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Originally published In Press as doi:10.1074/jbc.M407575200 on August 10, 2004

J. Biol. Chem., Vol. 279, Issue 42, 43427-43436, October 15, 2004
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An Alternatively Spliced Isoform of PSD-93/Chapsyn 110 Binds to the Inwardly Rectifying Potassium Channel, Kir2.1*

Mark L. Leyland{ddagger}§ and Caroline Dart¶

From the Departments of {ddagger}Biochemistry and Cell Physiology and Pharmacology, University of Leicester, P.O. Box 138, Leicester LE1 9HN, United Kingdom

Received for publication, July 7, 2004 , and in revised form, August 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inwardly rectifying potassium (Kir) channels are prime determinants of resting membrane potential in neurons. Their subcellular distribution and surface density thus help shape neuronal excitability, yet mechanisms governing the membrane targeting and localization of Kir channels are poorly understood. Here we report a direct interaction between the strong inward rectifier, Kir2.1, and a recently identified splice variant of postsynaptic density-93 (PSD-93), a protein involved the subcellular targeting of ion channels and glutamate receptors at excitatory synapses. Yeast two-hybrid screening of a human brain cDNA library using the carboxyl terminus of Kir2.1 as bait yielded cDNA encoding the first two PDZ domains of PSD-93, but with an extended N-terminal region that diverged from other PSD-93 isoforms. This clone represented the human homologue of the mouse PSD-93 splice variant, PSD-93{delta}. Reverse transcription-polymerase chain reaction analysis showed diffuse low level PSD-93{delta} expression throughout the brain, with significantly higher levels in spinal cord. In vitro binding studies revealed that a type I PDZ recognition motif at the extreme C terminus of the Kir2.1 mediates interaction with all three PDZ domains of PSD-93{delta}, and association between Kir2 channels and PSD-93{delta} was confirmed further by the ability of anti-Kir2.1 antibodies to coimmunoprecipitate PSD-93{delta} from rat spinal cord lysates. Functionally, coexpression of Kir2.1 and PSD-93{delta} had no discernible effect upon channel kinetics but resulted in cell surface Kir2.1 clustering and suppression of channel internalization. We conclude that PSD-93{delta} is potentially an important regulator of the spatial and temporal distribution of Kir2 channels within neuronal membranes of the central nervous system.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inwardly rectifying potassium channels comprise a family of integral membrane proteins whose diverse cellular functions include maintenance of the resting membrane potential, control of neuronal excitability and heart rate, and potassium homeostasis (1–3). Underlying these functions is the property of inward rectification, the ability to allow potassium to move easily into the cell at membrane potentials negative to the potassium equilibrium potential (EK) but to restrict potassium outflow at potentials positive to EK. This asymmetry in the current-voltage relation results from the channel's susceptibility to voltage-dependent block by intracellular polyamines and magnesium ions (4, 5) and ensures that, while being extremely active around EK, they pass little or no current at membrane potentials positive to –40 mV. Thus, Kir1 channels maintain a tight control on the resting membrane potential but close in the face of significant membrane depolarization (such as that generated by the cardiac or neuronal action potential) to protect the cell from excessive K+ loss. This pivotal role in the regulation of the membrane potential makes Kir channels attractive candidates for modulation by neurotransmitters and hormones, since even small fluctuations in Kir channel activity have profound effects on the resting membrane potential and, hence, cellular excitability (2, 6).

Members of the classical strong inwardly rectifying Kir2 subfamily are expressed widely in neuronal tissues. Within the brain, they show a differential distribution: Kir2.1, -2.2, and -2.3 are abundant in the olfactory bulb, cerebral cortex, striatum, and hippocampus, whereas Kir2.2 is the only subunit that shows significant levels of expression in the brainstem, thalamus, and cerebellum (7, 8). Kir2.1 and -2.2 additionally show overlapping expression in regions of the midbrain where Kir2.3 is largely absent. Of all the Kir2 family members, Kir2.4 exhibits the most restricted subunit distribution, being found only in neurons of cranial nerve motor nuclei in the midbrain and brainstem (9). It is assumed that the differential distribution of Kir2 channel subunits throughout the brain underlies specific functions in the regulation of excitability within different neuronal populations. Additionally, the subcellular localization of Kir2 channels defines their physiological function. Kir2.3 channels, for example, are specifically localized to postsynaptic membranes of the dendritic spines of granule cells within the forebrain, where they are believed to contribute to the resting membrane potential and the regulation of excitatory synaptic transmission (10). However, despite their potential importance, mechanisms involved in the control of subcellular localization and distribution of Kir2 channels within the central nervous system are incompletely understood.

A potential method of targeting Kir2 family members to specific neuronal membranes is through interaction with selected members of the membrane-associated guanylate kinase (MAGUK) protein family (10–15). In mammals, these intracellular scaffolding proteins comprise a family of four main members: PSD-95/SAP90, its closely related homologue PSD-93/ chapsyn 110, SAP102, and SAP97/hDlg (16). These proteins are characterized by the presence of three N-terminal PDZ domains (so-called because they were originally recognized as repeats in the proteins PSD-95, discs large, and zona occludens-1), an Src homology 3 domain and an enzymatically inactive guanylate kinase-like region. All of these domains mediate specific protein-protein interactions and have been implicated in the assembly of extensive signaling protein complexes at junctional sites such as synapses (17–21).

Kir2 channels seem to be able to interact with several different members of the neuronal MAGUK family. Kir2.1 and Kir2.3 are able to bind PSD-95/SAP90 via a type I PDZ domain recognition motif located at the channel's extreme C terminus (11, 12), and Kir2.3 can additionally associate with PSD-93/chapsyn 110 (10). Kir2.1, -2.2, and -2.3 also interact with SAP97 in the cerebellum and nonneuronal tissues such as the heart (13, 14). Recent detailed proteomic analysis of Kir2-associated proteins in brain revealed additional interactions with the family of CASK-like MAGUK proteins CASK/Lin-2, Dlg2, Dlg3, and Pals2 (14, 15). These MAGUK proteins differ from the PSD-95/SAP-90 family by possessing a single class II N-terminal PDZ domain, which makes it unlikely that they bind directly to Kir2 channels but are most likely recruited to Kir2 complexes through association with SAP-97. This ability to interact with different MAGUKs may reflect a fundamental requirement for differential targeting of Kir2 channels within cells. PSD-95 and PSD-93, for example, are found predominantly at postsynaptic sites within neurons, whereas SAP102 is also found in axons and some presynaptic terminals (22). SAP-97 localizes to presynaptic terminals and axons and is unusual among the MAGUKs in that it is less tightly associated with the membrane (23). Additionally, PSD-95 and PSD-93 mediate surface ion channel and receptor clustering, whereas SAP-102 and SAP-97 do not (13, 22). It is believed that differences in subcellular localization and surface distribution arise from diverse targeting signals located in the N-terminal regions of MAGUK proteins (22, 24–26).

We are interested in the molecular mechanisms involved in the subcellular targeting and localization of members of the Kir2 subfamily of strong inward rectifiers. Here we report the result of a yeast two-hybrid screen of human brain cDNA library using a C-terminal portion of Kir2.1 (residues 307–428) as bait. This screen identified an alternatively spliced isoform of PSD-93/chapsyn 110 as a binding partner for Kir2.1. This variant exhibits an extended N-terminal region that diverges from other PSD-93 isoforms just before the first PDZ domain and represents the human homologue of the recently identified mouse PSD-93 splice variant, PSD-93{delta} (27). We find that this new N-terminal variant of PSD-93 is preferentially expressed within the spinal cord and go on to investigate the potential functional consequences of Kir2.1 interaction with PSD-93{delta}. We demonstrate that whereas it has no discernible effect upon channel kinetics, association between Kir2.1 and PSD-93{delta} promotes cell surface Kir2.1 clustering and suppresses ion channel internalization. We propose that PSD-93{delta} acts as an anchoring protein for Kir2.1, ensuring its stable surface expression and potentially controlling its location and proximity to other proteins within neuronal membranes of the central nervous system.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies, Polyacrylamide Gel Electrophoresis, and Immunoblotting—The following primary antibodies were used: mouse monoclonal anti-PDZ (Upstate Biotechnology, Inc., Lake Placid, NY), rabbit polyclonal anti-PSD-93 (Chemicon), rabbit polyclonal anti-glutathione S-transferase (GST) (Sigma), rat monoclonal high affinity anti-HA (Roche Applied Science), and rabbit polyclonal anti-Kir2.1 (a kind gift from Dr. R. Norman, Department of Medicine, University of Leicester, UK). Horseradish peroxidase-, Texas Red-, and fluorescein isothiocyanate-conjugated anti-rabbit, anti-mouse, and anti-rat secondary antibodies were from Jackson ImmunoResearch Laboratories, Inc. Anti-PSD-93{delta} polyclonal antibodies were generated to a synthetic peptide MNAYLTKQHSCSRGSDGMDI, corresponding to amino acids 1–20 of PSD-93{delta}. The peptide was conjugated to keyhole limpet hemocyanin and used to immunize rabbits. Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis on 10% polyacrylamide-Tris gels and transferred electrophoretically onto nitrocellulose membranes (Hybond ECL; Amersham Biosciences). Immunoblotting was carried out as described previously (28).

Plasmid Constructs—pGEX-Kir2.1 (aa 421–428) has been described previously (28). Individual PDZ domains from PSD-93{delta}, PDZ1 (aa 130–227), PDZ2 (aa 228–322), and PDZ3 (aa 456–549) were amplified by PCR and inserted between the BamHI and EcoRI sites of pGEX-2T. Hexahistidine (His)-tagged Kir2.1 C terminus was constructed by amplifying the region encoding aa 307–428 by PCR and inserting this fragment between the NdeI and BamHI sites of pET15b. The same PCR product was also inserted into pGBT9 to create the GAL4 binding domain construct pGBT9-Kir2.1C. The Kir2.1 construct in pcDNA3, EGFP-Kir2.1, and HA-Kir2.1 expression constructs have been described previously (28–30). PSD-93{alpha} and PSD-93{beta} in the mammalian expression vector pGW1 were a generous gift from Professor David Bredt (University of California, San Francisco CA). Full-length PSD93{delta} was constructed by PCR amplification of the unique N-terminal region using the following primer pair: 5'-GGGGGTACCGCCACCATGAATGCATACCTCACCAAGCAACACAGC-3' and 5'-GGGGCTAGCCTGTGCTGGGCTGAACCAACCATGTGG-3'. Following digestion of the PCR product with KpnI and NheI, this fragment was ligated into KpnI/NheI-digested pGW1-PSD-93{beta}. All PCR-amplified DNA constructs were verified by DNA sequencing.

Yeast Two-hybrid Library Screen—The C-terminal region of Kir2.1 (aa 307–428) was amplified by PCR and inserted in frame with the GAL4 binding domain of plasmid pGBT9 (BD Biosciences) to create pGBT9-Kir2.1C. This construct was used to screen a human brain cDNA library in the activation domain plasmid pACT2 as described by the manufacturer (BD Clontech). pGBT9-Kir2.1C and the pACT2 library plasmids were used to cotransform the yeast strain Y190 using the lithium acetate protocol. Approximately 1 x 106 transformants were selected on synthetic minimal medium that lacked tryptophan, leucine, and histidine and contained 25 mM 3-amino-1,2,4-triazole. His+ colonies were selected and assayed for {beta}-galactosidase activity using the colony filter lift assay. Plasmid DNA from His+ LacZ+ colonies were isolated by standard methods. {beta}-Galactosidase liquid assays were performed in yeast strain Y187 as described by the manufacturer (Clontech).

Cells and Cell Transfection—HEK293 were grown in minimal essential medium supplemented with 1% nonessential amino acids and 10% (v/v) fetal bovine serum. All media and reagents were from Invitrogen. No antibiotics were used. Cells were transiently transfected using LipofectAMINE transfection reagent (Invitrogen) according to the manufacturer's protocol. Transfections were performed in 6-well culture plates with cells at 80% confluence.

Recombinant Protein Expression, in Vitro Binding, and GST Pull-down Assays—GST fusion proteins were produced by inducing Escherichia coli DH5{alpha} cells containing the appropriate plasmid with 1 mM isopropyl-{beta}-D-thiogalactopyranoside for 4–5 h at 37 °C. Cells were pelleted by centrifugation and resuspended in 0.5 ml of ice-cold phosphate-buffered saline (PBS). The cells were lysed by sonication, Triton X-100 was added to a final concentration of 1% (v/v), and the lysates were cleared by centrifugation (10,000 x g for 10 min at 4 °C). The supernatant was added to a suspension of glutathione-Sepharose beads (Amersham Biosciences) and incubated with mixing at 4 °C for 30 min. Glutathione-Sepharose beads were washed 3 times in PBS and GST fusion proteins eluted with glutathione elution buffer (20 mM reduced glutathione, 120 mM NaCl, 100 mM Tris-HCl, pH 8). Purity was determined by SDS-PAGE followed by Coomassie Blue staining. His-tagged Kir2.1C was expressed in E. coli BL21 (DE3) pLysS by induction with isopropyl-{beta}-D-thiogalactopyranoside as described above. Pellets were resuspended in buffer A (20 mM Tris-HCl, 100 mM NaCl, pH 8.0) containing 8 M urea. Following sonication, the lysate was cleared by centrifugation. Supernatants were incubated with Talon metal affinity resin (Clontech) for 15 min at room temperature, and bound His-Kir2.1C was refolded by washing with serial dilutions of urea (8, 6, 4, 2, 0 M) in buffer A. Renatured His-tagged Kir 2.1C (aa 307–428) was resolved by SDS-PAGE (10%) and transferred onto nitrocellulose. Membranes were incubated overnight with block buffer and overlaid with equal quantities of GST and GST-tagged PDZ domain fusion proteins (1 h, 20 °C). Following extensive washing in blot buffer, membranes were exposed to anti-GST antibody (1:1000 in blot buffer, 1 h, 20 °C), washed, and incubated with horseradish peroxidase-conjugated anti-rabbit antibody (1:10,000 in blot buffer, 1 h, 20 °C).

To identify interactions between PSD-93 isoforms and Kir2.1, the GST fusion protein of the PDZ binding motif of Kir2.1 (residues 421–428) was incubated with lysates of HEK293 cells expressing PSD-93 isoforms. Extracts from transfected HEK293 cells were prepared 48 h post-transfection. Cells were washed briefly in PBS and incubated with 0.5 ml of lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 3 mM EDTA, pH 7.6) containing 1% Triton X-100 and 1% protease inhibitor mixture (Sigma) for 30 min on ice. The extract was cleared by centrifugation (10,000 x g at 4 °C for 15 min). The supernatant was added to 200 µlof glutathione-Sepharose beads, loaded with equal amounts of either GST or GST-Kir2.1 (aa 421–428), and incubated at 4 °C with gentle inversion for 1 h. The beads were pelleted by gentle centrifugation and washed three times with lysis buffer. The beads were resuspended in 200 µl of SDS-PAGE loading buffer and boiled, and proteins were separated by SDS-PAGE. Proteins were then transferred electrophoretically onto Hybond-ECL membranes and detected using anti-PDZ antibodies as above.

Reverse Transcription PCR—Forward primers to PSD-93 isoforms were designed using the region containing the unique N-terminal domains. These were as follows: PSD-93{alpha}, 5'-ATGTTCTTTGCATGTTACTGTGCACTC-3' (nucleotides 1–27); PSD-93{beta}, 5'-ATGATTTGCCACTGCAAAGTTGCTTGC-3' (nucleotides 1–27); PSD-93{delta}, 5'-ATGAATGCATACCTCACCAAGCAACAC-3' (nucleotides 1–27).

The reverse primer was common to all isoforms: 5'-CAATTGTCACAAAATAGGTCGTCTTCTACG-3' (bp 681–661 of PSD-93{delta}). Reverse transcription-PCR was performed with the Access reverse transcription-PCR system (Promega) using either 1 µg of rat or mouse brain total RNA (Ambion) or 1 µg of mouse brain total RNA panel (Abcam). Following reverse transcription (48 °C for 45 min followed by 94 °C for 2 min), DNA was synthesized by 40 cycles of PCR (30 s at 94 °C, 1 min at 50 °C, and 1 min at 68 °C) followed by a final extension at 68 °C for 7 min. Reaction products were separated by 1.4% agarose gel electrophoresis. Identity of the product was confirmed by sequencing.

Coimmunoprecipitation—Rat spinal cord was homogenized in ice-cold lysis buffer, and insoluble material was removed by centrifugation. The cleared lysate was incubated overnight with either 5 µg of rabbit nonimmune control serum or rabbit polyclonal anti-Kir2.1. Antibody complexes were isolated by the addition of Protein A-Sepharose (Amersham Biosciences) for 2 h at 4 °C. Following centrifugation, the beads were washed five times in ice-cold PBS, and bound proteins were eluted by the addition of SDS-PAGE sample buffer. Samples were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunoblotting.

Biotin Protection Assay—To investigate the effect of PSD-93{delta} coexpression on the rate of Kir2.1 internalization, we used a modified form of surface biotinylation termed biotin protection. HEK293 cells expressing HA-Kir2.1 alone or HA-Kir2.1 and PSD-93{delta} were plated out into poly-L-lysine-coated 6-well culture plates and allowed to reach 80% confluence. Cells were washed twice in PBS, and surface proteins were biotinylated by the addition of 1 ml of sulfo-NHS-SS-biotin (Pierce) membrane-impermeant disulfide-linked ("cleavable") biotin (0.5 mg/ml in PBS; Pierce) to each well followed by 30 min of incubation at 4 °C. Cells were washed briefly in PBS, and excess biotin was quenched with 50 mM Tris-HCl, pH 7.4, for 10 min at 4 °C. Prewarmed medium (minimal essential medium supplemented with 1% nonessential amino acids and 10% (v/v) fetal bovine serum) was added to each well, and cells were placed in an incubator at 37 °C to allow internalization to take place. At various time points (30, 60, 90, or 120 min), cells were removed from the incubator, and internalization was stopped by placing the cells on ice. Any remaining (not yet internalized or recycled) surface biotin was stripped from the cell surface by incubation in a solution containing 50 mM glutathione, 75 mM NaCl, 75 mM NaOH, and 10% fetal bovine serum for 5 min. The glutathione solution was then removed, and excess glutathione was quenched by the addition of 50 mM iodoacetamide and 1% bovine serum albumin in PBS for 2 x 10 min. Cells were subsequently lysed, and internalized ("protected") biotinylated proteins were recovered by incubation with strepavidin-agarose (Sigma; 20–30 µl of beads per 400 µl of lysates incubated with gentle agitation for 2 h at 4 °C). The recovered internalized proteins were separated by SDS-PAGE, and the amount of HA-Kir2.1 that had been internalized over the various time points was assessed by immunoblotting with anti-HA.

Immunocytochemistry—HEK293 cells were plated onto poly-L-lysine-coated coverslips and transiently transfected with the appropriate plasmids. Cells expressing EGFP-Kir2.1 and PSD-93 isoforms were fixed and permeabilized 48 h post-transfection in a solution containing 80 mM Na2HPO4, 20 mM NaH2PO4, 2% paraformaldehyde, and 0.1% Triton X-100. Fixed cells were incubated overnight at 4 °C with mouse monoclonal anti-PDZ diluted 250-fold in PBS containing 10% (v/v) goat serum. The following morning, the coverslips were washed 5 x 10 min in PBS and then incubated in goat anti-mouse secondary antibody conjugated with Texas Red in PBS plus 10% goat serum for 2 h at room temperature. Coverslips were washed in PBS for 3 x 10 min and mounted onto microscope slides using fluorescent mounting medium (Dako Ltd.) before viewing. Confocal images were obtained using a PerkinElmer UltraViewTM imaging system equipped with a krypton/argon laser and x 60 oil immersion lens with numerical aperture of 1.0.

Electrophysiology—Whole-cell currents were recorded from HEK293 cells typically 24–48 h post-transfection using an Axopatch 200B amplifier (Axon Instruments). Currents recorded in response to voltage steps were filtered at 5 kHz (–3 dB, 8-pole Bessel), digitized at 10 kHz using a DigiData 1320A interface (Axon Instruments), and analyzed using pCLAMP software. Electrodes were pulled from borosilicate glass (outer diameter 1.5 mm, inner diameter 1.17 mm; Clarke Electromedical, Pangbourne, UK) and fire-polished to give a final resistance of 5 megaohms when filled. The pipette-filling solution contained 140 mM KCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.2. The external solution contained 70 mM KCl, 70 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, pH 7.25. The junction potential between pipette and external solutions was sufficiently small (<1.5 mV) to be neglected. As far as possible, analogue means were used to correct capacity transients. Up to 90% compensation was routinely used to correct for series resistance. All experiments were performed at room temperature (18–22 °C), and the results are expressed as mean ± S.E.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of PSD93{delta} as a Binding Partner of Kir2.1 by Yeast Two-hybrid Analysis—To isolate binding partners of Kir2.1, a carboxyl-terminal segment of the channel (amino acids 307–428) was subcloned into the GAL4 binding domain bait vector, pGBT9. This was used as bait to screen a human brain cDNA library (BD Clontech) by yeast two-hybrid analysis. A screen of 1 x 106 yeast colonies resulted in several hundred colonies exhibiting a positive phenotype. These colonies were screened by {beta}-galactosidase filter assay, and one of the positive clones (Y12) showed strong {beta}-galactosidase activity (development of blue color at ~40 min) in the presence of Kir2.1C (Fig. 1A). Y12 encoded a partial cDNA representing the first two PDZ domains of the MAGUK PSD-93 but with an extended N-terminal region that diverged from other PSD-93 isoforms just before PDZ1 (Fig. 1B). This clone represented a human homologue of the recently identified mouse alternatively spliced PSD93 isoform, PSD93{delta} (27). PSD93{delta} is highly conserved between humans, mice, and rats (Fig. 1C), with human PSD93{delta} exhibiting 87 and 88% identity with the rat and mouse homologues, respectively. Most of this difference is due to a small insert of 7 amino acids (aa 65–82) in the human variant that is lacking from both mice and rats.



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  		FIG. 1.
Yeast two-hybrid screening identifies PSD93{delta}, an alternatively spliced N-terminal isoform of PSD93, as a binding partner of Kir2.1. A, {beta}-galactosidase assays showing the specific interaction of clone Y12 with the C terminus of Kir2.1. For {beta}-galactosidase liquid assays, yeast clones transfected with the indicated plasmids were grown overnight in the appropriate selective media. {beta}-Galactosidase activity from these lysates was measured in triplicate. For {beta}-galactosidase plate assays, yeast clones transfected with the indicated plasmids were plated onto appropriate selective media and grown for 2 days at 30 °C. After transfer to filter paper, {beta}-galactosidase activity was measured as described under "Experimental Procedures." Time taken for colony to turn blue was as follows. +++, less than 60 min; –, no color after 6 h. B, domain structure of clone Y12. This clone contained two PDZ domains that were identical to the first two PDZ domains of PSD93 but possessed an extended N-terminal region not present in other PSD-93 isoforms. C, alignment of PSD93{delta} isoforms from mice, rats, and humans. Identical residues are shaded.

 
PSD93{delta} Binds to the Extreme C Terminus of Kir2.1—To verify the interaction between PSD-93{delta} and the carboxyl terminus of Kir2.1 (Kir2.1C), a GST fusion protein incorporating the last eight amino acids of Kir2.1 (aa 421–428) was tested for its ability to isolate full-length PSD-93{delta} from lysates of HEK293 cells that had been transfected with cDNA encoding PSD-93{delta}. The extreme carboxyl terminus of Kir2.1 harbors a type I PDZ domain recognition motif (X(S/T)X(V/I)) that has been shown to mediate binding to PSD-95/SAP90 and the related MAGUK SAP97 (11–14). We found that GST fusion proteins of this extreme C-terminal region of Kir2.1 isolated PSD-93{delta} from HEK293 lysates, whereas GST alone failed to interact with PSD-93{delta} (Fig. 2A).



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  		FIG. 2.
PSD93{delta} binds to the C terminus of Kir2.1 via PDZ domains 1–3. A, PSD93{delta} binds to the extreme C terminus of Kir2.1. Lysates were prepared from HEK293 cells transfected with PSD93{delta} and incubated with glutathione-Sepharose beads charged with equal amounts of GST or GST-Kir2.1C (aa 421–428). Bound proteins were separated by SDS-PAGE, transferred onto nitrocellulose, and immunoblotted with anti-PDZ or anti-PSD-93. 1% of the input lysate was used as a control. B, overlay assay to show binding of Kir2.1 C terminus to all three PDZ domains of PSD93{delta}. Renatured His-tagged Kir2.1 (amino acids 307–428) protein was resolved by SDS-PAGE and transferred onto nitrocellulose membrane. Membranes were overlaid with equal quantities of GST or GST-PDZ fusion proteins and immunoblotted with anti-GST. All three PDZ domains show binding to Kir2.1C with the strongest affinity shown by PDZ2 and PDZ3.

 
Kir2.1C Is Able to Interact with All Three PDZ Domains of PSD-93{delta}—Binding of PSD-93{delta} to a region on the extreme carboxyl terminus of Kir2.1 containing a PDZ binding motif suggests that, as with other many other ion channels and receptors, Kir2.1 interacts with the PDZ domains of the MAGUK protein. Previous studies have indicated that Kir2 channel interaction with MAGUKs is usually mediated via PDZ1 and/or PDZ2 (11, 13). Our initial clone isolated by the yeast two-hybrid screen encoded for the extended N-terminal region and the first two PDZ domains of PSD93{delta}, indicating that either of these may be involved in the interaction with Kir2.1. To test the contribution of each of the PDZ domains of PSD-93{delta} to Kir2.1 binding, a far Western overlay assay was performed. Equal amounts of His-Kir2.1C (amino acids 307–428) were resolved by SDS-polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane, and incubated with equal amounts of GST or GST fusion proteins of the individual PDZ domains of PSD-93{delta}. Fusion protein binding was detected by immunoblotting with anti-GST antibodies. Fig. 2B shows that all three PDZ domains of PSD-93{delta} interacted with membrane-bound Kir2.1C, whereas GST alone failed to bind. Band intensity was strongest for GST fusion proteins of PDZ2 and PDZ3, indicating that Kir2.1C may have a higher affinity for these domains over PDZ1.

PSD-93{delta} Is Preferentially Expressed in Spinal Cord—The existence of different splice variants of PSD-93 has led to the suggestion that they may be differentially expressed within tissues. To investigate this, we utilized the reverse transcription-PCR using primers specific for PSD-93{alpha}, -{beta}, and -{delta} to amplify PSD-93 isoforms from spinal cord and different regions of the brain. Fig. 3A shows PSD-93{alpha} to be robustly expressed in a number of brain regions including the cerebral cortex, striatum, hippocampus, and olfactory bulb. Lower levels of expression are seen in the midbrain, brainstem, and spinal cord, with extremely low levels detected in the cerebellum. PSD-93{beta} follows a similar expression pattern but exhibits lower levels of expression in the striatum and seems to be virtually absent from spinal cord. These results are in keeping with previous findings (35, 37). In contrast, PSD-93{delta} shows diffuse, low level expression throughout the brain but is significantly enriched in spinal cord. Immunoblots of rat brain and spinal cord lysates with anti-PSD93{delta} polyclonal antiserum show the presence of a band of ~116 kDa that comigrates with the band found in HEK293 cells transfected with cDNA encoding full-length PSD-93{delta} (Fig. 3B).



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  		FIG. 3.
PSD-93{delta} is preferentially expressed in spinal cord. A, reverse transcription-PCR analysis of PSD-93 isoform {alpha}, {beta}, and {delta} expression in spinal cord and different regions of the brain. Following PCR amplification with isoform-specific primers, the products were separated on a 1.4% agarose gel. The sizes of the bands are as follows: PSD-93{alpha}, 570 bp; PSD-93{beta}, 591 bp; PSD-93{delta}, 675 bp. Mouse brain total RNA was used as a positive control. No amplification of specific product was obtained in the absence of reverse transcriptase. PSD-93{delta} shows the highest level of expression in spinal cord. B, Western blot analysis showing expression of PSD-93{delta} in spinal cord and brain. Left, proteins present in lysates from untransfected HEK293 cells or HEK293 cells transfected with cDNA encoding PSD-93{delta} were separated by SDS-PAGE and immunoblotted with antisera against PSD-93{delta}. Only HEK293 cells expressing PSD-93{delta} show the presence of a band at ~116 kDa. Right, lysates prepared from rat brain or spinal cord also show the presence of a ~116-kDa band when blotted with anti-PSD-93{delta}. C, in vivo association of PSD-93{delta} with Kir2.1. Immunoprecipitation of proteins from rat spinal cord lysates was performed using rabbit polyclonal antibodies directed against Kir2.1 or rabbit nonimmune control serum as described under "Experimental Procedures." Precipitated proteins were separated by SDS-PAGE, transferred onto nitrocellulose membrane, and immunoblotted with anti-PSD-93.

 
Kir2.1 Associates with PSD-93{delta} in Spinal Cord—To determine whether Kir2.1 forms a complex with PSD-93{delta} within intact cells, we performed a coimmunoprecipitation using polyclonal antisera to Kir2.1. Lysates of rat spinal cord were used, since this tissue showed the highest expression levels of PSD-93{delta}. Fig. 3C shows that antibodies directed against Kir2.1 coimmunoprecipitate PSD-93{delta} from rat spinal cord lysates, consistent with the association of these two proteins in vivo. Coimmunoprecipitations from rat brain also showed the association of both Kir2.1 and Kir2.3 with PSD93{delta} (data not shown).

PSD93{delta} Promotes Clustering of Kir2.1 at the Cell Surface— Having demonstrated in vitro and in vivo binding of Kir2.1 to PSD-93{delta}, we investigated the role this interaction plays in subcellular channel distribution. Previous studies have shown that sequences located at the N terminus of MAGUK proteins are critical for membrane targeting and surface distribution of associated proteins (22, 24, 25), and our initial findings show that PSD-93{delta} has an N-terminal region that is divergent from other PSD-93 isoforms. To investigate whether PSD-93{delta} was able to aggregate Kir2.1 channels at the cell surface, we transfected HEK293 cells with cDNA encoding EGFP-Kir2.1 alone or together with PSD-93{delta}. Fig. 4A shows a confocal image of HEK293 cells transfected with only EGFP-Kir2.1. EGFP fluorescence shows the protein distributed evenly throughout the plasma membrane. Fig. 4, B and D, shows confocal images of HEK293 cells transfected with EGFP-Kir2.1 and PSD93{alpha} as a positive control. The distribution of PSD93{alpha} was detected by staining with anti-PDZ and visualized by the addition of Texas Red-conjugated secondary antibody. Coexpression of the ion channel and MAGUK results in the co-clustering of both EGFP-Kir2.1 (B) and PSD93{alpha} (D) at, or in close apposition to, the plasma membrane. Fig. 4, C and E, show confocal images of HEK293 cells transfected with EGFP-Kir2.1 and PSD93{delta}. Again, the distribution of PSD93{delta} was detected by staining with anti-PDZ and visualized by the addition of Texas Red-conjugated secondary antibody. EGFP-Kir2.1 (C) and PSD93{delta} (E) both appear at the cell periphery and colocalize in large clusters that appear similar to those induced by PSD-93{alpha}.



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  		FIG. 4.
PSD93{delta} promotes cell surface clustering of Kir2.1. A, confocal image of HEK293 cells expressing EGFP-Kir2.1 alone. EGFP fluorescence shows that Kir2.1 is evenly distributed in the plasma membrane. B and D, confocal image of HEK293 cells expressing EGFP-Kir2.1 and PSD93{alpha}. EGFP fluorescence now shows Kir2.1 to be localized to discrete punctuate regions at or near the membrane (B). The subcellular distribution of PSD93{alpha}, detected by staining with anti-PDZ and visualized by addition of Texas Red secondary antibody, shows that PSD93{alpha} localizes to the same membrane regions as EGFP-Kir2.1 (D). C and E, confocal images of HEK293 cells expressing EGFP-Kir2.1 and PSD93{delta}. EGFP fluorescence again shows Kir2.1 clustering at or near the membrane (C). PSD93{delta} colocalizes to the same membrane regions as EGFP-Kir2.1 (E). F and G, confocal images of HEK293 cells expressing Kir2.1 channels tagged with an extracellular HA epitope (HA-Kir2.1) alone (F) or expressing HA-Kir2.1 and PSD-93{delta} (G). The cells are fixed but not permeabilized, and the membrane distribution of the HA-Kir2.1 was determined by staining with extracellular anti-HA and visualized by the addition of a fluorescein isothiocyanate-conjugated secondary antibody. Expression of HA-Kir2.1 alone results in the channel being evenly distributed in the plasma membrane (F), whereas coexpression of HA-Kir2.1 and PSD-93{delta} results in the formation of ion channel aggregates at the plasma membrane (G).

 
To ensure that the aggregates of EGFP-Kir2.1 seen in the presence of PSD-93{delta} were inserted into the membrane and not, for example, in vesicle pools just beneath the surface, we repeated the experiments outlined above using Kir2.1 channels expressing an extracellular HA epitope tag (28). Fig. 4F shows a confocal image of HEK293 cells transfected with HA-Kir2.1 alone. The cells are fixed but not permeabilized, and the membrane distribution of HA-Kir2.1 was visualized by staining with extracellular anti-HA and a fluorescein isothiocyanate-conjugated secondary antibody. Expression of HA-Kir2.1 alone resulted in the even distribution of the channel in the plasma membrane. Coexpression of HA-Kir2.1 and PSD-93{delta} once again resulted in the formation of large ion channel aggregates at the plasma membrane (Fig. 4G).

Effect of Kir2.1-PSD-93{delta} Association on Ion Channel Function—PSD-93{delta} clearly has a profound effect upon the spatial distribution of Kir2.1 channels at the cell surface. Previous reports have indicated that coexpression of MAGUKs with inwardly rectifying potassium channels can directly affect the current recorded from these channels (12, 32). To determine what effect, if any, association with PSD-93{delta} has on ion channel function, we used the conventional whole-cell clamp technique to record membrane currents from single HEK293 cells that had been transiently transfected with cDNAs encoding either EGFP-Kir2.1 alone or EGFP-Kir2.1 and PSD-93{delta}. Currents were recorded in response to voltage steps from a holding potential of –17 mV (the equilibrium potential for K+ (EK) under these recording conditions: [K+]i = 140 mM, [K+]o = 70 mM) to test potentials ranging from +60 mV to –100 mV in 10-mV increments. Voltage steps negative to EK (–17 mV) elicited substantial inward currents, whereas steps positive to EK (–17 mV) produced only small outward currents, with no current at all passing at depolarizing steps positive to +30 mV (Fig. 5, A and B). These currents are consistent with the expression of the strong inward rectifier Kir2.1. No significant whole cell currents were recorded from nontransfected cells under these conditions. The presence of PSD-93{delta} within the cells had no significant effect upon whole-cell Kir2.1 current amplitudes (Fig. 5B) or the relationship between whole-cell conductance and voltage (Fig. 5C).



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  		FIG. 5.
Characterization of whole-cell Kir2.1 currents in the presence of PSD-93{delta}. A, membrane currents recorded from a single HEK293 cell expressing EGFP-Kir2.1 alone and eGFP-Kir2.1 and PSD-93{delta}, in response to voltage steps from a holding potential of –17 mV to test potentials ranging from +60 to –100 mV in 10-mV increments. Extracellular [K+] was 70 mM; intracellular [K+] was 140 mM. Outward currents are defined as being positive and are shown as upward deflections; inward currents are defined as being negative. B, mean current-voltage relation for cells expressing eGFP-Kir2.1 alone (open circles) or eGFP-Kir2.1 and PSD-93{delta} (filled circles), n = 6 each. At –100 mV, the magnitude of the inward current was 589 ± 62 pA/picofarads for cells expressing eGFP-Kir2.1 alone and 696 ± 84 pA/picofarads for cell expressing both EGFP-Kir2.1 and PSD-93{delta}. C, relationship between chord conductance and membrane potential for cells expressing EGFP-Kir2.1 alone (open circles) or EGFP-Kir2.1 and PSD-93{delta} (filled circles). Chord conductance was computed as gK = IK/(V – EK). Its relation to membrane potential may be fitted by a Boltzmann relation with the relative conductance g'K = (1 + exp((V – V0.5)/k))–1, where V0.5 (mV) gives the voltage at which g'K = 0.5, and k (mV) factor is a of affecting the steepness the relationship. For cells expressing EGFP-Kir2.1 alone, V0.5 = –13.3 ± 1.9 mV; k = 17.3 ± 0.3 mV (n = 5). For cells expressing EGFP-Kir2.1 and PSD-93{delta}, V0.5 = –9.6 ± 2.7 mV; k = 17.2 ± 0.3 mV (n = 5).

 
PSD-93{delta} Stabilizes Kir2.1 at the Cell Surface—Previous studies have suggested that MAGUK proteins not only function to control the spatial localization of ion channels and receptors but also stabilize them at the plasma membrane, possibly by limiting the rate of endocytosis (33, 34). To determine whether association with PSD-93{delta} suppressed the rate of Kir2.1 internalization, we used a modified form of surface biotinylation termed biotin protection. HEK293 cells expressing HA-Kir2.1 alone or HA-Kir2.1 and PSD-93{delta} were incubated in membrane-impermeant disulfide-linked ("cleavable") biotin to biotinylate surface proteins. Cells were then returned to the incubator to allow normal internalization to take place. At various time points (30, 60, 90, or 120 min), cells were removed from the incubator, and biotinylated proteins still present at the cell surface (not yet internalized or recycled) were "stripped" of biotin by incubation in a membrane-impermeant reducing agent. Cells were subsequently lysed, and internalized ("protected") biotinylated proteins were recovered by incubation with strepavidin-agarose. The recovered internalized proteins were separated by SDS-PAGE, and the amount of HA-Kir2.1 that had been internalized over the various time points was assessed by immunoblotting with anti-HA. Fig. 6A shows that in the absence of PSD-93{delta}, HA-Kir2.1 undergoes steady internalization, with the amount of "protected" HA-Kir2.1 gradually increasing with time. In contrast, in the presence of PSD-93{delta}, there is no perceivable difference in the levels of protected HA-Kir2.1 between cells that were stripped immediately following biotinylation (time point 0) and those that were returned to the incubator for 120 min (Fig. 6B), indicating that little internalization occurred during the course of the experiment. These data are summarized in Fig. 6C. Thus, whereas association with PSD-93{delta} does not affect channel function, it seems to regulate the channel's spatial and temporal distribution within the cell membrane.



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  		FIG. 6.
Association with PSD-93{delta} suppresses internalization of Kir2.1. A, in the absence of PSD-93{delta}, HA-Kir2.1 undergoes steady internalization. HEK293 cells expressing HA-Kir2.1 alone were incubated in membrane-impermeant disulfide-linked ("cleavable") biotin to biotinylate surface proteins. Cells were then returned to the incubator to allow internalization to take place for 30, 60, 90, or 120 min, following which proteins still present at the cell surface were "stripped" of biotin. Cells were subsequently lysed, and internalized ("protected") biotinylated proteins were recovered by incubation with strepavidin-agarose. The recovered internalized proteins were separated by SDS-polyacrylamide gel electrophoresis, and the amount of HA-Kir2.1 that had been internalized over the various time points was assessed by immunoblotting with anti-HA. B, in the presence of PSD-93{delta}, levels of protected HA-Kir2.1 remain constant, indicating that little internalization occurred during the course of the experiment. C, time course for the amount of biotinylated HA-Kir2.1 recovered in cells expressing HA-Kir2.1 alone (filled circles) or HA-Kir2.1 and PSD-93{delta} (open circles). The error bars show S.E. (n = 3 separate experiments).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we identify an alternatively spliced isoform of PSD-93/chapsyn 110 as a protein that interacts with the strong inwardly rectifying K+ channels, Kir2.1. As a means of identifying potential Kir2 binding partners within the mammalian central nervous system, we screened a human brain cDNA library using the yeast two-hybrid system and a carboxyl-terminal construct of Kir2.1 (Kir2.1C) as bait. The screen yielded cDNA encoding the first two PDZ domains of the neuronal MAGUK PSD-93, but with a novel extended N-terminal region that diverged from other PSD-93 isoforms just before the first PDZ domain. This clone represented the human homologue of the mouse alternatively spliced PSD-93 isoform, PSD-93{delta}, a variant recently identified as a structural component in cholinergic synapses of autonomic ganglia (27).

PSD-93 is best characterized as a key component of the postsynaptic density at glutamergic synapses and has previously been shown to associate with NMDA receptors (35), {delta}2 glutamate receptors (36), neuronal nitric-oxide synthase (37), voltage-gated potassium channels Kv1.4 (38), and inwardly rectifying potassium channels Kir2.3 (10). Interestingly, we find that the pattern of PSD-93{delta} expression is different from the two major brain isoforms of PSD-93, PSD-93{alpha} and PSD-93{beta}. In agreement with previous reports, we find abundant PSD-93{alpha} and PSD-93{beta} in the cerebral cortex and hippocampus and low levels in the brainstem region (35, 37). In contrast, PSD-93{delta} shows diffuse, low level expression throughout the brain and seems to be entirely absent from regions such as the olfactory bulb. The region showing the highest level of PSD-93{delta} expression is spinal cord, which in contrast expresses relatively modest levels of PSD-93{alpha} and -{beta}. This suggests that whereas we may not have isolated a primary targeting protein for Kir2 channels in the brain, we may have identified a variant that achieves this role in the spinal cord. The ability of antibodies directed against Kir2.1 to coimmunoprecipitate PSD-93{delta} from rat spinal cord lysates is supportive of association of these proteins in this tissue.

Abundant PSD-93 expression of unknown isoforms has been reported previously in the superficial dorsal horn of spinal cord, a primary center for the transmission and processing of pain signals (39, 40). Here PSD-93 plays a familiar role in targeting N-methyl-D-asparate receptors (NMDARs) to postsynaptic membranes. Genetic deletion of PSD-93 lowers the surface expression of NMDAR subunits NR2A and NR2B in dorsal horn neurons and significantly reduces NMDAR-dependent persistent inflammatory or nerve injury-induced neuropathic pain (39). A variant of PSD-93 that is expressed predominantly in spinal cord is therefore an attractive potential target for the treatment of chronic pain. Kir2.3 channels localize to postsynaptic membranes of excitatory synapses in mouse forebrain, probably through association with PSD-95 and/or PSD-93 (10). Since NMDAR activity is voltage-dependent due to Mg2+ block, Inanobe et al. (10) suggested that the presence of Kir channels at these synapses and their ability to modulate the resting membrane potential may impact upon NMDAR function. It is tempting to speculate that Kir2 channels and NMDAR localize to postsynaptic membranes of dorsal horn neurons through interaction with PSD-93{delta}, but little information exists regarding the distribution of Kir channels within the spinal cord. mRNA for Kir2.1, 2.2, and 2.4 has been reported to be present in spinal cord (8, 9), but we are unaware of detailed distribution studies.

In common with other members of the PSD-95/SAP-90 MAGUK family, PSD-93{delta} possesses three N-terminal PDZ domains and C-terminal Src homology 3 and guanylate kinase-like regions. Interaction between Kir2.1 and PSD-93{delta} occurs via a type I PDZ domain recognition motif located at the extreme C terminus of the ion channel. This was confirmed by the ability of a GST fusion protein of the final eight amino acids of Kir2.1 (aa 421–428), which includes a type I PDZ binding motif ((S/T)X(V/I)), to isolate full-length PSD-93{delta} from cell lysates. Interaction between Kir2.1 and the PDZ domains of PSD-93{delta} was verified by overlay of membrane-bound Kir2.1C with GST fusion proteins of the individual PDZ domains. All three PDZ domains of PSD-93{delta} interacted with membrane-bound Kir2.1C, with band intensity being strongest for GST fusion proteins of PDZ2 and PDZ3, indicating that Kir2.1C may have a higher affinity for these domains over PDZ1. This was unexpected, since the C-terminal sequences of Kir2.1, Kir2.2, and Kir2.3 all contain a glutamate at position –3 which is thought to be necessary for binding to PDZ1 and PDZ2 but should not favor binding to PDZ3 (41). Previous work has shown that Kir2.1 binds to PDZ2 of PSD-95, whereas Kir2.2 interacts strongly with PDZ2 of SAP97 (11, 13). This study is the first to demonstrate an interaction with PDZ3 of a MAGUK protein and raises questions about the factors that define interaction specificity. Analysis of PDZ3 binding to the protein CRIPT suggested that amino acids upstream of the last 4 amino acids of the PDZ binding motif do affect ligand binding (41). This is supported by the observation that mutations N-terminal to the PDZ domain recognition motif affect the interaction of Kir2.3 with PSD-95 (12). We have also found that Kir2.1 binds to PDZ3 of SAP97 (42) and that mutations outside of the last 4 amino acids abolish this interaction.2

Whereas PDZ domains regulate the interaction between the Kir2.1 and PSD-93{delta}, sequences located at the N terminus of MAGUK proteins seem critically important for membrane targeting and surface distribution (22, 24, 25). The PDZ domains, Src homology 3 and guanylate kinase regions of PSD-93 are highly conserved among all of the splice variants, but the different isoforms show considerable divergence in their N-terminal domains (27). The most abundant MAGUK at excitatory synapses is PSD-95, a protein closely related to PSD-93 and one that is also subject to extensive N-terminal alternative splicing (26). One of its splice variants, PSD-95{alpha}, has N-terminal cysteine residues at positions 3 and 5 that must be palmitoylated, the reversible post-translational addition of the lipid palmitate, for the MAGUK protein to be correctly localized and clustered at postsynaptic sites (24). N-terminal cysteines are also present in splice variants of PSD-93. PSD-93{alpha} (Cys3 and Cys5) and PSD-93{beta} (Cys5 and Cys7) can be palmitoylated, but in contrast to PSD-95, palmitoylation is not a prerequisite of postsynaptic clustering. This instead depends upon a signal located somewhere in the first 30 amino acids of the PSD-93 protein (25). However, as with PSD-95, palmitoylation of the N-terminal cysteines on PSD-93{alpha} and -{beta} is required for ion channel aggregation at the plasma membrane, at least in heterologous cells (22). PSD-93{delta} contains two pairs of N-terminal cysteines for potential palmitoylation (Cys33/35 and Cys96/98 of the human isoform), and when coexpressed in HEK293 cells, PSD-93{delta} induces the formation of large cell surface aggregates of Kir2.1 channels in a similar manner to PSD-93{alpha} and -{beta} isoforms. Although the cysteine pairs in PSD-93{delta} are much further from the free N terminus than seen in other palmitoylated MAGUKs, both pairs contain hydrophobic consensus sequences that have been found to be necessary for efficient palmitoylation. This suggests that palmitoylation of this PSD-93{delta} may also be important for effective Kir2.1 clustering, although this will require further investigation. Aside from promoting cell surface channel clustering, we find that the association between Kir2.1 and PSD-93{delta} profoundly suppresses the rate of Kir2.1 internalization, effectively stabilizing the channel in the membrane. A role for MAGUKs in the regulation of channel and receptor surface expression is supported by the finding that deletion of the C-terminal PDZ binding motif of NR2B subunits increases the rate of NMDAR internalization in neurons (34), and by a recent study that shows a reduction of surface NR2A and NR2B expression in spinal dorsal horn neurons of PSD-93 knockout mice (39). The rate of internalization of Kv1.4 and Kv4.2 channels is also reduced when coexpressed with PSD-95 (33, 43), and at least in the case of Kv1.4, surface stabilization seems to intimately linked with channel clustering, since PSD-95 mutants that bind but do not cluster Kv1.4 allow rapid channel endocytosis (33). Interestingly, whereas PSD-93{delta} suppresses the rate of Kir2.1 internalization, it does not seem to affect the overall level of channel expression at the cell surface. This is demonstrated by the fact that whole-cell Kir2.1 currents recorded in the presence of PSD-93{delta} were indistinguishable from those recorded in its absence. Similar results have been obtained for Kv1.4 and Kv4.2 (33, 43) and suggest that pathways involved in the insertion or removal of K+ channels into or out of the plasma membrane may be more tightly regulated and synchronized than previously thought.

In conclusion, we demonstrate that PSD-93{delta}, a recently identified novel splice variant of neuronal MAGUK protein PSD-93 interacts with the strong inwardly rectifying K+ channel Kir2.1. Analysis of its tissue distribution suggests that PSD-93{delta} is not a major MAGUK protein in the brain but instead is enriched primarily within spinal cord, and it may thus act as an important regulator of the subcellular location of Kir2 channels within specific regions of the central nervous system.


   FOOTNOTES
 
* This work was supported by the British Heart Foundation, The Royal Society, and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Leicester, University Rd., Leicester LE1 7RH, United Kingdom. Tel.: 116-252-3455; Fax: 116-252-3369; Email: ml27{at}le.ac.uk.

1 The abbreviations used are: Kir, inward rectifier potassium channel; PSD-93, postsynaptic density-93 protein; chapsyn 110, channel-associated proteins of the synapse; PDZ domain, postsynaptic density-95/discs large/zona occludens-1 domain; HA, haemagglutinin; PBS, phosphate-buffered saline; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; MAGUK, membrane-associated guanylate kinase; His, hexahistidine; aa, amino acids; NMDA, N-methyl-D-aspartate; NMDAR, NMDA receptor. Back

2 M. L. Leyland and C. Dart, unpublished observations. Back



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