated from the human tissues indicated. C: bovine RNA blot with lanes containing 4 µg of poly(A+) RNA from neural retina and isolated retinal pigment epithelium (RPE) sheets. A-C, bottom: hybridization of the same respective blots with a beta -actin probe.

Localization of Kir2.4 Transcripts in the Retina

To determine the cellular distribution of hKir2.4 in the retina, we performed in situ hybridization on embedded adult monkey and bovine retinal sections using sense and antisense probes recognizing the 3'-untranslated region or the COOH-terminal coding sequence. Figure 4 shows a Nomarski photomicrograph of monkey peripheral retina hybridized with antisense probes corresponding to the COOH-terminal coding region of hKir2.4. A strong hybridization signal was observed in the outer nuclear layer, which contains the cell bodies of rod and cone photoreceptors, in the inner nuclear layer, which includes the cell bodies of bipolar, amacrine, horizontal, and Müller cells, and also in some of the cell bodies located in the ganglion cell layer (Fig. 4A). In situ hybridization with the complementary sense probe demonstrated no detectable signal (Fig. 4B). A similar pattern of hybridization was observed with the antisense and sense probes for the 3'-untranslated region of the hKir2.4 in monkey and bovine retina sections, confirming the specificity of hybridization to Kir2.4 transcripts (data not shown). Hence, Kir2.4 transcript appears to be present in most retinal neurons and possibly Müller cells as well.


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Fig. 4.   Localization of Kir2.4 message in monkey retina. In situ hybridization to sections of peripheral monkey retina with antisense (A) and sense (B) digoxigenin-labeled hKir2.4 riboprobes representing the COOH-terminal coding region is shown. Specific expression is seen in the outer nuclear layer (ONL), inner nuclear layer (INL), and ganglion cell layer (GCL). OS, photoreceptor outer segments. A similar pattern of hybridization was also observed for hKir2.4 riboprobes synthesized using the 3'-untranslated region (data not shown).

Chromosomal Localization of the Kir2.4 Gene

Southern blot hybridization of a 32P-labeled hKir2.4 probe with Pst I-digested genomic DNA from a human-rodent somatic cell hybrid panel was carried out to identify human-specific genomic fragments that were scored in the human-rodent hybrid panel (data not shown). Three human-specific fragments of 5.3, 3.2, and 3 kb, a mouse-specific band of 1.35 kb, and a Chinese hamster band of 1.8 kb were detected. All three human-specific fragments showed perfect segregation with human chromosome 19. These results of discordance analysis indicated the segregation of the hKir2.4 gene with the human chromosome 19. Regional assignment of the hKir2.4 gene was accomplished by dual-color FISH experiments using the probe for hKir2.4 and a probe specific for 19p. The results indicated that hKir2.4 maps to human chromosome 19q13.1-q13.3 (Fig. 5).


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Fig. 5.   Chromosomal localization of human Kir2.4. The metaphase chromosome spread indicates the location of Kir2.4 by fluorescence in situ hybridization (FISH) to human chromosome 19q. The human Kir2.4-specific signal (p17B cDNA) is indicated by the arrowheads, and the 19p-specific probe (P1 clone RMCP009) is indicated by the straight lines.

Electrophysiology of Heterologously Expressed hKir2.4 in Xenopus Oocytes

Xenopus oocytes injected with hKir2.4 cRNA generally developed large negative membrane potentials, indicating the expression of functional Kir channels. Oocytes injected with 0.1-10 ng of hKir2.4 cRNA had an average zero-current potential (V0) of -72 ± 26 mV (mean ± SD, n = 115) when recorded 1-3 days later, compared with -44 ± 15 mV (n = 13) for water-injected controls. Figure 6A depicts representative whole cell currents recorded in a water-injected (top) and an hKir2.4 cRNA-injected (bottom) oocyte bathed in 2 mM K+ Ringer. Currents were evoked by a series of voltage steps from a holding potential of 0 mV to voltages ranging from +20 mV to -180 mV. Continuous current-voltage (I-V) relationships, obtained from currents evoked by 4-s voltage ramps in these same oocytes, are shown in Fig. 6B, and the algebraic difference between these two curves is depicted in Fig. 6C. Endogenous currents in the water-injected oocyte were relatively small and exhibited mild outward rectification. In contrast, the currents in the oocyte injected with hKir2.4 cRNA exhibited strong inward rectification at voltages negative to about -90 mV. At larger negative potentials, the inward current exhibited inactivation. Replacement of Na+ in bath with equimolar N-methyl-D-glucamine (NMDG) virtually eliminated this inactivation (not shown), indicating that, like other cloned and native Kir channels (24), hKir2.4 is blocked by extracellular Na+ in a voltage- and concentration-dependent manner. In addition, the oocyte expressing hKir2.4 had small but significant outward currents that peaked at ~20 mV positive to the reversal potential.


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Fig. 6.   Electrophysiology of recombinant hKir2.4. A: family of currents elicited by voltage steps in hKir2.4 cRNA-injected (top) and water-injected (bottom) oocytes. B: steady-state current-voltage (I-V) relationships produced by voltage ramps in the same oocytes as in A. C: difference I-V relationship obtained by a point-by-point subtraction of the data in B.

Dependence on extracellular K+ concentration. As has been well documented for other Kir channels (24, 45), the magnitude of hKir2.4 currents was strongly dependent on the extracellular K+ concentration ([K+]o). Figure 7A shows two families of hKir2.4 currents recorded in the same oocyte superfused first with 2 mM K+ and then with 98 mM K+. Elevation of [K+]o produced more than a 10-fold increase in inward current. Figure 7B summarizes the results of experiments on six hKir2.4 cRNA-injected oocytes, each bathed with a series of [K+]o concentrations (Na+ replacement). Increasing [K+]o produced a marked increase in inward but not outward currents. The I-V relationship also became more linear at higher [K+]o as a result of a decrease in voltage-dependent block by extracellular Na+. As has been found with other strong inwardly rectifying K+ channels (22), the inward slope conductance of hKir2.4 was roughly proportional to the square root of [K+]o (Fig. 7C). A plot of V0 as a function of log [K+] yielded a slope of 49 mV per decade change in [K+] (Fig. 7D), which is somewhat less than the 58 mV predicted by the Nernst equation. Because V0 was not affected by the substitution of extracellular Na+ with NMDG+ (not shown), this lower value does not reflect a significant Na+ permeability of hKir2.4 channels but probably resulted from the presence of endogenous Cl- channels. Hence, we conclude that hKir2.4 is a K+-selective channel.


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Fig. 7.   Dependence of Kir2.4 currents on extracellular K+ concentration ([K+]o). A: voltage-clamped currents recorded from a Xenopus oocyte injected with Kir2.4 cRNA in 2 mM K+ solution (top) and 98 mM K+ solution (bottom). B: whole cell I-V relationships in solutions of increasing [K+]o. Each symbol represents the mean of 5 measurements. C: relationship between slope conductance (g) at -150 mV and [K+]o. Data are from the same oocytes as in B. The smooth curve is the least-squares fit of the data to the equation g = 0.0045 × ([K+]o)0.74 + 0.0047. D: relationship between zero-current potential (V0) and [K+]o. Symbols represent means ± SD (n = 9), and the straight line is the least-squares regression fit of the data with a slope of 49 mV per decade change in [K+]o.

Time course of [K+]o-induced changes in hKir2.4 current. When the solution bathing the oocytes expressing hKir2.4 was switched from 2 mM to 98 mM K+, the inward current typically increased in a biphasic manner (Fig. 8A, top). The first phase, which occurred within the first minute of the solution change, coincided with the change in V0 (Fig. 8A, bottom) and reflected the time course of the change in [K+] outside the oocyte. This was followed by a slower, second phase of current increase that continued for the next 5 min, resulting in nearly a doubling of current (Fig. 8B). This phenomenon appears to be particular to the hKir2.4 channel because in identical experiments on oocytes injected with cRNA encoding Kir7.1, exposure to high [K+]o produced only a single, rapid increase in inward current (data not shown). After the bathing solution was briefly returned to 2 mM K+, the oocyte was exposed to 98 mM K+ a second time. The inward current rapidly increased to a level that was slightly greater than that before the superfusion of 2 mM K+ and then continued to increase slowly. These and similar results in other oocytes are consistent with the idea that exposure to 98 mM K+ triggers an increase in macroscopic hKir2.4 conductance by a mechanism that is not rapidly reversible. Therefore, in subsequent experiments testing the effects of Cs+, Ba2+, or H+, each test measurement was bracketed by measurements in control solution to correct for time-dependent increases in hKir2.4 current.


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Fig. 8.   Time course of hKir2.4 current activation by high [K+]o. A: changes in current (top) and V0 (bottom) plotted as a function of time. Each line represents current evoked by a voltage ramp from -175 to +50 mV. Before superfusion with 98 mM K+, the oocyte had been continuously exposed to 2 mM K+. B: I-V relationships taken at the times indicated by the arrows in A at 0.75, 5.25, and 18 min.

Inhibition by Cs+. Inwardly rectifying K+ channels are generally blocked to various degrees by extracellular Cs+ and Ba2+. To determine the pharmacological sensitivity of the hKir2.4 channel, we measured currents in hKir2.4 cRNA-injected oocytes bathed with various concentrations of blocker ions. Figure 9A shows a family of currents recorded in an oocyte bathed with 100 µM Cs+. Currents were evoked by voltage steps from a holding potential of 0 mV to membrane potentials in the range between +30 and -130 mV. Stepping the membrane potential from 0 mV to more negative potentials produced a rapid block with a time course that was faster than the capacitive transient (<2 ms). Figure 9B summarizes the results of experiments on five oocytes and plots normalized steady-state currents measured in the presence and absence of Cs+ as a function of membrane voltage. The block by Cs+ was strongly voltage dependent, with the fraction of current blocked increasing as the membrane was made more negative. At potentials negative to -100 mV, the fractional block decreased, perhaps as a result of Cs+ permeation through the channel. To quantify the concentration and voltage dependence of Cs+ block, we plotted the ratio of the steady-state currents measured in the presence and absence of Cs+ as a function of Cs+ concentration. Figure 9C shows a family of dose-response curves for the Cs+-induced block of hKir2.4 current measured at various potentials. The symbols represent mean values, and the smooth curves are the least-squares fits of the data to the first-order equation
I<SUB>B</SUB><IT>&cjs0823;  I</IT><SUB>0</SUB><IT>=1−{</IT>[B]<IT>&cjs0823;  </IT>(<IT>K</IT><SUB>D</SUB><IT>+</IT>[B])<IT>}</IT> (1)
where IB/I0 is the fraction of unblocked current in the presence of a particular concentration of blocker ([B]) and KD is the concentration at which the block is half-maximal. The average KD for the Cs+-induced block of hKir2.4 currents was 83.5 ± 16.3 µM at -100 mV, and it increased roughly 10-fold to 989 ± 157 µM at -60 mV (n = 5). The voltage dependence of the Cs+ block was quantified by fitting the data to the equation (52)
log[<IT>K</IT><SUB>D</SUB>(<IT>V</IT>)]<IT>=</IT>log[<IT>K</IT><SUB>D</SUB>(<IT>O</IT>)]<IT>+</IT>(<IT>&dgr;zFV</IT>)<IT>&cjs0823;  </IT>(<IT>2.303RT</IT>) (2)
where KD(V) is the KD at membrane potential V, KD(0) is the KD at 0 mV, delta  is the electrical distance or fraction of the transmembrane electrical field sensed by the blocking ion, z is the valence of the ion, and F, R, and T have their usual meaning. Figure 9D plots the average value of log KD obtained from the data in Fig. 9C as a function of voltage, and the straight line is the least-squares fit of the data to Eq. 2. The results indicate a 10-fold change in KD per 35-mV change in membrane voltage and imply that the fractional distance of the binding site in the electric field is 1.61. A value of delta  > 1 is generally interpreted to signify the occupancy of a multi-ion channel with more than one blocking ion (19).


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Fig. 9.   Concentration and voltage dependence of the Cs+-induced block of hKir2.4 current. A: family of hKir2.4 currents recorded in the presence of 100 µM Cs+. Currents were evoked by 1-s voltage steps from a holding potential of 0 mV to voltages ranging from -130 to +30 mV. Note that the current at -130 mV was of smaller magnitude than the current at -90 mV. B: I-V relationships in the presence of various Cs+ concentrations. Symbols represent the mean values of currents normalized to the current at -175 mV in the absence of Cs+ (n = 5); SD are smaller than the size of the symbols. C: dose-response curves for the Cs+-induced block at various potentials. The solid lines are best fits of the data to Eq. 1. Data are means ± SD. D: voltage dependence of apparent concentration at which block is half-maximal (log KD). The straight line is the best fit of the data to Eq. 2 with delta = 1.64 and KD(0) = 49.1 mM. Data are means ± SD.

Inhibition by Ba2+. Compared with the block by Cs+, the Ba2+-induced block of hKir2.4 currents was more weakly voltage dependent. Figure 10A shows that 100 µM Ba2+ in the bath produced a time-dependent block of inward currents. Figure 10B summarizes the results of experiments on five oocytes and plots the normalized steady-state I-V relationships in the presence of various concentrations of Ba2+. The block by Ba2+ was mildly voltage dependent, resulting in a slight curvature in the I-V relationship at voltages more negative than -80 mV. Figure 10C summarizes the concentration and voltage dependence of the Ba2+-induced block of hKir2.4 currents. The symbols represent mean values of normalized current, and the smooth curves are the least-squares fits of the data to Eq. 1. The KD at -100 mV was 116.0 ± 8.9 µM, and it increased slightly at more positive potentials, as can be seen by the rightward shift in the dose-response curve. Figure 10D plots the average value of log KD as a function of voltage, and the straight line is the least-squares fit of the data to Eq. 2. The results indicate a 10-fold change in KD per 166-mV change in membrane voltage and imply that the fractional distance of the binding site in the electric field is 0.18. Hence, Ba2+ appears to act at some superficial site near the outside of the channel.


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Fig. 10.   Concentration and voltage dependence of the Ba2+-induced block of hKir2.4 current. A: family of hKir2.4 currents recorded in the presence of 100 µM Ba2+. Currents were evoked by 1-s voltage steps from a holding potential of 0 mV to voltages ranging from -130 to +30 mV. B: I-V relationships in the presence of various Ba2+ concentrations. Symbols represent the mean values of currents normalized to the current at -175 mV in the absence of Ba2+ (n = 5); SD are smaller than the size of the symbols. C: dose-response curves for the Ba2+-induced block at various potentials. The solid lines are best fits of the data to Eq. 1. Data are means ± SD. D: voltage dependence of apparent KD. The straight line is the best fit of the data to Eq. 2 with delta = 0.16 and KD(0) = 480 µM. Data are means ± SD.

pH sensitivity. As mentioned in Identification and Sequence Analysis of hKir2.4, analysis of the predicted amino acid sequence of hKir2.4 revealed that it contains residues in the extracellular linker between the M1 and H5 domains that correspond to residues in human Kir2.3 that are thought to confer extracellular pH sensitivity (8). To test the possibility that hKir2.4 channels are also sensitive to changes in external pH, we measured hKir2.4 currents while superfusing oocytes with solutions buffered to a range of pH values. We carried out these experiments with 10 mM K+ in the bath so that possible effects of extracellular pH on endogenous currents could be detected as a change in V0. In addition, Na+ was omitted (NMDG substitution) to avoid possible complications arising from interactions between protons and the Na+ binding site. Figure 11A shows currents recorded in response to a series of voltage steps from a holding potential of 0 mV to membrane potentials in the range from +50 to -150 mV. Compared with currents recorded in our standard solution buffered to pH 7.4, hKir2.4 currents were significantly smaller at pH 6.5 and larger at pH 8.5. In contrast, manipulations expected to alter intracellular pH, such as exposure to 10 mM NH4+, 50 mM acetate, or CO2/HCO3- at constant extracellular pH, had no significant effect on hKir2.4 currents. The time course of currents following voltage steps was not affected by pH, indicating that modulation of hKir2.4 currents by extracellular protons was essentially voltage independent. This conclusion is supported by the finding that the fraction of current increased or decreased by the extracellular pH change was essentially independent of voltage (Fig. 11B) and suggests that the regulation of hKir2.4 channel involves the binding of protons to some external site on the channel protein.


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Fig. 11.   Modulation of hKir2.4 currents by changes in extracellular pH (pHo). A: family of currents measured in a single oocyte bathed with solutions buffered to pH 6.5, 7.4, and 8.5. Currents were evoked by voltage steps from a holding potential of 0 mV to membrane potentials in the range from +50 to -150 mV. B: steady-state I-V relationships measured in the same oocyte as in A. C: summary of the pHo dependence of hKir2.4. For each oocyte, the current at -120 mV at each pHo was normalized to the current measured with pHo 7.4 solution. Data are means ± SD (n = 5). The smooth curve is the best fit of the data to the Hill equation IpHX/IpH7.4 = A[pKanH/(pKanH + pHonH)] + C, where X is the test pHo, A is the maximum normalized current, nH is the Hill coefficient, and C is a constant.

The pH dependence of hKir2.4 currents is summarized in Fig. 11C. The steady-state current measured at -120 mV at each extracellular pH was normalized to the current recorded at pH 7.4 in the same oocyte. The symbols represent mean values, and the smooth curve is the least-squares fit of the data to the Hill equation with a Hill coefficient of 1 and a pKa of 7.14, which is near the middle of the physiological pH range.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have described the cloning, expression pattern, chromosomal localization, and functional characterization of a retinal inwardly rectifying K+ channel. Analysis of the predicted amino acid sequence indicated that it is a member of the Kir2 subfamily of K+ channel subunits. Subsequent to our initial report (25), the rat homolog of this gene (rKir2.4) was identified from a rat brain cDNA library (46). The human and rat homologs of Kir2.4 share a high degree of identity (92%), with some differences in their amino and carboxy-terminal regions. Because the amino acid substitutions between these homologs are mostly conserved or lie outside known functional motifs, it seems unlikely that these differences have any functional significance.

The heterologous expression of hKir2.4 in Xenopus oocytes resulted in functional channels with properties similar to those of other members of the Kir2 subfamily, including strong inward rectification, a conductance that is roughly proportional to the square root of the extracellular K+ concentration, and susceptibility to block by extracellular Cs+ and Ba2+. As reported previously for rKir2.4 (46), the block of hKir2.4 currents by Cs+ was more voltage dependent than the block by Ba2+. Specifically, a 10-fold change in KD was produced by a 35-mV change in membrane potential for the Cs+ block, but an equivalent change in the KD for the Ba2+ block required a 166-mV change in membrane potential. We also confirmed that, compared with other Kir2 channels, hKir2.4 channels required a 20-fold higher concentration Ba2+ to block 50% of the inward current; this result suggests a binding site with an unusually low affinity for this ion. In contrast, the concentration dependence of the Cs+ block of hKir2.4 channels at -80 mV indicated an affinity similar to that of Kir2.1 channels (24, 46). In this regard, our results differ from those of Topert et al. (46), who reported a Cs+ affinity for rKir2.4 channels that is lower by a factor of 30-50 compared with that for other Kir2 channels. The reason for this difference between hKir2.4 and rKir2.4 channels is not known.

A previously unrecognized property of the Kir2.4 channel is its modulation by changes in extracellular pH in the physiological range. We found that extracellular alkalinization enhanced, whereas extracellular acidification diminished, macroscopic hKir2.4 currents, with an apparent pKa of 7.14. This behavior is similar to that of Kir2.3 (HIR), for which the molecular determinant of pH sensitivity has been shown to be a histidine residue (H117) in the M1-to-H5 linker that influences one of two titratable cysteine residues (114 and 146) located in the M1-to-H5 and H5-to-M2 linkers (8). It has also been shown that extracellular Zn2+ blocks Kir2.3 currents in a voltage-independent and pH-sensitive manner, which indicates that Zn2+ binds to the same residue as H+. It seems likely that the modulation of hKir2.4 by extracellular pH involves a similar mechanism, because its predicted amino acid sequence contains the same critical residues in corresponding locations (C127, H130, and C159). In support of this idea, we have found that hKir2.4 currents are also blocked by extracellular Zn2+ in micromolar concentrations (data not shown).

Another remarkable characteristic of hKir2.4 channels expressed in oocytes is that exposure to high [K+]o solution triggers a slow increase in macroscopic current. This property has not been described for any other Kir2 channel (including rKir2.4), but it superficially resembles the high [K+]-induced activation of Kir1.1 (9) and Kir4.2 channels (33) expressed in oocytes. Although the activation of these other cloned channels is thought to involve allosteric regulation by external K+, the mechanism by which hKir2.4 currents are activated may be different, because the conductance increase was not rapidly reversed on return to low [K+]o (see Fig. 8).

Northern blot analysis of human RNA indicated the presence of Kir2.4 transcripts only in the retina, with no detectable signal in any other tissues. To our knowledge, this is the first report of an inwardly rectifying K+ channel with this particular tissue distribution. Our results contrast with those of Topert and colleagues (46), who recently reported that Kir2.4 transcript is expressed predominantly in human and rat brain but not in significant amounts in the retina. This discrepancy may be due to differences in both the selection of probes and the hybridization conditions. Whereas we hybridized under very stringent conditions (65°C in Express-Hyb solution) using a highly selective probe synthesized using the 3'-untranslated region of hKir2.4 cDNA, Topert et al. used a probe representing the coding region of Kir2.4 cDNA and hybridized under lower stringency conditions (42°C in Express-Hyb solution). Hence, it is possible that Topert et al. detected transcripts for other K+ channel subtypes sharing homology with Kir2.4. Regardless, our finding that hKir2.4 probe hybridizes to bovine as well as human retinal poly(A+) mRNA strengthens our conclusion that Kir2.4 transcript is expressed in the retina.

The hybridization of hKir2.4 probe revealed 3- and 5-kb bands in Northern blots of human retinal mRNA, suggesting the possibility that different isoforms of Kir2.4 may be expressed in the retina. Topert et al. (46) reached a similar conclusion from their observation that their probe hybridized to two transcripts, 3 and 4 kb in size. Although there is no evidence for alternative splicing of other members of the Kir2 subfamily, alternatively spliced variants of ROMK (Kir1.1) channels with varying expression pattern and channel properties have been demonstrated (5).

In situ hybridization to sections of monkey retina indicated the presence of Kir2.4 transcripts in the perinuclear region of most cells in the neural retina. On the basis of this expression pattern, one might expect that retinal neurons should exhibit inwardly rectifying K+ channels with properties similar to those of the cloned Kir2.4 channel, i.e., strong inward rectification, high selectivity to K+, block by Cs+ and Ba2+, and sensitivity to changes in extracellular pH. The inwardly rectifying K+ current is a major component of the Müller cell membrane properties (30), but recent evidence strongly suggests that the underlying conductance is comprised of Kir4.1 channels (21). Inwardly rectifying K+ currents displaying many of the same properties as Kir2.4 have been documented in horizontal cells, where they help shape light-evoked responses (50). Takahashi and Copenhagen (44) have demonstrated that the inwardly rectifying K+ current in isolated catfish horizontal cells is enhanced by intracellular alkalinization, but there is no published information on the sensitivity of this current to changes in extracellular pH. Further studies are needed to determine the relationship between Kir2.4 and the inwardly rectifying K+ channel in horizontal cells.

Apart from horizontal cells, no other retinal neuron displays the classic inwardly rectifying K+ current. Another current exhibiting inward rectification, the hyperpolarization-activated current, or Ih, has been identified in photoreceptor inner segments (18, 28, 53), amacrine cells (14), bipolar cells (23), and retinal ganglion cells (43). The channels underlying these currents, however, are distinct from Kir channels in that they are blocked by Cs+ but not by Ba2+ and that they are permeable to both K+ and Na+. How does one reconcile the presence of Kir2.4 transcript in retinal neurons that apparently lack inward rectifier K+ currents? One possible explanation is that Kir2.4 channels are localized to synaptic terminals or are expressed in such low abundance that their contribution to whole cell currents is relatively small and therefore difficult to detect. Another possibility is that Kir2.4 channel subunits assemble with an inhibitory subunit. Negative interactions have been demonstrated between Kir2.2 and Kir2.2v subunits (29) and between Kir4.1 and Kir3.4 subunits (47); in both cases, the expression of homomeric channels with one of type of subunit alone (Kir2.2 or Kir4.1) produces inwardly rectifying currents, but coexpression of one subunit with the other produced no current. It will be interesting to learn what other Kir channel subunits are expressed in retinal neurons and whether they can have inhibitory interactions with Kir2.4.

Extracellular pH in the retina changes as a function of light adaptation (54) and hypoxia (55). Our finding that hKir2.4 currents are steeply extracellular pH dependent with a pKa of about pH 7.14 implies that the function of horizontal cells (or any other retinal cell expressing Kir2.4) may be modulated under physiological and pathophysiological conditions.

Mutations in the K+ channel genes have been shown to cause several inherited diseases (10, 36), including long QT syndrome (48, 49), Bartter syndrome (38), and episodic ataxia/myokymia syndrome (6). In addition, mutations in the H5 pore region of the Kir3.2 result in degeneration of cerebellar granule cells and cause ataxia in weaver mice (32). The importance of Kir2.4 channels in determining membrane properties and its expression in retinal cells suggest that mutations in Kir2.4 might lead to hereditary retinal disease. The localization of hKir2.4 gene to human chromosome 19 at q13.1-13.3 makes it an attractive candidate for inherited eye diseases that map to this chromosomal region. The genetic loci for recessive optic atrophy with ataxia (31), dominant cone-rod dystrophy (CORD2) (11), and a form of retinitis pigmentosa (RP11) (1) have been mapped to 19q13. Although the mutations in the cone-rod homeobox (CRX) gene have been identified in families with cone-rod dystrophy (15, 40) and Leber congenital amaurosis (41), the original CORD2 family did not reveal a CRX mutation, indicating that mutations in another gene at 19q13 may also cause this disease. PCR-based analysis, however, indicates that Kir2.4 gene is not present in the YAC Contig spanning RP11 locus (S. S. Bhattacharya, personal communication). Whether Kir2.4 is a candidate gene for CORD2 or any other genetic disorder remains to be evaluated.


    ACKNOWLEDGEMENTS

We thank Dr. Peter F. Hitchcock, Deborah C. Otteson, and Mitchell Gillett for help with in situ hybridization experiments and Dr. Alan Goldin (University of California, San Diego) for kindly providing the pBSTA plasmid.


    FOOTNOTES

This work was supported by National Eye Institute Grants EY-08850 and EY-07703 (to B. A. Hughes), the Michigan Eye Bank (to G. Kumar), Fight for Sight (to G. Kumar), a Research to Prevent Blindness Lew Wasserman Award (to A. Swaroop), and the Foundation Fighting Blindness (to A. Swaroop and B. A. Hughes).

Address for reprint requests and other correspondence: B. A. Hughes, Dept. of Ophthalmology, Univ. of Michigan, Kellogg Eye Center, 1000 Wall St., Ann Arbor, MI 48105 (E-mail: bhughes{at}umich.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 22 January 2000; accepted in final form 3 April 2000.


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DISCUSSION
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