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J. Biol. Chem., Vol. 275, Issue 36, 28276-28284, September 8, 2000

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Complex Structure and Regulation of Expression of the Rat Gene for Inward Rectifier Potassium Channel Kir7.1^*

Nobuhiro Nakamura, Yoshiro Suzuki, Yugo Ikeda, Michitaka Notoya, and Shigehisa Hirose

From the Department of Biological Sciences, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan

Received for publication, May 3, 2000, and in revised form, June 26, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Genomic organization of the rat inward rectifier K⁺ channel Kir7.1 was determined in an attempt to clarify how multiple species of its mRNA are generated in a tissue-specific manner and how its expression is regulated. The rat Kir7.1 gene spans >40 kilobases (kb) and consists of eight exons; the first four exons encode the 5'-untranslated region that is unusually long (~3 kb). The coding region is located in exons 5 and 6. In the testis, exon 4 is processed as four exons (4a-4d), whereas it is recognized as a single exon in the small intestine. The three major species of rat Kir7.1 mRNA (1.4, 2.2, and 3.2 kb) were found to arise from alternative usage of the two promoters and polyadenylation signals and by alternative splicing of the 5'-noncoding exons. The splicing pattern of the 5'-noncoding exons is quite complex and highly tissue-specific, suggesting that complex mechanisms may operate to regulate the Kir7.1 expression. Deletion and mutational analysis of the promoter activity indicated that the rat Kir7.1 gene is regulated by cAMP through a CCAAT element. The cAMP induction was also demonstrated using the rat follicular cell line FRTL-5 endogenously expressing Kir7.1.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Kir7.1 is a member of the inward rectifier K⁺ channel (Kir)¹ family with two transmembrane spans and a pore-forming hairpin loop (2TM/1P) (1, 2). The functional Kir channels are formed by association of four such 2TM/1P subunits and are characterized by their ability to conduct large inward currents at potentials negative to the K⁺ equilibrium potential and small outward currents at more positive potentials. The presence of inward rectifier K⁺ channels was recognized as early as 1949 (3), and since then the channels have been characterized mainly by a combination of electrophysiological and pharmacological techniques. In the past 7 years, however, triggered by the first molecular cloning of the ATP-regulated inward rectifier ROMK1 (4) and the classical strong rectifier IRK1 (5), a large number of Kir family members have been cloned, expressed in recombinant systems, characterized, and classified into seven subfamilies, Kir1-Kir7, based on their sequence similarities and functional properties such as the sensitivity to ATP (Kir1 and Kir6) and coupling with G proteins (Kir3). The above mentioned ROMK1 and IRK1 are now called Kir1.1 and Kir2.1, respectively. In addition to the molecular dissection aimed at clarifying the structure-function relationship, cloning of Kir family members also helped the determination of their tissue distributions by Northern blot analysis, in situ hybridization, and immunohistochemistry, which in turn, together with the electrophysiological properties, provided useful information concerning their physiological roles. The functions of the Kir family members include maintenance of the resting membrane potential, regulation of the duration of action potential, coupling of cellular metabolism with membrane excitability, and secretion and absorption of K⁺ ions across plasma membranes of epithelial cells to maintain K⁺ homeostasis.

Kir7.1, the latest member of the Kir family, was first described by Krapivinsky et al. (6) in 1998 by computer-assisted data base search combined with molecular cloning for full-length cDNA. Using similar approaches, other groups including ours have also identified the same channel independently (7-9). Kir7.1 shares the 2TM/1P membrane topology with other members but is unique in exhibiting very low single channel conductance, which is due to the replacement with methionine of the positively charged arginine residue in the pore region that is conserved among other Kir family members (6, 10). Kir7.1 occurs in a wide variety of tissues with high expression being found in the choroid plexus (8, 9), thyroid gland (9), kidney (6-9, 11), small intestine (6, 7, 9), and testis (6, 8). As for physiological functions, Kir7.1 has been suggested to help set membrane potential by providing a steady background K⁺ current (6) and to be involved in the transepithelial transport of potassium (8, 9) based, respectively, on its uniquely low single channel conductance and on its high expression in ion-transporting epithelial cells. We further suggested its functional coupling with Na⁺,K⁺-ATPase based on their colocalization, revealed by immunohistochemistry, in the epithelial cells of the choroid plexus and small intestine and the follicular cells of the thyroid gland (9).

For better understanding of the physiological roles of Kir7.1, it is also necessary to know how its levels and activity are regulated to allow the channel to fulfill the needs of particular tissues and cells. In the present study, as a first step to address this question, we isolated the rat Kir7.1 gene, determined its structure and the splicing patterns of the transcript, and showed that the expression of the Kir7.1 gene is under the control of cAMP. Interestingly, deletion and mutational analysis of the promoter region indicated that the cAMP sensitivity is conferred through an inverted CCAAT element rather than the CRE or AP-1 sites as recently recognized in the promoters of the CFTR (12), tryptophan hydroxylase (13), and H ferritin (14, 15) genes. Another question we addressed here is how multiple species of Kir7.1 mRNA are generated, since three major species of the Kir7.1 message have been demonstrated to occur in the rat in a tissue-specific manner: 2.2-kb species in the testis and 3.2- and 1.4-kb species in other tissues.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Library Screening-- A rat testis cDNA library constructed in ZAP II (CLONTECH, Palo Alto, CA) was screened using an EcoRI fragment of rat Kir7.1 cDNA (9). Filters were prehybridized in a solution containing 50% formamide, 5× SSC, 5× Denhardt's solution, and 0.1% SDS for 1 h at 42 °C and then hybridized overnight at 42 °C in the same solution containing ³²P-labeled probe. Filters were washed twice in 2× SSC and 0.05% SDS at room temperature for 20 min and once in 1× SSC and 0.1% SDS at 55 °C for 2 h. Positive inserts were subcloned into pBluescript II SK (Stratagene, San Diego, CA) by the in vivo excision and recircularization process in accordance with the manufacturer's protocol and sequenced.

Genomic Library Screening-- A rat genomic library constructed in EMBL3 SP6/T7 (CLONTECH) was screened as described above. Eight positive inserts were digested with XbaI and NheI or DraI and StuI, and the digests were subcloned into pBluescript II SK using a XbaI or EcoRV restriction site, respectively, and sequenced.

Cloning of the 5'-Flanking Region of the Rat Kir7.1 Gene-- To isolate the 5'-flanking region of the rat Kir7.1 gene, a rat GenomeWalking kit (CLONTECH), which is a tagged rat genomic library, was subjected to nested PCR using Advantage Genomic Polymerase (CLONTECH). The first round PCR was performed using a primer for the tagged sequence of the library, AP1 (5'-GTAATACGACTCACTATAGGGC-3'), rat Kir7.1-specific primer UP-4R (5'-CCTCTAACCTAGCGATACAGTAGTAAGCG-3', antisense), and the following conditions: six cycles of 94 °C for 25 s and 72 °C for 4 min and then 32 cycles of 94 °C for 25 s and 67 °C for 4 min, with a final elongation of 67 °C for 4 min. The second round PCR was performed using the primers AP2 (5'-ACTATAGGGCACGCGTGGT-3', for the tagged sequence nested to AP1) and UP-5R (5'-TGTCGACCTTCAGAGCTGCATCTTCAGGCG-3', antisense), and the following conditions: five cycles of 94 °C for 25 s and 72 °C for 4 min and then 22 cycles of 94 °C for 25 s and 67 °C for 4 min, with a final elongation of 67 °C for 4 min. PCR products were gel-purified, subcloned, and sequenced.

Reverse Transcriptase (RT)-PCR-- RNA was isolated from rat brain, kidney, small intestine, and testis, and poly(A)⁺ RNA was purified using an mRNA purification kit (Amersham Pharmacia Biotech). Single strand cDNA was synthesized from 1 µg of mRNA using SuperScript II reverse transcriptase (Life Technologies, Inc.) and an oligo(dT)_12-18 primer. To isolate alternative spliced forms with different 5'-UTR sequences in small intestine, PCR amplification was performed using the primers S1 (5'-AGGCGTTGGTCCACTTTCCT-3', sense) and A1 (5'-AAGATACACAAGACCTCTTTGAGCAC-3', antisense) and the following conditions: 94 °C for 3 min and then 30 cycles of 94 °C for 1 min, 60 °C for 1 min, and 72 °C for 2 min, with a final elongation of 72 °C for 15 min. To determine tissue-specific usage of 3'-UTR exons, PCR amplification was performed using the primers S4 (5'-CGCCTGCAGTTCCTCTCAGCAATGCAA-3', sense) and A3 (5'-TGAAACTAGAGATACAGACTGT-3', antisense) and the following conditions: 94 °C for 3 min and then 30 cycles of 94 °C for 30 s, 60 °C for 50 s, and 72 °C for 90 s, with a final elongation of 72 °C for 7 min. PCR products were gel-purified, subcloned, and sequenced.

3'-Rapid Amplification of cDNA Ends (RACE)-- 3'-RACE was performed using a 5'/3'-RACE kit (Roche Molecular Biochemicals). cDNA was synthesized from 2 µg of rat small intestine or testis poly(A)⁺ RNA using avian myeloblastosis virus reverse transcriptase and an oligo(dT)-anchor primer (5'-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTT(A/C/G)-3'). The cDNA was amplified using an anchor-specific primer (5'-GACCACGCGTATCGATGTCGAC-3'), and the sense primer P2 (5'-CGCCTGCAGGTCTCTGCTGTACTCTAT-3') and reamplified with the same anchor-specific primer and the primer S4. PCR products were gel-purified, subcloned, and sequenced.

Determination of the Transcription Start Site-- To determine the transcription start site of the rat Kir7.1 gene, the CapSite^TM hunting method (16) was used in accordance with the manufacturer's protocol (Nippon Gene, Tokyo, Japan). CapSite cDNA® from rat small intestine or testis, in which the 5'-terminal m⁷GpppN cap structure of mRNA was removed and recapped by the 3'-end of a specific 38-mer oligonucleotide (rOligo; 5'-CAACGCAATGTTCCATGCGGTGTCGCATACTACGCATT-3') was subjected to nested PCR. The first round PCR was performed using the rOligo-specific primer 1RC (5'-CAAGGTACGCCACAGCGTATG-3') and rat Kir7.1-specific primer A6 (5'-TCAACTTGGCTGAGGTGACATT-3', for amplification of small intestine CapSite cDNA), A1 (for small intestine CapSite cDNA), or A4 (5'-TCTCCTCTGTTAGCTGCCAGT-3', for testis CapSite cDNA). The second round PCR was performed using the rOligo-specific primer 2RC (5'-GTACGCCACAGCGTATGATGC-3') and rat Kir7.1-specific primer A7 (5'-ATAATGGCACAGAGGATACAGCAC-3', for small intestine), A8 (5'-CTTTACAATTCCTGCTGTCCAT-3', for small intestine), or A5 (5'-TGTCATGGACAGTGATGATCA-3', for testis). The reactions were performed using the following conditions: 94 °C for 3 min and then 35 cycles of 94 °C for 30 s, 62 °C for 50 s, and 72 °C for 60 s, with a final elongation of 72 °C for 7 min. PCR products were subcloned and sequenced. The transcription start sites were determined by identification of the boundary sequence between rOligo and rat Kir7.1 mRNA sequence.

DNA Sequencing-- DNA was sequenced by the dideoxynucleotide chain-termination method using a SequiTherm Long-Read cycle sequencing kit (Epicentre Technologies, Madison, WI) and an automated laser fluorescent DNA sequencer (model 4000, LI-COR; Lincoln, NE). DNA sequences were analyzed using the computer program GENETYX-MAC (Software Development, Tokyo, Japan). Exon-intron boundaries were identified by comparing the genomic and cDNA sequences.

Construction of Secreted Alkaline Phosphatase (SEAP) Reporter Vector-- All DNA fragments of the promoter regions 1 and 2 were amplified by PCR using rat genomic DNA as the template, subcloned into the pBluescript II SK vector, and verified by sequencing. The promoter region 1 from nt +76 to 1067 (numbered with respect to the transcription initiation site) was amplified using the upstream primer tagged with the XhoI restriction site (5'-TCCCTCGAGATCTATCATCTCTACAGTCTA-3') and the downstream primer tagged with the HindIII restriction site (5'-GGCAAGCTTCGTTTAGTTTGTTGTCAAGTG-3'). The amplified fragment was subcloned into the XhoI and HindIII sites of the pBluescript II SK vector (designated pBS-P1) or pSEAP2-Basic vector (CLONTECH, designated pP1). The pP1 vector was digested with XhoI and BglII or ApaI, end-filled with Klenow fragments (Takara) and recircularized to generate plasmids containing segments of the promoter region from nt +76 to 551 or 315, respectively (designated pP1b or pP1c). The pBS-P1 vector was digested with XhoI and HpaI, end-filled, and recircularized. And then the segment of the promoter region from nt +76 to 668 was excised with KpnI and HindIII and subcloned into the same sites of the pSEAP2-Basic vector (designated pP1a). The promoter region 2 from nucleotide +64 to 969 (numbered with respect to the transcription initiation site) was amplified using the upstream primer tagged with the HindIII restriction site (5'-TGGAAGCTTTTACTTCAAACAAACAGTGT-3') and the downstream primer tagged with the EcoRI restriction site (P2R1; 5'-AAGGAATTCGGAAGCTAGCAGACTCTTGTC-3'). The amplified fragment was subcloned into the HindIII and EcoRI sites of pSEAP2-Basic vector (designated pP2). The pP2 vector was digested with HindIII and EcoRV, end-filled, and recircularized to generate a plasmid containing a segment of the region from nucleotide +64 to 357 (designated pP2b). The segments of the region from nucleotide +64 to 551, 188, or 91 were amplified using the upstream primers tagged with the XhoI restriction site (5'-TGGCTCGAGCCTTTGCAATTAAGAAAGGGG-3'; P2F3, 5'-TGGCTCGAGGCTTTCTCCAGAGTTGTTAAG-3'; or 5'-TGGCTCGAGCCTGCAGACTAAGTGACCCAAT-3', respectively) and the downstream primer P2R1. The amplified fragments were subcloned into the XhoI and EcoRI sites of pSEAP2-Basic vector (designated pP2a, pP2c, and pP2d, respectively). To change nucleotides 96 to 99 (5'-TTGG-3') to 5'-GCTT-3' in the pP2c plasmid, site-specific mutagenesis was performed as follows. First PCR was performed using P2F3 and the mutagenic primer (5'-GTCTGCAGGGGGGAAGCTTAGCTCTGATCA-3'). Second PCR was performed using the amplified fragment of the first PCR and P2R1, and the resulting fragment was subcloned into the XhoI and EcoRI sites of the pSEAP2-Basic vector (designated pP2c-mut).

Analysis of Promoter Activity-- COS-7 (in Dulbecco's modified Eagle's minimal essential medium containing 10% fetal bovine serum) and CHO-K1 cells (in Ham's modified F-12 medium containing 5% fetal bovine serum) were seeded on 35-mm plates to give 70-90% confluency at the transfection. The appropriate SEAP reporter vector (2 µg) was co-transfected with the pEGFP-C2 vector (CLONTECH, 1 µg) using LipofectAMINE Plus reagent (Life Technologies) according to the manufacturer's instructions. Plasmid DNA (3 µg total) with Plus reagent (12 µl) and LipofectAMINE reagent (8 µl) were first added to Opti-MEM I (Life Technologies) in separate tubes to a total volume of 200 µl each. The solutions were combined and incubated at room temperature for 15 min. An additional 1.6 ml of Opti-MEM I was added, and the mixture was applied to one dish of cells, which was then returned to the tissue culture incubator. After a 3-h incubation at 37 °C, the medium was replaced with 1 ml of standard culture medium with or without 1 mM 8-Br-cAMP. After 48-h transfection, the medium was harvested for measurement of SEAP activity, and the cells were lysed with phosphate-buffered saline for measurement of concentration of green fluorescent protein. SEAP activity was measured using a Great EscAPe SEAP fluorescence detection kit (CLONTECH) according to the manufacturer's instructions. The culture medium containing SEAP (50 µl) was diluted with an equal volume of 1× dilution buffer and incubated at 65 °C for 30 min to inactivate the endogenous alkaline phosphatase activity. The sample was mixed with assay buffer (194 µl) and incubated at room temperature for 5 min. Next, the sample was incubated with 1 mM 4-methylumbelliferyl phosphate (6 µl) at room temperature for 1 h, and then the fluorescence was measured at 460 nm when excited at 360 nm using a CytoFluor-4000 plate reader. Concentration of green fluorescent protein was determined by measuring the green fluorescence in cell lysates at 530 nm when excited at 480 nm. The SEAP activity was normalized for transfection efficiency by the green fluorescent protein concentrations and then averaged from 3-6 independent measurements. Results are expressed as the means ± S.E. Statistical analysis was performed using Student's t test.

Cell Culture-- FRTL-5 cells (ATCC CRL 8305; American Type Culture Collection, Manassas, VA), a strain of rat thyroid follicular cells, were grown in Coon's modified Ham's F-12 medium supplemented with 5% (v/v) calf serum and a six-hormone mixture (6H medium) containing somatostatin (10 ng/ml), hydrocortisone (10 nM), transferrin (5 µg/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), insulin (10 µg/ml), and TSH (1 milliunit/ml). For measuring the effects of TSH, insulin, and 8-Br-cAMP on the levels of Kir7.1, the cells were washed twice with Hanks' balanced salt solution and then maintained in culture medium without TSH and insulin (4H medium) for a period of 4 days before experiments. Under this condition, TSH, insulin, and 8-Br-cAMP were added to the cells in 4H medium at 1 milliunit/ml, 10 µg/ml, and 1 mM, respectively.

Northern Blot Analysis-- Poly(A)⁺ RNA was isolated from the rat stomach, small intestine, and testis, using an mRNA purification kit. Poly(A)⁺ RNA (3 µg/lane) was electrophoresed and transferred to a Magna nylon membrane (Micron Separations Inc., Westborough, MA) and hybridized with a ³²P-labeled rat Kir7.1 cDNA probe. In the case of determining the effects of TSH and cAMP on the Kir7.1 message levels, total RNA was isolated from FRTL-5 cells using the method described previously (9). Total RNA (30 µg/lane) was electrophoresed and transferred to a Magna nylon membrane and hybridized with ³²P-labeled rat Kir7.1, Na⁺,K⁺-ATPase ₁ subunit, or -actin cDNA. Hybridization was performed in the solution containing 50% formamide, 5× SSPE, 2× Denhardt's solution, 0.5% SDS, and 0.1 mg/ml heat-denatured salmon sperm DNA at 42 °C for 16 h, and the membrane was washed twice in 2× SSC and 0.05% SDS at room temperature for 20 min and once in 0.1× SSC and 0.1% SDS overnight at 55 °C. The membranes were exposed to an imaging plate, and the results were analyzed using a BAS-2000 image analyzer (Fuji Film, Tokyo, Japan).

Western Blot Analysis-- FRTL-5 cells were washed six times in Hanks' balanced salt solution, scraped from culture dishes, and pelleted by centrifugation at 1000 × g for 10 min. Pellets were resuspended in 500 µl of lysis buffer (10 mM sodium phosphate, pH 7.4, 1% (v/v) Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 2 mM EDTA, 50 mM NaF, 1 mM benzamidine, 10 mM leupeptin, 1 mM pepstatin, and 1 mM phenylmethanesulfonyl fluoride) and homogenized immediately. The extract was then centrifuged at 20000 × g for 1 h. The supernatants (10 µg of protein) were separated by SDS-polyacrylamide gel electrophoresis and electroblotted to Immobilon-P membrane (Millipore Corp., Bedford, MA). After blocking in TBST (150 mM NaCl, 0.05% Tween 20, and 10 mM Tris-HCl, pH 8.0) containing 5% nonfat milk for 1 h at room temperature, the blot was incubated with a previously characterized anti-Kir7.1 peptide antiserum (9) at a 1:1000 dilution overnight at 4 °C, incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Tago, Burlingame, CA) at a 1:3000 dilution for 4 h at 4 °C, and then developed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium chloride (BCIP/NBT) as chromogenic substrates.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Gene Structure-- A rat genomic DNA library, constructed in EMBL3, was screened for the Kir7.1 gene using, as a probe, the previously cloned cDNA (9). Eight positive clones (Fig. 1A) were isolated and sequenced. By comparison of the nucleotide sequences of the genomic clones with the already known cDNA sequence, most of the exon-intron organization was defined including the exons coding for the entire open reading frame and 3'-untranslated region (Fig. 1A, exons 5-8). However, the number of exons that encode the 5'-untranslated region and their positions could not be determined because of lack of the 5'-UTR sequence information; although our cDNA clone covered the entire 3'-UTR, it contained only a partial sequence of the 5'-UTR. To determine the complete 5'-UTR sequence, therefore, we isolated cDNA clones from a rat testis cDNA library and sequenced them (Fig. 1B). Furthermore, the 5'-most sequence of cDNA was determined by nested PCR amplification of cap site cDNA prepared from rat testis and small intestine mRNA (Figs. 1B, 4, and 5). Again, comparison of the cDNA and genomic DNA sequences allowed us to determine the complete exon-intron organization of the rat Kir7.1 gene (Fig. 1A). The cDNA sequences suggested alternative splicing of Kir7.1 pre-mRNA in the 5'-UTR as described later in detail (Fig. 1B).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Schematic map of the rat Kir7.1 gene and its transcripts. A, eight genomic DNA clones isolated from a rat genomic DNA library and exon-intron organization of the rat Kir7.1 gene. The 5'-flanking region (GW-1) was isolated using a GenomeWalking kit. Clones containing a part of intron 6 and exon 7 of 12 nucleotides (AAAAAAAAAAAG) were not isolated (broken line). Introns are indicated by horizontal lines, and exons are indicated by boxes and numbered 1-8. The filled and open boxes indicate the coding and noncoding exon sequences, respectively. In introns 2 and 3, repetitive DNA elements (ID elements) are present in the reverse direction. Above the genomic map is shown a partial restriction enzyme map for XbaI. B, multiple species of rat Kir7.1 transcripts. These structures were deduced from combinations of cDNA sequence information, which were obtained by cDNA library screening, RT-PCR, 3'-RACE, and CapSite hunting. In the small intestine, at least three isoforms are generated by alternative usage of promoters, alternative splicing, and alternative polyadenylation. In the testis, at least two forms are generated by alternative splicing in exons 1 (1a and 1b) and 4 (4a-4d). Exons 2a and 5a are specific for type 2 transcript. The predicted size of each isoform is shown at the left. The relatively large difference between the observed (~2.2 kb) and calculated (1.9 kb) length of testis transcripts may be due to the presence of a long poly(A) tail in the case of the testistranscripts.

The gene consists of eight exons and spans >40 kb (Fig. 1A). The coding region is located in exons 5 and 6. The exon-intron boundaries (Fig. 2) agree with the consensus dinucleotide sequences for splice donor (GT-) and acceptor (-AG) sites except the donor sites following exons 2a (CT-) and 6 (GC-) and the acceptor sites of introns 4 (-AC) and 7 (-TG). There are three polyadenylation signals in the 3'-UTR, two of which are actually used (Figs. 1B and 8A). Rat ID elements, a class of short interspersed repetitive elements, are present in introns 2 and 3 (Fig. 1A). As described below, multiple mRNA species are generated by alternative usage of two transcription initiation sites and two polyadenylation signals and by alternative splicing of pre-mRNA.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Exon-intron organization and boundary sequences of the rat Kir7.1 gene. The sizes of the exons and introns and nucleotide sequences around the splice sites are indicated. Exon sequences are in uppercase letters, and intron sequences are in lowercase letters. Consensus splice acceptor and donor sequences (34) are shown at the bottom. N.D., not determined.

An interesting duplication of a short segment of the rat Kir7.1 gene was found within its intron (Fig. 3). About 800 nucleotides upstream of exon 6, there is a sequence of ~200 nucleotides that is highly identical to that of the intron 5/exon 6 boundary with complete conservation of the splice acceptor site sequence (Fig. 3, sequence a). If sequence a is used instead of sequence b during splicing, such a transcript generates a C-terminally truncated variant form of Kir7.1, since sequence a contains an in-frame stop codon. However, such a splice variant was not detected despite our intensive search by RT-PCR using mRNA preparations from the rat kidney, intestine, and testis.

View larger version (63K): [in this window] [in a new window]   Fig. 3.   Duplication of a short region around the acceptor site of exon 6. In the intron between exons 5 and 6, there is a short segment (~200 bp, line a and sequence a) that is very similar to the sequence around the splice acceptor site of exon 6 (line b and sequence b). The lower panel compares the sequences. Identical nucleotides are indicated by asterisks. The amino acid sequences corresponding to sequences a and b are shown above and below the nucleotide sequences, respectively. The splice acceptor site of exon 6 is indicated by a filled box.

Transcription Initiation Site and Potential Regulatory Elements-- The transcription start site of the rat Kir7.1 gene was determined by CapSite hunting (16), which consists of the following four steps: 1) isolation of poly(A)⁺ RNA, 2) replacement of the cap structure (m⁷Gppp) at the 5' end of mRNA with an oligoribonucleotide (rOligo), 3) RT-PCR using primers complementary to rOligo and the cDNA of interest, and 4) nested PCR to increase specificity. The primers used in the present study are indicated in Figs. 4B and 5B. Sequencing of the nested PCR products (Figs. 4A and 5A) revealed that there are two transcription start sites: one defining the 5'-end of exon 1 and the other 105 nucleotides downstream of the 5'-end of exon 4d. The relationship of exon 4 and exons 4a-4d will be discussed later.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Determination of transcription start site and sequence analysis of its 5'-flanking region (promoter 1). A, identification of the cap site by nested PCR. CapSite hunting method (16) was used to determine the transcription start site of the rat Kir7.1 gene. CapSite cDNA from rat small intestine or testis was subjected to nested PCR. The first round PCR was performed using the rOligo-specific primer 1RC and rat Kir7.1-specific primer A6 (for amplification of small intestine CapSite cDNA) or A4 (for amplification of testis CapSite cDNA), whose positions are shown in B. The second round PCR was performed using the rOligo-specific primer 2RC and rat Kir7.1-specific primer A7 (for small intestine, lane 1) or A5 (for testis, lane 3), whose positions are also shown in B. For negative control, the second round PCR was performed using the primer 2RC alone (lanes 2 and 4). The resulting products were analyzed by 1.3% agarose gel electrophoresis and visualized by ethidium bromide staining. DNA size marker (in bp) is shown on the left. B, positions of the primers used for nested PCR (arrowheads) and products of CapSite hunting (double-headed arrows). The structure of CapSite cDNA is shown by a combination of boxes (closed box representing rOligo sequence that is artificially introduced as primer sites and open numbered boxes representing the corresponding 5'-noncoding exons). C, nucleotide sequence of the promoter region 1. The transcription start site, determined by sequencing the products of CapSite hunting, is indicated by an arrow, and the downstream sequence transcribed as exon 1 is shaded. This start site is used in both small intestine and testis, but in the case of small intestine a second start site (Fig. 5) is also used as schematically summarized in Fig. 1B. A potential TATA box is located at position 80 relative to the transcription start site. A putative CCAAT-box and binding sequences for GATA-1, AP-1, Oct-2, HNF-5, MyoD, C/EBP, and glucocorticoid receptor (GR) are indicated.

An approximately 1-kb fragment of the 5'-flanking region (promoter 1) of the gene was obtained using a rat GenomeWalking kit and sequenced (Fig. 4C). It contained a TATAA box located at 80 and consensus motifs for several transcription factors, some of which are shown in Fig. 4C. Between the TATAA box and transcription start site, there is a unique repeat of 26 adenines.

The promoter 2 region, upstream from the second transcription start site in exon 4d, was also analyzed and found to contain a TATAA box (33), which is preceded by a CCAAT box (75) and a canonical CCAAT sequence (ATTGG, 100). These locations of the TATAA and CCAAT motifs strongly suggest that they are functional. Putative binding sites for transcriptional factors such as AP-1, GATA-1, and CREB/ATF are shown in Fig. 5C.

View larger version (48K): [in this window] [in a new window]   Fig. 5.   Determination of alternative transcription start site and analysis of its 5'-flanking region (promoter 2). A, cap site analysis by nested PCR using, as template, CapSite cDNA from rat small intestine. The first round PCR was performed using primers 1RC and A1, whose positions are shown in B. The second round PCR was performed using primers 2RC and A8 (lane 1). For negative control, the second round PCR was performed using 2RC alone as primers (lane 2). The resulting products were analyzed by 1.3% agarose gel electrophoresis and visualized by ethidium bromide staining. DNA size marker (in bp) is shown on the left. B, positions of the primers used (arrowheads) and length of the product of CapSite hunting (double-headed arrow). C, nucleotide sequence of the promoter region 2. By sequencing the PCR-amplified CapSite cDNA, a second start site (arrow) is located in exon 4d. The potential TATA box is located at position 33 relative to the transcription start site. The putative CCAAT-box (at 75) and its canonical form (ATTGG, at 100) and binding sequences for GATA-1, AP-1, Oct-R, CF-1, and ATF-1 (CRE; cAMP-responsive element) are indicated. Shaded boxes represent exons 4b, 4c, and 4d.

Deletion and Mutational Analysis of Promoters of the Rat Kir7.1 Gene-- To determine whether the putative promoter regions identified above are functional, we performed reporter gene analysis using the SEAP reporter system. A series of deletion mutants of the fragments containing the promoter 1 (1067 to +76; numbers refer to the positions relative to the transcription start site) and promoter 2 (969 to +64) sequences were constructed, inserted into the pSEAP2-Basic reporter vector, and assayed for the SEAP activity in the culture medium of transiently transfected COS-7 and CHO-K1 cells. In the case of promoter 1, no significant basal transcriptional activity was observed in either COS-7 or CHO cells (Fig. 6). When 8-Br-cAMP was applied to COS-7 cells transfected with the full-length construct pP1 that contains a CCAAT/enhancer-binding protein (C/EBP) binding site, a CAAT box, and an AP-1 binding site, there was a marked increase (Fig. 4C). Deletion constructs lacking the C/EBP binding site and CAAT box showed no response to 8-Br-cAMP, a membrane-permeable analogue of cAMP, although they contain the AP-1 binding site (pP1a, Fig. 6).

View larger version (27K): [in this window] [in a new window]   Fig. 6.   Promoter activity of the rat Kir7.1 gene in COS-7 and CHO-K1 cells. Left panel, schematic representations of DNA constructs containing various lengths of the promoter regions 1 (A) and 2 (B) of the rat Kir7.1 gene linked to the SEAP reporter gene. All constructs were co-transfected with pEGFP-C2 to normalize the transfection efficiency. Cells were incubated for 48 h in the presence or absence of 1 mM 8-Br-cAMP. Right panel, normalized SEAP activities of the indicated constructs measured in COS-7 and CHO-K1 cells untreated or treated with 8-Br-cAMP. The activity is expressed as the value relative to that obtained with promoterless control plasmid, pSEAP2-Basic, in the absence of 8-Br-cAMP. Open and closed bars indicate the activities in COS-7 cells untreated or treated with 8-Br-cAMP, respectively. Shaded and hatched bars indicate the activities in CHO-K1 cells untreated or treated with 8-Br-cAMP, respectively. In the mutated construct (pP2c-mut), the inverted CCAAT sequence (5'-ATTGG-3') was changed to 5'-AGCTT-3' (the position is indicated by ×). Values are the means ± S.E. of 3-6 independent experiments. The asterisk in A indicates a significant difference between 8-Br-cAMP-treated and nontreated cells (p < 0.05, Student's t test).

The full-length promoter 2 construct, pP2, exhibited significant levels of basal activity in both COS-7 and CHO cells, which were strongly stimulated by cAMP as typically seen in CHO cells (Fig. 6). When analyzed in CHO cells, deletions from nucleotide 969 to 188 (pP2a-c) showed marked increases in the basal and cAMP-stimulated activities, suggesting the presence of suppressor sequences in the deleted region (969 to 189). Further deletion to nucleotide 91 resulted in lowering of the promoter activity to a very low level (Fig. 6, pP2d). As mentioned above, the promoter 2 region contains the following transcription regulatory elements: a TATAA box (33), a CCAAT box (75), a canonical CCAAT sequence (ATTGG, 100), and a CRE sequence (ACGTCA, 200). We first considered that the CRE-like sequence at nucleotide 200 might be the element responsible for the cAMP sensitivity, but its deletion did not reduce the sensitivity (Fig. 6B; pP2c), leaving us with the canonical CCAAT sequence at nucleotide 100 as the most likely candidate. Mutation analysis indicated that this is the case. A mutation in the candidate region, which changes the sequence ATTGG to AGCTT, decreased the basal and cAMP-stimulated promoter activity to a level seen with a promoter deletion to 91 (Fig. 6, pP2c-mut). These results indicate that both promoters 1 and 2 are highly cell type-specific and dependent on cAMP levels.

Multiple Species of mRNA Generated by Alternative Usage of Promoters and Polyadenylation Signals and by Alternative Splicing of Noncoding Exons-- Northern blot analyses indicated that there are three major transcripts (1.4, 2.2, and 3.2 kb) of the rat Kir7.1 gene in a tissue-specific manner (Fig. 7 and Ref. 8). The testis expresses exclusively the 2.2-kb form, and the other two forms of 1.4 and 3.2 kb are predominantly transcribed in the small intestine, stomach, kidney (8), lung (8), brain (8), and thyroid (9). To determine how these transcripts are generated, we performed cDNA cloning, RT-PCR, and 3'-RACE. We first focused on the testis-specific 2.2-kb transcript and isolated several testis cDNA clones and found that they all use the first polyadenylation signal (ATTAAA) present 32 nucleotides downstream from the stop codon TAA in exon 6 (Figs. 1B and 8). The possibility of using the second polyadenylation signal in exon 8 was eliminated by RT-PCR (Fig. 8B). Analysis of the cDNA clones also revealed the presence of two splice variants with similar sizes: one skipping exons 1b, 4b, 4c, and 5a and the other skipping exons 4d and 5a (Fig. 1B). Cap site analysis indicated that only promoter 1 is used in the testis. The calculated length (1.9 kb) of the testis transcript is shorter than the size (2.2 kb) estimated by Northern analysis, suggesting the presence of a relatively long poly(A) tail (1.9 kb + poly(A)  2.2 kb).

View larger version (83K): [in this window] [in a new window]   Fig. 7.   Multiple forms of rat Kir7.1 mRNA and their tissue-specific expression. Poly(A)+ RNA (3 µg) from the indicated tissues was analyzed by Northern blotting using 32P-labeled rat Kir7.1 cDNA spanning the coding region. Transcripts of three different sizes (about 1.4, 2.2, and 3.2 kb) were detected in a tissue-specific manner. Here, 1.4- and 3.2-kb transcripts are seen in stomach and small intestine; these two bands are also present in other tissues of rat including the brain, thyroid, lung, and kidney as previously demonstrated (8, 9). The testis appears to express only the 2.2-kb species. The positions of size markers are indicated to the right.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Tissue-specific 3'-UTR sequences, derived from exons 6-8, of rat Kir7.1 transcripts. A, sequence of 3'-UTR of rat Kir7.1 mRNA determined by 3'-RACE. The Kir7.1 transcripts in the rat small intestine were found to be alternatively polyadenylated by use of either the polyadenylation signal near the end of exon 6 (dark box) or that of exon 8 (long dark box). When the proximal polyadenylation signal in exon 6 (ATTAAA) is used, the addition of a poly(A) tail occurs 24 or 46 nucleotides downstream (vertical arrowheads); in the case of the distal polyadenylation site, the signal is tandemly arranged (AATAATAAA) and followed by a poly(A) tail, 15 nucleotides downstream. In the testis, however, only the first polyadenylation signal is used. Boundaries of exons 6 and 7 and exons 7 and 8 are indicated by vertical lines. The translation termination codon (TAA) is indicated by stop. Open box indicates an AATAAA sequence that is not used as a basic polyadenylation signal. Waved lines indicate adenylate/uridylate-rich element (ATTTA and TTATTTATA) considered to be involved in message destabilization. Positions of primers for RT-PCR are marked by arrows. B, detection of the 3'-most exon 8 sequence in the rat Kir7.1 transcripts in the brain, kidney, and small intestine but not in the testis. One µg of mRNA from the rat brain, kidney, small intestine, or testis was reverse transcribed with oligo(dT) primer and subjected to PCR amplification using specific primers (S4 and A3, indicated in A). The resulting products were resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. The transcript containing the exon 8 sequence is represented by a 640-bp product (arrowhead), which is seen in brain, kidney, and small intestine samples but is not present in the testis transcripts. DNA size marker (in bp) is shown on the left.

In the small intestine, similar analyses (Figs. 4, 5, 8, and 9) revealed three types of transcripts, types 1-3, that can be grouped into two categories based on their sizes: large ones (3.1 and 3.3 kb) corresponding to the 3.2-kb band of Northern analysis and small ones (1.3 kb) corresponding to the 1.4-kb band. The large forms are transcribed by the use of the first promoter located 5' adjacent to exon 1 (Figs. 1B and 9). Although the type 1 transcript of 3.3 kb has a short 3'-UTR, it contains long exon 4, making it the largest transcript. Type 2 (3.1 kb) contains relatively long exon 5a and long 3'-UTR. The short form (type 3) is a product of a combination of the second promoter near exon 4d and the first polyadenylation signal in exon 6 (Figs. 1B, 5, and 9).

View larger version (31K): [in this window] [in a new window]   Fig. 9.   RT-PCR analysis of alternatively spliced messages in the rat small intestine. A, positions and combinations of primers used for RT-PCR amplification of Kir7.1 mRNA. B, analysis of splicing patterns of Kir7.1 transcripts in the rat small intestine by RT-PCR followed by agarose gel electrophoresis. One µg of mRNA from the rat small intestine was reverse transcribed with oligo(dT) primer and subjected to PCR amplification. The resulting products were resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining. A DNA size marker (in kb) is shown on the left. Two forms (lane 1) were obtained that had different 5'-UTR, type 1 (2198 bp) and type 2 (984 bp), using primers S1 and A1 that were designed to amplify the transcripts from the first promoter (promoter 1). Sequencing of the two bands revealed the splicing patterns of the 5'-noncoding exons as shown in Fig. 1B. To determine the 3'-UTR structures of types 1 and 2 transcripts, two primer sets were designed whose positions are shown in A: 1) S2 and A3 (lane 2) or A2 (lane 3) and 2) S3 and A3 (lane 4). S2 is specific for the type 1 transcript containing the exon 4d sequence, and S3 is specific for type 2 with exon 5a. No DNA band was detected using S2 and A3 (lane 2), indicating that type 1 uses the proximal polyadenylation signal and lacks the exon 8 sequence; lane 3 represents positive control. One major band was detected using S3 and A3 (lane 4), indicating that type 2 contains exon 8. Concerning the transcript from promoter 2 in exon 4d (type 3 transcript), its 5'-UTR structure was established by CapSite hunting (Fig. 5B), and the short nature of its 3'-UTR was established by PCR (lanes 2 and 3).

The RNA splicing patterns in the testis and small intestine are summarized in Fig. 1B. Noteworthy is the splicing concerning exon 4. In the small intestine, exon 4 is either incorporated or eliminated as a whole, resulting in the 3.3-kb type 1 or 3.1-kb type 2 mRNA (Fig. 1B) that cannot be distinguished by Northern analysis, whereas, in the testis, exon 4 is recognized as four discrete exons (exons 4a-4d) and three introns (gray boxes). Exon 4 in the small intestinal mRNA is not due to partially spliced pre-mRNA, since RT-PCR amplification of the region yielded the exon 4 sequence and not the separate 4a-4d sequences. In the testis, removal of intron 1 occurs in two ways using two splice donor sites and one acceptor site (Fig. 1B). In the small intestine, the lengths of exons 2 and 5 vary depending on the splicing using 1) two donor sites and one acceptor site and 2) one donor site and two acceptor sites, respectively (Fig. 1B).

Regulation of Expression of the Kir7.1 Gene by TSH-- Kir7.1 has previously been demonstrated to be highly expressed in the brain, thyroid gland, lung, stomach, kidney, small intestine, prostate, and testis (6-9). Immunohistochemistry (9) and in situ hybridization histochemistry (8) further established its epithelial cell localization in the choroid plexus and small intestine and follicular cell localization in the thyroid gland. We considered that cell lines derived from these tissues may, if available, be useful for studying the regulation of expression of the Kir7.1 gene and found that a rat follicular cell line, FRTL-5, is such a cell line expressing Kir7.1.

Fig. 10 shows that in FRTL-5 cells, the expression of Kir7.1 is stimulated by TSH. The addition of TSH to the culture medium increased the Kir7.1 mRNA levels time-dependently (t  8 h) (Fig. 10, upper panel). As previously reported by others (17), similar induction was observed in the Na⁺,K⁺-ATPase mRNA levels (Fig. 10, middle panel). The stimulatory effect was also confirmed at the protein level by Western blotting (Fig. 11A). The action of TSH is known to be mediated through the second messenger cAMP; we therefore examined the effects of 8-Br-cAMP, a membrane-permeable analog of cAMP. As expected, it exerted similar stimulatory effects on the Kir7.1 expression (Fig. 11B, lane 5). Insulin, a hormone known to increase the message levels of the TSH receptor and thyroglobulin in the thyroid follicular cells (18, 19), also increased the levels of Kir7.1 in the FRTL-5 cells (Fig. 11B, lane 3).

View larger version (112K): [in this window] [in a new window]   Fig. 10.   Time course of Kir7.1 mRNA induction by TSH in FRTL-5 cells. Confluent FRTL-5 cells, maintained in 4H medium for 4 days, were grown in the presence of 1 milliunit/ml TSH for various time intervals, at which time total RNA was extracted. Upper panel, Northern blot analysis of Kir7.1 mRNA using 30 µg of total RNA per lane. RNA size markers (in kb) are shown on the right. Induction by TSH of 3.2- and 1.4-kb transcripts is evident. Middle panel, Northern blot analysis of the message levels of Na+,K+-ATPase 1 subunit. As reported by Pressley et al. (17), the levels were also significantly increased by TSH as clearly seen at 36 and 48 h. Lower panel, Northern blot analysis of -actin mRNA performed as a loading control.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Effect of TSH, 8-Br-cAMP, or insulin on Kir7.1 protein levels in FRTL-5 cells. A, Western blot analysis of Kir7.1 in FRTL-5 cells treated with TSH for the indicated time periods. Confluent FRTL-5 cells, maintained in 4H medium for 4 days, were grown in the presence of 1 milliunit/ml TSH for various times, and total cell homogenates were prepared and subjected to Western blotting (10 µg of protein/lane). Kir7.1 protein (~54 kDa) was visualized with anti-Kir7.1 peptide antiserum at a dilution of 1:1000. B, effects of TSH (1 milliunit/ml, lane 2), insulin (10 µg/ml, lane 3), TSH plus insulin (6H, lane 4), and 8-Br-cAMP (1 mM, lane 5) on the Kir7.1 protein levels. FRTL-5 cells were treated with various agents for 48 h and processed for Western blotting as in A. 4H, basic culture medium for FRTL-5 cells containing the following four hormones: somatostatin, hydrocortisone, transferrin, and glycyl-L-histidyl-L-lysine; 6H, 4H plus two additional hormones, TSH and insulin.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The Kir family belongs to the superfamily of K⁺ channels, which comprises a diverse family of members classified into three major structural categories: 1) those possessing the 2TM/1P membrane topology as typically seen in the Kir family members, 2) those possessing four transmembrane spans and two pore regions (4TM/2P) in a single subunit, and 3) those possessing six transmembrane spans and a pore region (6TM/1P) as represented by voltage-gated Kv channels (20, 21). Each category or family has multiple subfamilies that also comprise multiple members. Despite a large number of cDNA sequences of K⁺ channels, information on their gene structures and transcriptional regulation is limited except the case of Caenorhabditis elegans, in which nearly 80 genes have been identified that most likely encode the above mentioned three classes of K⁺ channels (22, 23). For example, among the 18 members of the Kir family so far identified in vertebrates by cDNA cloning (2, 24), only four of them have been characterized in their entire span and two of them only partially: Kir1.1 (the gene locus is referred to KCNJ1) (25), Kir2.1 (KCNJ2) (26), Kir3.1 (KCNJ3) (27), Kir3.4 (KCNJ5, partial) (28), Kir6.1 (KCNJ8) (29), and Kir7.1 (KCNJ13, partial) (30). In this study, we determined the complete exon-intron organization, transcription start sites, putative cis-acting elements, and splicing patterns of the gene for rat Kir7.1 in an effort to understand the evolutionary relationship among the Kir family members and regulation of its expression.

The positions and numbers of introns of Kir7.1 are quite different from those of the other Kir family genes so far characterized, including Kir1.1 (25), Kir2.1 (26), Kir3.1 (27), Kir3.4 (28), and Kir6.1 (29). This appears to be consistent with the results of phylogenetic analysis that indicated early divergence of Kir7.1 from the other members (6). The 5' region of the rat Kir7.1 gene is surprisingly complex, with 11 5'-noncoding exons (exons 1a, 1b, 2a, 2b, 3, 4a-4d, 5a, and 5b) and two promoter sites, and hence their expressions are quite complicated, giving rise to multiple transcripts that have differential tissue expression (Fig. 1). The total length of the 5'-noncoding exons is unusually long (3.2 kb). Another interesting unusual finding is that the 5'-noncoding exon 4 is processed as a single exon in the rat small intestine, whereas it is recognized as four exons (4a-4d) separated by three introns in the testis. The 3'-UTR is also interrupted by two introns, and the resulting 3'-noncoding exons 7 and 8 are used in a tissue-specific manner; exon 8 contains two ATTTA motifs that are considered to be associated with rapid mRNA turnover. This complexity may be related to tissue-specific control of message transport, translation, and lifetime.

The rat Kir7.1 gene is known to generate at least three transcripts, 1.4, 2.2, and 3.2 kb, in a tissue-specific manner (8), whereas the human counterpart yields a single species of 3.2 kb (6, 7, 9). The present analysis of the rat Kir7.1 gene revealed that the multiplicity in the message size is mainly due to alternative usage of multiple promoters and polyadenylation signals and that, quite unexpectedly, each species is a mixture of mRNA molecules with similar sizes but with different 5'- and 3'-UTR sequences generated by various combinations of the noncoding exons by alternative RNA splicing (Fig. 1). Although human Kir7.1 mRNA gives a single band (~3.2 kb) on Northern blot analysis, the band is likely to represent a number of RNA species containing, like the rat transcripts, distinct 5'-noncoding sequences. In fact, the available cDNA sequences for human Kir7.1 are not identical in their 5'-noncoding region and exhibit very complicated patchwork-like patterns of sequence similarity (accession numbers: AB013889, AJ007557, AJ006128), suggesting the presence of multiple 5'-noncoding exons and their alternative splicing. The presence of two promoters and multiple transcripts with unusually complex 5'-UTR structures implies that Kir7.1 is one of the key regulators of K⁺ homeostasis so that its levels should be controlled precisely not only transcriptionally but also posttranscriptionally to meet the demand of specific cells for K⁺. The complexity reported here for the structures of the rat Kir7.1 gene and its transcripts may constitute a useful basis for future studies on the tissue-specific regulation of expression of the Kir7.1 channel gene.

Although a limited number of the K⁺ channel family genes have been characterized, their promoters except that of Kir1.1 (ROMK1) have been shown to lack TATA consensus sequences as often seen in housekeeping genes. The rat Kir7.1 promoters P1 and P2, however, contain a classical TATA box at positions 80 (Fig. 4C) and 33 (Fig. 5C), respectively. Furthermore, promoter 2 contains a CAAT box and a canonical CAAT box in a reasonable context (at 75 and 100, respectively). This classical nature of the promoters makes the Kir7.1 gene unique among the family members. The Kir1.1 gene KCNJ1 also has the TATA and CAAT boxes (25), and, consistent with this classical nature of the promoter elements, its expression is highly restricted to the specific segments of the renal tubules in a splice variant-dependent manner (31, 32).

Sequence analysis of the promoter regions of the rat Kir7.1 gene for cis-acting elements revealed the presence of CRE and AP-1 sites, which prompted us to ask whether the expression of the Kir7.1 gene can be regulated by cAMP. Reporter gene assays using transient expression systems demonstrated that both promoters 1 and 2 are functional and their activities are enhanced by a cAMP analog, but deletion analysis suggested, contrary to our expectation, that the elements responsible for the cAMP-mediated regulation of promoters 1 and 2 are the binding site for C/EBP and/or CAAT element and the inverted CAAT sequence, respectively, rather than CRE and AP-1 sites. Using the relatively strong promoter, promoter 2, this possibility was confirmed by mutational analysis, in which the inverted CAAT sequence is altered by site-directed mutagenesis (Fig. 6, pP2c versus pP2c-mut). Similar cases of cAMP-mediated induction of non-CRE-containing promoters have been observed in several genes including those of CFTR (12), tryptophan hydroxylase (13), and H ferritin (14, 15). For example, Pittman et al. (12) demonstrated the requirement of an inverted CCAAT element and involvement of C/EBP for cAMP-mediated regulation of the CFTR gene. In the case of the human H ferritin gene, the CAAT-binding factor (NF-Y) has been shown to form a complex with the co-activator p300/CREB-binding protein on a single CAAT element (15). We next tried to extend the analysis of cAMP induction to the cells originally expressing Kir7.1 and observed, by using FRTL-5 (a rat thyroid follicular cell line), a significant induction of Kir7.1 by cAMP in both message and protein levels. Cyclic AMP is expected to exert its stimulatory effects not only at the transcription level but also at the posttranslational level, since the channel protein contains consensus phosphorylation sites for cAMP-dependent protein kinase (6, 8, 9). Similar cAMP-dependent regulation of expression and activity of Kir7.1 may also be operative in other cells expressing Kir7.1 such as the epithelial cells of the choroid plexus and small intestine (9). We previously suggested a functional coupling of Kir7.1 and Na⁺,K⁺-ATPase based on their abundance and colocalization in the ion-transporting epithelial cells and the same polarity of distribution in the apical or basolateral side of plasma membranes (9). In this context, the fact that the levels of Na⁺,K⁺-ATPase can be regulated by cAMP (17) and Na⁺,K⁺-ATPase has many cAMP-dependent protein kinase phosphorylation sites (33) appears to be noteworthy.

	ACKNOWLEDGEMENTS

We thank Dr. Hidenari Sakuta for discussion and Setsuko Satoh for secretarial assistance.

	FOOTNOTES

^* This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sport and Culture of Japan, Research Grant for Cardiovascular Diseases 11C-1 from the Ministry of Health and Welfare of Japan, and an SRF grant for biomedical research.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. Section 1734 solely to indicate this fact.

The nucleotide sequences reported in this paper have been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession numbers AB034241 and AB034242.

To whom correspondence should be addressed. Tel.: 81-45-924-5726; Fax: 81-45-924-5824; E-mail: shirose@bio.titech.ac.jp.

Published, JBC Papers in Press, June 27, 2000, DOI 10.1074/jbc.M003734200

	ABBREVIATIONS

The abbreviations used are: Kir, inward rectifier potassium channel; 8-Br-cAMP, 8-bromoadenosine 3',5'-cyclic monophosphate; CFTR, cystic fibrosis transmembrane conductance regulator; CRE, cAMP-responsive element; CREB, CRE-binding protein; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-PCR; SEAP, secreted alkaline phosphatase; TSH, thyroid-stimulating hormone; UTR, untranslated region; bp, base pair(s); kb, kilobase(s) or kilobase pair(s); CHO, Chinese hamster ovary; C/EBP, CCAAT/enhancer-binding protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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