Alternative Splicing of sur2 Exon 17 Regulates Nucleotide Sensitivity of the ATP-sensitive Potassium Channel*

William A. ChutkowDagger , Jonathan C. Makielski§, Deborah J. NelsonDagger , Charles F. BurantDagger , and Zheng Fan§parallel

From the Dagger  Departments of Medicine and Pharmacological and Physiological Sciences, the University of Chicago, Chicago, Illinois 60637 and the § Department of Medicine, the University of Wisconsin, Madison, Wisconsin 53792

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATP-sensitive potassium channels (KATP) are implicated in a diverse array of physiological functions. Previous work has shown that alternative usage of exons 14, 39, and 40 of the muscle-specific KATP channel regulatory subunit, sur2, occurs in tissue-specific patterns. Here, we show that exon 17 of the first nucleotide binding fold of sur2 is also alternatively spliced. RNase protection demonstrates that SUR2(Delta 17) predominates in skeletal muscle and gut and is also expressed in bladder, fat, heart, lung, liver, and kidney. Polymerase chain reaction and restriction digest analysis of sur2 cDNA demonstrate the existence of at least five sur2 splice variants as follows: SUR2(39), SUR2(40), SUR2(Delta 17/39), SUR2(Delta 17/40), and SUR2(Delta 14/39). Electrophysiological recordings of excised, inside-out patches from COS cells cotransfected with Kir6.2 and the sur2 variants demonstrated that exon 17 splicing alters KATP sensitivity to ATP block by 2-fold from approx 40 to approx 90 µM for exon 17 and Delta 17, respectively. Single channel kinetic analysis of SUR2(39) and SUR2(Delta 17/39) demonstrated that both exhibited characteristic KATP kinetics but that SUR2(Delta 17/39) exhibited longer mean burst durations and shorter mean interburst dwell times. In sum, alternative splicing of sur2 enhances the observed diversity of KATP and may contribute to tissue-specific modulation of ATP sensitivity.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

ATP-sensitive potassium channels (KATP),1 first described in cardiac myocytes (1, 2), are weak inwardly rectifying K+ channels, which are inhibited by intracellular ATP and activated by Mg-ADP (3, 4). KATP are found in a diversity of tissues, including cardiac, skeletal, and smooth muscles, brain, and the beta -cell of the pancreas (5, 6). In these cells, KATP couple metabolic stresses to membrane excitability (5) and therefore play a key role in regulating physiological events such as muscle contraction, control of vascular tone, and insulin secretion (7-9). KATP are also generally characterized by their sensitivity to inhibition by a class of antihyperglycemic drugs, sulfonylureas, and to activation by a number of K+ opening drugs such as the antihypertensive diazoxide and the vasodilative pinacidil (10, 11). The properties of KATP with respect to nucleotide block, activation, and drug responsiveness vary widely in the literature, especially with respect to the tissue specificity (4, 6, 9, 12, 13). The recent cloning of a number of KATP channel subunits (14-16) has established a molecular framework underlying the structural basis for functional diversity of KATP channels in various cells.

Current evidence indicates that KATP are hetero-octameric complexes (17, 18) consisting of at least two types of subunits as follows: a member of the small inward rectifier potassium channel family, Kir6.1 (ubiquitous KATP) or Kir6.2 (beta -cell inward rectifier), and a sulfonylurea receptor regulatory subunit, sur1 or sur2, which are members of the ATP binding cassette superfamily (ABC proteins) (14-16, 19-21). The coexpression of both types of subunits yielded K+ currents inhibitable by cytoplasmic ATP and sulfonylurea compounds (16, 20). When the same pore protein, Kir6.2, is expressed either with sur1 or sur2, the resulting channels exhibit identical conductive properties but differ in their ATP block profile and drug responses. Channels from sur1 were inhibited half-maximally with 10 µM ATP and were responsive to the potassium channel opener, diazoxide (20), whereas channels from sur2 demonstrated a half-maximal block at 100 µM ATP and were insensitive to diazoxide (16).

To date, five species of sur mRNA transcribed from two different sur genes have been isolated and shown to yield functionally distinct KATP when expressed with Kir6.2. sur1 (14) confers typical beta -cell KATP properties. sur2 identified by Inagaki et al. (16) resembles the cardiac type KATP. A variant of the sur2 carboxyl-terminal domain (CTD) arises from the excision of the final 129 coding bp of sur2 (along with 48 bp of the sur2 3'-untranslated sequence) and results in the expression of a unique CTD from the use of 129 bp of the 3'-untranslated sequence as coding sequence. This splice variant is described as SUR2B. SUR2B yields recombinant KATP channels with restored diazoxide sensitivity (22) and resembles a smooth muscle type KATP (16, 20, 22). The expression pattern of these sur species corresponds to their tissue-predictive functional properties. A third sur2 variant lacking exon 14, named SUR2A (by coincidence, as Isomoto et al. (22) denoted the original sur2 as SUR2A), has been identified only in heart (15). Each of the identified sur2 variants results from a single exon alternative splicing event. Examination of the mouse sur2 genomic structure reveals that the sur2 intron/exon boundaries2 are comparable to the published genomic structure of human SUR1, with 40 exons available for the mRNA coding region.3 Here, we adopt a system of sur2 nomenclature based on the exons involved in alternative splicing to clarify the identified sur2 variants, as well as future yet to be described variants resulting from alternative splicing. The alternate carboxyl terminus usage of exon 40 replacing exon 39, SUR2B, is denoted as SUR2(40). Likewise, we identify the sur2 isoform described by Inagaki et al. (16, 19, 20) that uses exon 39 as SUR2(39), and our previously identified cardiac-restricted variant, SUR2A, is thus designated SUR2(Delta 14/39).

Exon 17 encodes the 13 amino acids next to the Walker A motif containing exon 16 of the first nucleotide binding fold (NBF). The Walker A motif is highly conserved in many ATP-binding proteins (21, 24) and is required for the functional integrity of the NBF. In sur1, mutations of the conserved lysine residue (Lys-719 in sur1) in the Walker A motif of NBF1 produce a decrease in ATP binding to the sur (25). This invariant lysine residue has been associated with facilitation of ATP hydrolysis (26-28). Electrophysiological experiments also implicate NBF1 of sur1 in the ATP sensitivity to ATP block (29). Given the close proximity of exon 17 to the Walker A motif within the NBF, we hypothesized that the alternate use of exon 17 would affect the nucleotide sensitivity of sur2/Kir6.2 recombinant KATP channels. Indeed, we describe here that exon 17 influences the ATP sensitivity by changing the bursting pattern of the recombinant channel.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RT-PCR Analysis of Exon 17

Total RNA was isolated from whole organ mouse tissues by TRIzol extraction (Life Technologies, Inc.). The RNA was primed with the sur2 gene-specific primer (5'-CTGACC CCCACTCAGGTTGATGCC-3'), or with oligo(dT), and reverse-transcribed with Superscript II (Life Technologies, Inc.) following the manufacturer's instruction. Single-stranded cDNA (1 µl) was used as template for polymerase chain reaction amplification using Klentaq polymerase (CLONTECH) under the following conditions: 35 cycles of 96 °C × 30 s melting, 58 °C × 30 s annealing, 72 °C × 1 min extension. The primers are as follows: forward (in exon 16) 5'-CA TCTCTTCTCCTTGCCATCCTT GG-3'; reverse (in exon 21) 5'- CCTCTCTCCAATTTC AGTTTGGTCTCC-3'.

Long RT-PCR for Detection of Midcoding Region Splicing and Alternative CTD Usage

---Total RNA was isolated by TRIzol extraction (Life Technologies, Inc.) from the indicated whole organ mouse tissues. The RNA was primed with oligo(dT) and reverse-transcribed with Superscript II (Life Technologies, Inc.).

Exon 14/Exon 39-40 Relationship-- Primers (forward, 5'-GAA GCTGGCGGAGGCTCAGAAGAG-3'; reverse, 5'- GATCGGGCCCACTTTTCGAG TGTGGACAGA TCGTA-3) were designed to reside 500 bp 5' to exon 14 and 3' of exon 40. These primers were used to amplify cDNA using Klentaq polymerase under the following conditions: 36 cycles of 96 °C × 30 s melting, 68 °C × 3 min annealing/extension. The products were purified by Qiaprep PCR purification (Qiagen) and digested with HaeII (New England Biolabs). The digested products were resolved on 0.8% SeaKem agarose gels (FMC), and the corresponding sur2 fragments with and without exon 14 were gel-purified using Qiaex II gel purification (Qiagen). The purified DNA fragments were digested with XbaI (New England Biolabs) and then resolved on 2.0% SeaKem agarose gels.

Exon 17/Exon 39-40 Relationship-- Primers were generated to reside entirely within exon 17 and exon 39 or were designed to exclude the two exons leaving 3-4 bp overhanging the splice junction at the 3' end of the primer (3' overhangs are underlined). The primers are as follows: exon 17, 5'-GTAAATGA ATCTGAGCCTTCTTTTGAAGCAACCCGAAG-3'; straddle 17, 5'-CCCTGGAAGG AAAAGTTTACTGGAACAACAG-3'; exon 39, 5'-TGGAGCAGGTTTGGACCAGTA TCAC-3'; straddle 39, 5'-CTGCAGTCAGAATGGTGTGAACCCGATGAG-3'. PCR conditions and agarose gel electrophoresis are as stated above.

RNase Protection Assay and S1 Nuclease Assay

Probe Generation (RNase)-- A 375-bp cDNA probe corresponding to the sur2 coding region bp 1879-2254 was generated by PCR with Pfu polymerase (Stratagene) and subcloned into Bluescript KS+ (Stratagene). Sequence integrity and orientation was confirmed by dye termination sequencing (Applied Biosystems, Perkin-Elmer). This template was used to generate labeled antisense RNA transcripts by in vitro transcription in the presence of 800 Ci/mmol alpha -[32P]CTP (Amersham Pharmacia Biotech) and T3 RNA polymerase (Promega). A beta -actin loading control probe was generated in a similar fashion using Tri-actin mouse 125 as a template (Ambion).

Probe Generation (S1 Nuclease)-- A full-length sur2 cDNA with exon 39 CTD was subcloned into a pcDNA3 vector. The construct was digested with Xba and primed with an antisense oligonucleotide directed to exon 39, 5'-TGGAGCAGGTTTGGACCAGTATAC-3'. Run-off primer extension was performed in the presence of 3000 Ci/mmol alpha -[32P]dCTP (Amersham Pharmacia Biotech) with Klenow polymerase (Ambion). The labeled probes were purified from the template by denaturing polyacrylamide electrophoresis, and the band containing the probe was excised and eluted from the gel overnight in TE at 37 °C. A beta -actin loading control probe was generated in a similar fashion using Tri-actin mouse 125 as a template (Ambion).

Solution Hybridization-- 40 µg of total RNA, isolated by TRIzol extraction, was coprecipitated with 5 × 105 cpm labeled probe, resuspended in hybridization solution (Ambion), and allowed to hybridize for 18 h at 44 °C. The hybridized RNA were digested with RNase A and T1 for 30 min (RNase protection assay) or with S1 nuclease at 37 °C for 30 min, and the protected products were resolved on a standard denaturing sequencing gel. The gel was autoradiographically exposed to Biomax MR film (Kodak) overnight at -80 °C or was exposed to a phosphorimaging cassette (Molecular Dynamics) for 3 h at room temperature for quantitation.

Cell Culture and Transfection

Full-length sur2 cDNA coding regions (bp 1-4644) were subcloned into a mammalian expression vector (pcDNA3, Invitrogen) using standard molecular biological techniques (30). COS-1 and COS-7 cells (ATCC), grown in Dulbecco's modified Eagle's medium/high glucose + 10% fetal calf serum (Life Technologies, Inc.), were plated at a density of 1 × 106 cells onto scored glass coverslips in 60-mm plates, 12-18 h prior to transfection. Each plate was transfected with 5 µg of total plasmid DNA, with a molar ratio of 4:4:1 SUR2-pcDNA3:mbeta -cell inward rectifier-pCMV6b:GFP-greenlantern (Life Technologies, Inc.), using the Superfect transfection system (Qiagen). The cells were used for recording 72 h post-transfection.

Electrophysiology-- Cells were selected for recording based on their green fluorescence at 525 nm when excited at 480 nm due to the coexpression of green fluorescent protein. Pipettes (1-3 megohms) were filled with "pipette solution" containing (mM) 140 KCl, 1 CaCl2, 0.22 MgCl2, 5 HEPES-KOH or HEPES-NaOH at pH 7.4. In some experiments, 130 NaCl + 10 KCl was iso-osmotically substituted for 140 NaCl as indicated in the text. Transfected cells were placed in a chamber bath perfused with "internal solution" containing (mM) 140 KCl, 0.2 MgCl2, 2 EGTA, 5 HEPES-KOH, pH 7.3; ATP, tolbutamide, and glyburide were added to the internal solutions as indicated in the text. The solutions superfusing the patch membrane were switched within 1 s using a fast solution switching system, DAD12 (ALA Instruments). Inside-out patches were recorded with an EPC-7 amplifier (List Electronics) for single channel and Axo200B (Axon Instruments) for multiple channel analysis. Single or multiple channel current recordings were filtered at 0.5-2 kHz, sampled at 5 kHz, and then stored digitally direct via the PCLAMP (Axon Instruments) and "Aquire" (Bruxton Corp.) software packages for subsequent offline data analysis.

In patches containing a single active channel, channel open activity was assessed by an open probability (Po) as follows. A 50% threshold criterion was used to detect an event, and all events were visually confirmed. The dwell time at each level was determined using FETCHAN software (Axon Inst.). From this, the Po was calculated using the equation derived by Spruce et al. (34). Patches containing multiple channels (<= 5 channels) were calculated in a similar fashion. In macro-patch recordings (>5 channels), an apparent open probability (N Po) was employed. The N Po was calculated as the average current in a 10-s time window divided by the known single channel current amplitude under the same recording conditions.

For single channel kinetic analysis, idealized current reconstructions for each recording were generated using "TAC/TACfit" program (Bruxton Corp.) based on a half-maximal amplitude threshold criterion. Open and closed time histogram analysis was performed on recordings demonstrating only a single level of channel activity. Burst durations were resolved at a critical time cutoff of 3.5 ms to distinguish a gap between two bursts from a closure within a burst (31, 32) (Equation 1).
a<SUB>1</SUB>[<UP>exp</UP>(<UP>−</UP>t<SUB><UP>cutoff</UP></SUB>/&tgr;<SUB>1</SUB>)]=a<SUB>2</SUB>[<UP>exp</UP>(<UP>−</UP>t<SUB>d</SUB>/&tgr;<SUB>2</SUB>)−<UP>exp</UP>(<UP>−</UP>t<SUB><UP>cutoff</UP></SUB>/&tgr;<SUB>2</SUB>)] (Eq. 1)
where tau 1 and tau 2 are the time constants for closed intervals within a burst and between bursts; a1 and a2 are the areas of exponential fits corresponding to tau 1 and tau 2, and td, the "dead time" of underestimated events, corresponds to double the sampling rate (400 µs). Distributions of open and closed time events were plotted against a logarithmic time scale with event durations log-binned at 50-µs resolution. Exponential fits to the histograms were performed by a maximum likelihood fitting strategy. Values for kinetic rate constants were determined based on the relationships described by Sakmann and Trube (33), Gillis et al. (31), and Alekseev et al. (32).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

sur2 Exon 17 Exhibits Tissue-restricted Alternative Splicing-- Our initial characterization of sur2 included the description of an alternatively spliced coding region variant, SUR2(Delta 14/39), restricted to heart expression (15). Further examination of the PCR products spanning this alternatively spliced region suggested a novel coding region variant. Primers designed to amplify from the coding region 2126-2415 bp (exons 16-21) resolved a novel product from the full-length sur2 PCR product (Fig. 1A). This smaller variant demonstrated wide tissue expression distribution in mouse and was detected in every tissue examined. It was prominent in bladder, gut, kidney, and skeletal muscle and was weakly detected in all other tissues. The sequence of this product indicated it lacked 39 bp (13 amino acids), from bp 2190 to 2229 of the sur2 coding region, corresponding precisely to a single exon, exon 17, based on the human SUR1 genomic structure. Subsequent mapping of the mouse sur2 genomic intron/exon boundaries demonstrated that the splice junction for this species indeed corresponded to exon 17, with appropriate flanking splice donor/acceptor sequences (data not shown). Previous work indicated that the beginning of exon 17 "wobbled" by ±1 amino acid, which was attributed to an alternate recognition of the splice acceptor site at the intron/exon junction.3 This is a distinct event from the alternative splicing of exon 17 we present here.


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Fig. 1.   Expression of alternatively spliced sur2 exons in various mouse tissues. A, RT-PCR spanning exons (Ex) 16-21. Exon 17 containing transcripts (Ex 17+) are represented by a 289-bp product, whereas exon 17 lacking variants (Ex Delta 17) resolves as a 250-bp product. Both species are present in every tissue examined. B, in solution hybridization RNase protection. An antisense probe spanned exons 13-18, and the protected fragments are indicated. Controls for probe digestion are represented by the lanes probe + RNase (no target mRNA) and probe + sense (in vitro transcribed probe in the sense direction). The beta -actin species represents a 3-h exposure, and the protected sur2 species represents an 18-h exposure. C, in solution hybridization S1 nuclease protection. A single-stranded, labeled cDNA probe spanned exons 35-39, and the protected carboxyl-terminal fragments are indicated. The heart and skeletal muscle lanes, as well as the beta -actin species, represent a 3-h exposure, whereas the other protected sur2 species represent an 18-h exposure.

Solution hybridization and RNA protection with a labeled antisense RNA probe spanning exons 13-18 (bp 1879-2254 of the coding region) was employed both to confirm the RT-PCR splicing data and to quantitate the relative mRNA tissue expression level for each of the three identified splice variants in the mouse (Fig. 1B). Digestion temperatures and RNase concentrations were varied to determine the optimal protection/digestion conditions, and relevant controls were included with each gel to ensure proper probe digestion (Fig. 2B, lanes 3 and 4). Individual variants were indicated by their predicted protected fragment sizes. A 375-nt protected fragment was present ubiquitously and represented an mRNA species containing both exons 14 and 17 which is predicted to be the full-length sur2 transcript. The exon 17 deleted variant (Delta 17), 312 nt, was alternatively spliced in a number of tissues but was predominant in bladder, heart, and skeletal muscle, and the exon 14 deleted SUR2(Delta 14), a 243-nt fragment, was present in heart alone. No evidence was seen that an sur2 variant exists that lacks both exons 14 and 17. This would be presented as a 179-nt fragment by RNase protection or as a 460-bp product in the previously reported RT-PCR (15). Quantitation of expression levels by phosphorimaging indicate that SUR2(Delta 17) was expressed in highest levels in heart, skeletal muscle, and bladder when normalized to the beta -actin loading control (n = 3 for each tissue, quantitation data not shown). In heart, full-length sur2 and SUR2(Delta 14) were expressed at nearly similar levels. SUR2(Delta 17) was the predominant species observed in skeletal muscle and in gut. In bladder, full-length sur2 was expressed at nearly equal levels as SUR2(Delta 17). Modest levels of sur2 were present in all tissues and were expressed at least 5-10-fold greater than SUR2(Delta 17).


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Fig. 2.   Relationship between midcoding region exon usage and carboxyl-terminal usage. A, PCR restriction digestion to evaluate exon 14 and CTD usage. Primers defined PCR products that spanned bp 1639-5240 of the full-length sur2 transcript, amplified from oligo(dT)-primed mouse heart cDNA. The products were digested and resolved by agarose gel electrophoresis in two steps as indicated in the schematic. Exon 39 and exon 40 species resolved as 760- and 581-bp fragments, respectively. B, exon 17 and carboxyl-terminal usage. Primer combinations and their corresponding sur2 targets are depicted in the schematic. Each lane of the resolved PCR products indicates the primer set employed and the sur2 species amplified according to the diagram. The presence of a 2-2.2-kilobase pair amplified product indicates the existence of the particular sur2 variant; the lack of a product indicates its absence. The 9th to 14th lanes represent a relevant positive and negative control for each primer using 10 ng of the indicated cDNA template.

Tissue-restricted Expression of CTD with Exon 39 and Exon 40-- Alternate sur2 CTD usage was recently demonstrated by Isomoto et al. (22). We have adopted a nomenclature based on exon usage, thus isoforms SUR2A and SUR2B correspond to exons 39 and 40, respectively. Exon 40 was demonstrated to be present in all tissues and is highly homologous to the sur1 CTD. S1 nuclease protection analysis of the carboxyl-terminal alternate exon usage was employed to corroborate the results reported by Isomoto et al. (22), as well as quantitatively determining which major carboxyl-terminal isoform was expressed in any single tissue. A single-stranded 32P-labeled antisense cDNA probe spanning exons 35-39 was generated and hybridized to total RNA in solution. Exon 39 variants resolved as a 378-nt fragment, whereas exon 40 species, which could not protect the last 131 nt of the probe, resulted in a 247-nt protected fragment (Fig. 1C). A second probe corresponding to a 125-bp stretch of beta -actin served as a loading control. Quantitation of protected fragments by phosphorimaging (n = 2 for heart and skeletal muscle) indicated that the heart contained both exon 39 and 40 sur2 variants in nearly equal amounts, while skeletal muscle expressed approximately 2-fold greater levels of exon 39 compared with exon 40 (Fig. 1C). Weak expression of exon 39 was also noted in adipose tissue and in bladder but at much lower levels than the corresponding expression of the exon 40 variants. The exon 40 variant was present in all tissues.

Exon 14 Splicing Only Occurs with Carboxyl Tail Exon 39-- Demonstrating three mid-coding region sur2 splice variants (full-length, Delta 14, and Delta 17) and two carboxyl-terminal tails (exons 39 and 40) suggests that six possible sur2 variations could be generated if the mid-coding region splice events occur independently of alternate carboxyl-tail usage. To determine if exon 14 splice variants occurred with both exon 39 and exon 40, long PCR was employed spanning exons 9-40 (Fig. 2A). Exon 14 and exon Delta 14 amplified products could be identified and separated by a unique HaeII restriction site present in exon 14, 500 bp 3' to the sense primer. From these amplified products, HaeII digestion yielded PCR products differing by 500 bp. The digested products were separated by gel electrophoresis, and the exon 14 and exon Delta 14 fragments were excised from the agarose gel. The presence or absence of exon 17 (± 35 bp), present only within exon 14+ variants, resolved as a nearly indiscernible doublet indicated as 14+ in Fig. 2A. These two exon 17 species, as both possessed exon 14, were copurified for the subsequent digestion step. The gel-purified fragments were digested a second time with a unique XbaI site 5' to exon 39. Exon 39 and 40 variants resolved as 760- and 581-bp fragments, respectively. The results demonstrated that exon 14 containing sur2 variants were present with both exon 39 and 40 CTD tails, but the SUR2(Delta 14) variant, which was expressed in heart alone, only existed with the exon 39 CTD.

Exon 17 Splicing Occurs Independently with Respect to Splicing of the CTD-- A similar PCR/restriction digestion approach was not available to determine the relationship between exon 17 splicing and the alternate carboxyl terminus, as no unique restriction sites existed in exon 17. Instead, primers were designed to reside entirely within exons 17 and 39 or to straddle these exons and thus exclude them (Fig. 2B). The straddling primers annealed to the splice junction asymmetrically; a majority of the 5' end of each primer annealed to one exon leaving 3-4 bp of the primer's 3' end to anneal to the exon following the splice junction. By this design, these primers could only anneal to those sur2 variants lacking either exon 17 or 39 to provide a productively primed template for polymerase extension (schematic, Fig. 2B). The four designed primers were used in combination to test each of the four possible splice forms for exons 17/Delta 17 and 39/40, resulting in a 2-2.2-kilobase pair amplified product (depending on the primer pair) if the variant was expressed in the tissue or no amplified product if the tissue lacked the variant. Heart and skeletal muscle were tested, as most of the other tissues were known to contain only the exon 40 CTD (Fig. 1 and Ref. 22). As such, these tissues must contain either 17/40 or Delta 17/40 as determined by the earlier RT-PCR and the S1 nuclease (Fig. 1). Fat and bladder, which also express minor levels of exon 39, were not examined at this time. As seen in Fig. 2B, both skeletal muscle and heart expressed all four of the possible exon 17 variants.

In summary, five sur2 isoforms are generated by the alternative splicing of the coding region: SUR2(39), SUR2(Delta 14/39), SUR2(Delta 17/39), SUR2(40), and SUR2(Delta 17/40). Their tissue expression pattern is summarized in Fig. 3.


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Fig. 3.   Summary of sur2 alternative splicing. A, a summary of the expression patterns for each sur2 variant in mouse tissues. * indicates these tissues have not yet been tested for mid-coding region/CTD combinations, but the individual exon splicing events have been determined. B, representative schematic of the sur2 topology and the locations of the identified alternatively spliced exons. C, the amino acid sequence of NBF1. The alternatively spliced exons are highlighted in gray, and the Walker A and B motifs are boxed.

Single Channel Currents Resulting from the Coexpression of sur2 Splice Variants with mKir6.2-- Coexpression of four of the five sur2 variants and mouse Kir6.2 in COS-1 or COS-7 elicited currents with identical single channel conductance (Fig. 4, A and B), and kinetic properties similar to cardiac and skeletal muscle KATP (7, 32, 34). One variant, SUR2(Delta 14/39), failed to generate current. The current-voltage relationship for each variant demonstrated weak inward rectification and an estimated reversal potential of approximately -58 mV (Fig. 4B), a value close to the K+ equilibrium potential of -65 mV. The single channel conductance was approximately 30 pS for all variants at a membrane potential of 0 mV. In addition, each coexpressed variant exhibited characteristic KATP channel inhibition at 1 mM cytoplasmic ATP. Application of 100 µM tolbutamide or 1 µM glyburide to exon SUR2(39) or SUR2(Delta 17/39) expressing variants showed roughly equal channel block (data not shown). These results demonstrate that the four sur2 variants possessing exon 14 yield typical KATP.


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Fig. 4.   Electrophysiological recordings or COS-7 cells expressing sur2 splice variants cotransfected with Kir6.2. A, current traces of single channels recorded from KATP generated by coexpressing mouse Kir6.2 and the indicated mouse sur2 variants. C and O represent closed and open channel levels, respectively. The holding potential was 0 mV for all traces. [K+]i/[K+]o = 140/10 mM/mM. Outward current is plotted as an upward deflection. B, single channel current I-V relationships for reconstituted KATP. The current amplitudes were measured from all points amplitude histograms. Each data point represents 1-6 patches.

Exon 17 Splicing Alters ATP Sensitivity-- The four exon 14-containing sur2 variants were inhibited by ATP in a dose-dependent manner, with a 2-fold difference in ATP sensitivity based on the presence or absence of exon 17. ATP in the presence of 0.2 mM MgCl2 was rapidly applied to the cytoplasmic side of the patch membrane at concentrations varying from 1 to 1000 µM. To avoid bias from any channel run-down, the concentration of ATP was stepped in both increasing and decreasing concentrations. Fig. 5A shows representative current records for the four functional sur2 variants. The open probability, or the averaged current at each ATP concentration, was normalized to that in the absence of ATP. The normalized current values, or relative current, represent the ATP block and are summarized in Fig. 5B. The concentration-dependent block of ATP was quantified by fitting the mean data points with a sigmoidal Hill equation (Fig. 5B). Half-maximal inhibitory concentrations, K1/2, and the Hill coefficients, H, for ATP block of channel activity were determined and are listed in Table I. The data show significant differences between variants with exon 17 and variants without exon 17. Deletion of exon 17 increased the K1/2 for ATP block approximately 2-fold, regardless of CTD usage. The Hill coefficients for all variants are approximately 1 and the differences among them are insignificant.


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Fig. 5.   Dose-dependent responses of ATP for sur2 splice variants expressed in COS-6 cells. A, representative current recordings of ATP block for reconstituted KATP from the coexpression of Kir6.2 and the indicated sur2 variants. Currents were filtered at 200 Hz. The holding potential was set at 0 mV. [K+]i/[K+]o = 140/10 mM/mM. Outward current is plotted as an upward deflection. i = 0 indicates the level where all channels are closed, and individual channel levels are marked to indicate the number of channels per patch. ATP was applied to the internal face of the membrane at the micormolar concentrations indicated. The fast downward deflections in the data traces correspond to solution change artifacts. 1 µM ATP was included in the internal solutions whenever the ATP concentration is not provided. B, dose-response relationships of ATP block for each sur2 variant coexpressed with Kir6.2. The curves represent fits of the experimental data to the Hill equation: Irel = 1/{1 +([ATP]/K1/2(ATP))H}, where Irel is the current amplitude relative to the peak current at 1 µM ATP, K1/2(ATP) is the concentration for half-maximal ATP block, and H is the Hill coefficient. The data points are the mean ± S.D. from the number of experiments listed in Table I. * indicates a significant difference of p < 0.05 between the relative current levels for the exon 17+ variants versus the exon Delta 17 variants, as determined by the paired Student's t test.

                              
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Table I
ATP block of KATP expressed from SUR2 splice variants and Kir6.2
Levels of significance were determined using the paired Student's t test. Both half-maximal inhibition (K1/2) and the Hill coefficients were determined from the best fits of the data to the Hill equation as demonstrated in Fig. 5B. For comparison * versus * and ** versus **. Both are p < 0.01.

Next, we explored whether the differences in single channel gating could account for the reduced ATP sensitivity. Given the observation that CTD usage did not affect ATP sensitivity, we restricted the study of single channel kinetics to variants with exon 39. The single channel behavior of SUR2(39) was compared with SUR2(Delta 17/39) at low (1 µM) and high (50 µM) internal ATP, in symmetrical K+ (140 mM) at a membrane potential of -60 mV. Both variants exhibited kinetics characteristic of striated muscle KATP, resolving into long bursts of channel openings that contained brief intraburst closures separated by interburst gaps (Fig. 6A) (7, 34, 35). Open and closed time constants were obtained using a maximal likelihood fit to the dwell time histograms. The distribution of open and closed times within a burst for each variant were accurately described with a single exponential function. The averaged time constants, tau O and tau C1, for SUR2(39)-tau O = 3.19 ± 0.09 ms and tau C1 = 0.37 ± 0.02 ms (n = 7) and SUR2(Delta 17/39)-tau O = 3.30 ± 0.10 ms and tau C1 = 0.36 ± 0.04 ms (n = 14) were not significantly different from each other nor did they differ from the corresponding averaged tau O and tau C1 values measured in the presence of 50 µM internal ATP, which were SUR2(39)-tau O = 3.12 ± 0.15 ms and tau C1 = 0.35 ± 0.01 ms (n = 8) and SUR2(Delta 17/39)-tau O = 3.04 ± 0.19 ms and tau C1 = 0.37 ± 0.01 ms (n = 8).


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Fig. 6.   Kinetic properties for SUR2(39) and SUR2(Delta 17/39). A, representative single channel current records for excised inside-out patches of the indicated sur2 exon 17 variant coexpressed with Kir6.2 in COS-7 at -60 mV. [K+] were equal in the pipette and bath (140/140 mM/mM). Patches were either excised into 1 µM ATP or first excised into 1 µM ATP and rapidly switched to 50 µM ATP. B, representative open time, closed time, and burst distributions from a single recording for each sur2 variant at the indicated ATP concentrations. Closed time histograms of the gaps between burst represent distributions exceeding the critical tcutoff of 3.5 ms and are fit to two exponents. Inset, closed time histograms representing distributions before imposition of the critical tcutoff. Burst distributions represent open state durations defined by gaps exceeding the critical tcutoff of 3.5 ms and fit to two exponents.

The distributions of gaps between bursts for both variants required at least two exponents to fit (Fig. 6B), in close correspondence to previously reported data from cardiac and skeletal KATP which has been described by a four-state linear sequential model (32, 35). The suitability of such a model, consistent with the model we derived from our duration histogram analysis (Fig. 7A), was tested independently by subjecting our raw single channel recordings to a maximum likelihood computational method for the modeling of an aggregated Markov process, as described by Qin et al. (36). The resulting four-state linear sequential model generated by this method was identical to the model based on our histogram analysis and to the previously described models (32, 35). The rate constants for this kinetic scheme, summarized in Fig. 7A, were calculated from the parameters determined by the maximal likelihood fits to the dwell time histograms (Fig. 6B). At 1 µM ATP, SUR2(39) differed significantly from SUR2(Delta 17/39) for rate constants directed away from the open state: k23 = 5.8 s-1 versus 3.4 s-1 (p < 0.05) and kO2 = 6.9 s-1 versus 5.4 s-1 (p < 0.05). At 50 µM ATP, both variants exhibited significant increases in the rate constants directed away from the open state (kO2 and k23), but the magnitude of the change was similar: kO2 for SUR2(39) increased 2.9-fold versus a 2.3-fold increase in kO2 for SUR2(Delta 17/39), and k23 for SUR2(39) increased 4.6-fold versus a 3.8-fold increase in k23 for SUR2(Delta 17/39). An appreciable difference between two variants was reflected in the rate constant directed toward the open state, k2O, at 50 µM ATP. Whereas k2O at 1 µM ATP showed no difference between the variants, a 2-fold decrease in k2O was observed for SUR2(39) at 50 µM ATP. The k2O for SUR2(Delta 17/39) remained unchanged with increases in the ATP concentration. The difference in k2O was reflected both in the calculated mean burst duration and in the mean interburst gap duration for each variant (Fig. 7B). The mean burst duration of SUR2(Delta 17/39) in 1 µM ATP, 1.5-fold longer than that of SUR2(39) (220 ± 14 ms versus 166 ± 13 ms), did not differ from its duration at 50 µM ATP. In contrast, the mean burst duration for SUR2(39) decreased 2-fold between 1 and 50 µM ATP. The mean interburst gap durations did not differ significantly between the two variants at 1 µM ATP. The mean interburst gap duration for SUR2(39) increased 7.2-fold at 50 µM ATP (from 45 ± 15 to 325 ± 58 ms), whereas SUR2(Delta 17/39) increased only 1.9-fold (32 ± 5 to 63 ± 26 ms).


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Fig. 7.   Summary of single channel kinetics. A, rate constants corresponding to the intraburst and interburst transitions were calculated using the set of parameters that define the open and closed distributions (as represented in Fig. 6B) based on the relationships defined by Gillis et al. (31) and adapted by Alekseev et al. (32). B, average burst and interburst gap durations were calculated based on the reciprocal values of the rate constants leading away from the given states, as determined by Sakmann and Trube (33): sigma burst = (k1O + kO1)/(k1O ·kO1) and sigma interburst (C3 and C2) = (k23 + k32)/(k32·k2O). Error bars represent ± S.E., and the brackets between each column indicate a statistically significant difference as determined by the paired Student's t test of p < 0.05 (n = 6-14 patches).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Contribution of Alternative Splicing of sur2 to KATP Channel Diversity-- KATP channel properties vary widely in different cell types, particularly with regard to nucleotide block and drug sensitivity (3, 4, 37, 38). This diversity may meet the various physiological and pathophysiological roles of KATP in the wide range of tissues. Many factors such as pH, cell membrane composition, and other nucleotides affect ATP and pharmacological sensitivity of KATP and contribute to the observed diversity in tissue response (3, 4, 9, 39-41). In addition, channel subunit isoform composition may also intrinsically regulate KATP response. The cloning of cDNAs for KATP subunits, sur1 (14), sur2 (15, 16), Kir6.1 (19) and Kir6.2 (20), and the demonstration of distinct nucleotide and pharmacological sensitivities between recombinant KATP (16, 20) indicated that a degree of the observed KATP diversity may result from the assembly of different channel subunit isoforms. Specifically, the sur subunit has been found to confer characteristic features for KATP including drug sensitivity and ADP activation. The distinct tissue expression patterns reported for sur1 and sur2 isoforms likely contribute to observed tissue-specific KATP channel diversity. Furthermore, Isomoto et al. (22) demonstrated that the alternative CTD usage of SUR2(40) results in recombinant KATP with altered response to the antihypertensive drug diazoxide. They argued that the properties of the reconstructed channel correspond to the native smooth muscle KATP. Here, we identify alternative splicing of exon 17, which alters nucleotide sensitivity by 2-fold. The alternate usage of exon 17 in sur2 follows a consistent pattern with previously observed tissue-specific differences in ATP sensitivities for KATP and as such may be a contributing factor to the overall tissue differences in ATP sensitivities. For example, cardiac myocytes, which express sur1, full-length sur2, and SUR2(Delta 17), ATP block over the range 20-100 µM has been reported (4, 42); vascular smooth muscle, likely only expressing full-length sur2, approx 30 µM (43); kidney, expressing full-length sur2 and SUR2(Delta 17), approx 100 µM (44); skeletal muscle, which predominantly expresses SUR2(Delta 17), approx 130 µM (7); and the beta -cell, which expresses sur1, approx 10-20 µM (3). It is important to note, however, that Kir6.2 is expressed primarily in the pancreas and in heart (20), suggesting that sur2 must associate with another Kir, possibly Kir6.1, in other tissues and thus may possess significantly different ATP inhibition properties. Indeed, when SUR2(40) has been coexpressed with Kir6.1 the resulting sulfonylurea-sensitive channels were unlike traditional KATP in that they did not spontaneously open when excised into an ATP/free bath solution (45).

KATP molecular heterogeneity is further extended with the identification of five different sur2 variants (Fig. 2). The exon selection and relative transcriptional levels occur in a tissue-specific pattern with Delta 14 usage restricted to heart and Delta 17 predominant in skeletal muscle and gut and high in bladder. Interestingly, as exon 39/40 usage has already proven to discriminate diazoxide sensitivity, it is conceivable that exon 17 and Delta 17 may prove to be targets for unique drug interactions, thus providing KATP drug/tissue specificity for some tissues, such as bladder or gut.

Previously, we described the identification of an sur2 variant lacking exon 14, SUR2(Delta 14/39) (15). In this study we tested the functional expression of this variant with Kir6.2 and found that it yielded undetectable currents. This information is important in the following two respects: first, it reveals that the peptide segment for exon 14 may be critical to form functional channels; second, this variant has the potential to serve as a negative suppressor of the sur2 and so KATP expression. However, we did not find an obvious suppressing effect in the test of the coexpression of this variant with SUR2(39) using transient transfection.4 Direct methods to assay the levels and subcellular expression patterns of SUR2(Delta 14/39) will be needed to confirm this result.

The approach of chimeric SUR/Kir fusion proteins has provided strong evidence that the KATP complex consists of a hetero-octamer, with the sur and Kir subunits in equal stoichiometry (17, 18). Of potential consequence, sur1 and sur2 demonstrate overlapping expression in heart and possibly skeletal muscle (16, 46), and sur2 and SUR2(Delta 17) variants show overlapping expression in most tissues (Fig. 1, A and B). If the subunit isoforms and variants are coexpressed in the same cell, it is possible that individual KATP subunits could coassemble into heterogeneous KATP octameric complexes. This would significantly expand the KATP subunit combinations possible. Considering that our tissue distribution data were collected from the whole organs that contained different types of cells, it still remains to be determined whether the variants are indeed coexpressed in a single cell.

As both full-length and SUR2(Delta 17) mRNA species are present in most tissues where sur2 is expressed, the alternative splicing of exon 17 may provide an intriguing mechanism to regulate KATP sensitivity with respect to the cellular metabolic environment. Recently, Akao et al. (47) demonstrated that the Kir6.2 subunit of the cardiac KATP is regulated at the transcriptional level in response to induced cardiac ischemia, and we have observed altered sur2 transcript levels in cardiac and skeletal muscle tissues of rats subjected to fasting.2 Additionally, Xie and McCobb (48) observed an example of environmentally controlled splice regulation by demonstrating that stress hormones could regulate the alternative splicing of a K+ channel transcript. This suggests transcriptional or post-transcriptional regulation (including exon usage) may have a role for altering, or resetting, overall ATP sensitivity of KATP current in cells experiencing periods of metabolic stress. Thus, by expressing different sur2 variants in different cells within a tissue, or within a single cell to form chimeric channels which may also involve a dynamic regulation of subunit expression levels, the alternative splicing of sur2 reported here is a significant mechanism employed in vivo to broaden the repertoire of KATP.

ATP Block and the Underlying Kinetic Mechanism of Reconstructed KATP-- A novel finding in theses studies is the alternation in ATP block in response to exon 17 usage. We found that the K1/2 for ATP block were around 40 µM for full-length sur2 and 90 µM for SUR2(Delta 17) variants. Interestingly, the CTD usage did not significantly affect ATP sensitivity. The K1/2 for exon 17 containing sur2 are similar to those previously reported (16, 22, 46). The Hill coefficient, which is determined by the nature of the ATP binding process, reportedly varies between 1 and 2 in both native striated muscle KATP and recombinant KATP (5, 17, 22). Here, under uniform conditions, we found that the Hill coefficient for all four variants was approximately 1, suggesting that the overall ATP binding to block the channels follows an apparent single site binding process.

The ATP block of striated muscle KATP has been described with a four-state linear sequential model (32, 35), similar to the model presented in Fig. 7. In this model the transition O left-right-arrow  C1 is ATP-independent, whereas the transitions C3 left-right-arrow  C2 left-right-arrow  O are ATP-dependent processes. In agreement with the observation in native cardiac KATP (35), our data indicated that the four rate constants, k32, k23, k2O, kO2, all varied with internal ATP concentration, with k32 the least variable. The ATP dependence of the rate constants leading to the open state, k32 and k2O for the SUR2(39) variant, indicated possible cooperativity of several ATP-binding sites or the regulation of ATP block by a second binding event at a different site. Negative cooperation was suggested for ATP binding to the sur1 regulating ATP block, possibly occurring at the Kir6.2 subunit (49). The 2-fold reduction in ATP sensitivity associated with the deletion of the exon 17 segment which we observed can be attributed to an elimination of k2O sensitivity to ATP. The absence ATP sensitivity of k2O and the relative insensitivity of k32 to ATP for SUR2(Delta 17) channels suggests that SUR2's ATP-dependent regulation of ATP block is ablated by the removal of exon 17 segment.

To date, including this report, three isoforms of recombinant KATP have been described at the single channel level as follows: SUR2(39), SUR2(Delta 17/39), and sur1 coexpressed with Kir6.2 (20, 50). A fourth channel has been described, an engineered truncation of Kir6.2 lacking its carboxyl-terminal 26 residues (Kir6.2Delta c26), which expressed as an ATP-sensitive channel in the absence of the sur subunit (51). From a casual inspection of the four different recombinant KATP channel burst lengths, the burst duration sequence is SUR2(Delta 17/39) > SUR2(39) > sur1 > Kir6.2Delta c26, in the absence of ATP (Fig. 7 (20, 51)). This sequence indicates the importance of sur in the determination of the burst kinetics of this channel.

Structural Basis of Exon 17 Affecting KATP Function-- The peptide segment encoded by exon 17 is a part of the NBF1 structure and is in close proximity to the Walker A motif. Its location suggests an intimate association between this segment and the putative ATP binding pocket of NBF1. The highly conserved Walker A motif is believed to be a critical part of a site for ATP binding and hydrolysis (52, 53). A consensus structure model has been proposed for the nucleotide binding pocket of NBF-containing proteins which consists of a series of six alpha -coil/beta -strands (54). According to this model, the segment of exon 17 begins with the end of beta -strand 2 and encompasses a critical residue predicted to participate in nucleotide hydrolysis. Alternatively, sequence homology between sur2 and other ABC proteins suggests that the exon 17 segment may comprise a portion of the putative switch I region identified in G-binding proteins which is predicted to affect nucleotide binding and/or signal transduction upon nucleotide hydrolysis (23, 27). The NBF of some ABC proteins has been shown to possess ATPase activity (26, 28). Although Ueda et al. (25) have demonstrated ATP binding to sur1, intrinsic ATPase activity by an sur has yet to be directly demonstrated.

Our finding of the reduction in ATP sensitivity with the removal of exon 17 suggests that the exon 17 segment contributes to nucleotide responsiveness of the Kir6.2-sur2 complex. However, our electrophysiological data are insufficient to distinguish between the direct involvement of exon 17 structure in ATP binding causing channel block or an indirect effect on the mechanism of ATP block. Shyng et al. (29) proposed a model for sur as an ATP-supersensitizing switch. In this model, the sur subunit enhances the intrinsic ATP block of Kir. ATP hydrolysis at one or both of sur's NBFs uncouples the ATP hypersensitivity sur imposes on Kir. Our finding suggests that the capability of SUR2(Delta 17) of enhancing ATP sensitivity is significantly diminished or lost. In the context of the supersensitizing model, Delta 17 could result in reduced ATP affinity at NBF1 thus preventing the key substrate for the hypersensitizing switch from binding, or Delta 17 enhances the rate of ATP hydrolysis and facilitates an uncoupling to the desensitized state, or perhaps both. Alternatively, should exon 17 play a role in the signal transduction through which a hydrolytic signal induces an sur conformational change to the desensitized state, SUR2(Delta 17) might then resemble an sur locked into a desensitized conformation. To test the validity of these interpretations, it will be important to determine ATP binding and hydrolysis at NBF1 for the two splice forms.

In conclusion, we have described five distinct sur2 transcripts resulting from the alternative splicing of the single sur2 gene, and we demonstrated that the alternative splicing of exon 17 affects the ATP block sensitivity of recombinant KATP. Together with the tissue-specific expression, alternative splicing of sur2 may contribute a meaningful role in determining KATP physiological functions.

    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical support of D. L. McClelland and Shujie Yu.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DK-K08-02170 (to C. F. B.), R29 HL58133-01 (to Z. F.), RO1-HL57414 (to J. C. M.), RO1-GM36823, and RO1-GM54266 (to D. J. N.), and the Oscar Rennebohm Foundation (to J. C. M.).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.

To whom correspondence should be addressed: Parke-Davis Pharmaceutical Research, 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-1282; Fax: 734-622-5668; E-mail: charles.burant{at}wl.com.

parallel Current address: Dept. of Physiology and Biophysics, the University of Tennessee, Memphis, TN 38163.

2 W. A. Chutkow and C. F. Burant, unpublished observations.

3 G. Gonzalez, L. Aguilar-Bryan, and J. Bryan, GenBankTM accession number L78208.

4 W. A. Chutkow and Z. Fan, unpublished observations.

    ABBREVIATIONS

The abbreviations used are: KATP, ATP-sensitive potassium channel; CTD, carboxyl-terminal domain; SUR, sulfonylurea receptor; Kir, inward rectifying potassium channel; NBF, nucleotide binding fold; bp, base pair; nt, nucleotide(s); RT-PCR, reverse transcriptase-polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
  1. Noma, A. (1983) Nature 305, 147-148[Medline] [Order article via Infotrieve]
  2. Trube, G., and Hescheler, J. (1984) Pfluegers Arch. 401, 178-184[Medline] [Order article via Infotrieve]
  3. Ashcroft, S. J., and Ashcroft, F. M. (1990) Cell. Signalling 2, 197-214[CrossRef][Medline] [Order article via Infotrieve]
  4. Nichols, C. G., and Lederer, W. J. (1991) Am. J. Physiol. 261, H1675-H1686[Abstract/Free Full Text]
  5. Ashcroft, F. M. (1988) Annu. Rev. Neurosci. 11, 97-118[CrossRef][Medline] [Order article via Infotrieve]
  6. Quast, U. (1996) Naunyn-Schmiedebergs Arch. Pharmacol. 354, 213-225[Medline] [Order article via Infotrieve]
  7. Spruce, A. E., Standen, N. B., and Stanfield, P. R. (1985) Nature 316, 736-738[Medline] [Order article via Infotrieve]
  8. Cook, D. L., and Hales, C. N. (1984) Nature 311, 271-273[Medline] [Order article via Infotrieve]
  9. Quayle, J. M., Nelson, M. T., and Standen, N. B. (1997) Physiol. Rev. 77, 1165-1232[Abstract/Free Full Text]
  10. Hiraoka, M., and Fan, Z. (1989) J. Pharmacol. Exp. Ther. 250, 278-285[Abstract]
  11. Hiraoka, M., Fan, Z., Furukawa, T., Nakayama, K., and Sawanobori, T. (1993) Cardiovasc. Drugs Ther. 7 Suppl. 3, 593-598
  12. Ashcroft, F. M., and Rorsman, P. (1989) Prog. Biophys. Mol. Biol. 54, 87-143[CrossRef][Medline] [Order article via Infotrieve]
  13. Beech, D. J., Zhang, H., Nakao, K., and Bolton, T., B. (1993) Br. J. Pharmacol. 110, 573-582[Abstract]
  14. Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P., Boyd, A. E. R., Gonzalez, G., Herrera-Sosa, H., Nguy, K., Bryan, J., and Nelson, D. A. (1995) Science 268, 423-426[Medline] [Order article via Infotrieve]
  15. Chutkow, W. A., Simon, M. C., Le Beau, M. M., and Burant, C. F. (1996) Diabetes 45, 1439-1445[Abstract]
  16. Inagaki, N., Gonoi, T., Clement, J. P., Wang, C. Z., Aguilar-Bryan, L., Bryan, J., and Seino, S. (1996) Neuron 16, 1011-1017[Medline] [Order article via Infotrieve]
  17. Clement, J. P., IV, Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, U., Aguilar-Bryan, L., and Bryan, J. (1997) Neuron 18, 827-838[Medline] [Order article via Infotrieve]
  18. Shyng, S., and Nichols, C. G. (1997) J. Gen. Physiol. 110, 655-664[Abstract/Free Full Text]
  19. Inagaki, N., Tsuura, Y., Namba, N., Masuda, K., Gonoi, T., Horie, M., Seino, Y., Mizuta, M., and Seino, S. (1995) J. Biol. Chem. 270, 5691-5694[Abstract/Free Full Text]
  20. Inagaki, N., Gonoi, T., Clement, J. P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar-Bryan, L., Seino, S., and Bryan, J. (1995) Science 270, 1166-1170[Abstract]
  21. Higgins, C. F. (1995) Cell 82, 693-696[Medline] [Order article via Infotrieve]
  22. Isomoto, S., Kondo, C., Yamada, M., Matsumoto, S., Higashiguchi, O., Horio, Y., Matsuzawa, Y., and Kurachi, Y. (1996) J. Biol. Chem. 271, 24321-24324[Abstract/Free Full Text]
  23. Schlichting, I., Almo, S. C., Rapp, G., Wilson, K., Petratos, K., Lentfer, A., Wittinghofer, A., Kabsch, W., Pai, E. F., and Petsko, G. A. (1990) Nature 345, 309-315[CrossRef][Medline] [Order article via Infotrieve]
  24. Walker, J. E., Saraste, M., Runswick, M. J., and Gay, N. J. (1982) EMBO J. 1, 945-951[Medline] [Order article via Infotrieve]
  25. Ueda, K., Inagaki, N., and Seino, S. (1997) J. Biol. Chem. 272, 22983-22986[Abstract/Free Full Text]
  26. Ko, Y. H., and Pedersen, P. L. (1995) J. Biol. Chem. 270, 22093-22096[Abstract/Free Full Text]
  27. Manavalan, P., Dearborn, D. G., McPherson, J. M., and Smith, A. E. (1995) FEBS Lett. 366, 87-91[CrossRef][Medline] [Order article via Infotrieve]
  28. Carson, M. R., Travis, S. M., and Welsh, M. J. (1995) J. Biol. Chem. 270, 1711-1717[Abstract/Free Full Text]
  29. Shyng, S., Ferrigni, T., and Nichols, C. G. (1997) J. Gen. Physiol. 110, 643-654[Abstract/Free Full Text]
  30. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) in Molecular Cloning: A Laboratory Manual (Irwin, N., ed), 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  31. Gillis, K. D., Gee, W. M., Hammoud, A., McDaniel, M. L., Falke, L. C., and Misler, S. (1989) Am. J. Physiol. 257, C1119-C1127[Abstract/Free Full Text]
  32. Alekseev, A. E., Brady, P. A., and Terzic, A. (1998) J. Gen. Physiol. 111, 381-394[Abstract/Free Full Text]
  33. Sakmann, B., and Trube, G. (1984) J. Physiol. (Lond.) 347, 659-683[Abstract]
  34. Spruce, A. E., Standen, N. B., and Stanfield, P. R. (1987) J. Physiol. (Lond.) 382, 213-236[Abstract]
  35. Fan, Z., Nakayama, K., and Hiraoka, M. (1990) Pfluegers Arch. 415, 387-394[Medline] [Order article via Infotrieve]
  36. Qin, F., Auerbach, A., and Sachs, F. (1997) Proc. R. Soc. Lond. Ser. B. Biol. Sci. 264, 375-383[CrossRef][Medline] [Order article via Infotrieve]
  37. Dunne, M. J., and Petersen, O. H. (1986) FEBS Lett. 208, 59-62[CrossRef][Medline] [Order article via Infotrieve]
  38. Fan, Z., Nakayama, K., and Hiraoka, M. (1990) J. Physiol. (Lond.) 430, 273-295[Abstract]
  39. Fan, Z., and Makielski, J. C. (1997) J. Biol. Chem. 272, 5388-5395[Abstract/Free Full Text]
  40. Baukrowitz, T., Schulte, U., Oliver, D., Herlitze, S., Krauter, T., Tucker, S. J., Ruppersberg, J. P., and Fakler, B. (1998) Science 282, 1141-1144[Abstract/Free Full Text]
  41. Shyng, S. L., and Nichols, C. G. (1998) Science 282, 1138-1141[Abstract/Free Full Text]
  42. Terzic, A., Jahangir, A., and Kurachi, Y. (1995) Am. J. Physiol. 269, C525-C545[Abstract]
  43. Kajioka, S., Kitamura, K., and Kuriyama, H. (1991) J. Physiol. (Lond.) 444, 397-418[Abstract]
  44. Bleich, M., Schlatter, E., and Greger, R. (1990) Pfluegers Arch. 415, 449-460[Medline] [Order article via Infotrieve]
  45. Yamada, M., Isomoto, S., Matsumoto, S., Kondo, C., Shindo, T., Horio, Y., and Kurachi, Y. (1997) J. Physiol. (Lond.) 499, 715-720[Abstract]
  46. Tokuyama, Y., Fan, Z., Furuta, H., Makielski, J. C., Polonsky, K. S., Bell, G. I., and Yano, H. (1996) Biochem. Biophys. Res. Commun. 220, 532-538[CrossRef][Medline] [Order article via Infotrieve]
  47. Akao, M., Otani, H., Horie, M., Takano, M., Kuniyasu, A., Nakayama, H., Kouchi, I., Sasayama, S., and Murakami, T. (1997) J. Clin. Invest. 100, 3053-3059[Abstract/Free Full Text]
  48. Xie, J., and McCobb, D. P. (1998) Science 280, 443-446[Abstract/Free Full Text]
  49. Shyng, S., Ferrigni, T., and Nichols, C. G. (1997) J. Gen. Physiol. 110, 141-153[Abstract/Free Full Text]
  50. Alekseev, A. E., Kennedy, M. E., Navarro, B., and Terzic, A. (1997) J. Membr. Biol. 159, 161-168[CrossRef][Medline] [Order article via Infotrieve]
  51. Tucker, S. J., Gribble, F. M., Zhao, C., Trapp, S., and Ashcroft, F. M. (1997) Nature 387, 179-183[CrossRef][Medline] [Order article via Infotrieve]
  52. Carson, M. R., and Welsh, M. J. (1995) Biophys. J. 69, 2443-2448[Abstract]
  53. Higgins, C. F. (1992) Annu. Rev. Cell Biol. 8, 67-113[CrossRef]
  54. Yoshida, M., and Amano, T. (1995) FEBS Lett. 359, 1-5[CrossRef][Medline] [Order article via Infotrieve]


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