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The cardiac sarcolemmal ATP-sensitive potassium channel (KATP) consists of a Kir6.2 pore and a SUR2 regulatory subunit, which is an ATP-binding cassette (ABC) transporter. KATP channels have been proposed to play protective roles during ischemic preconditioning. A SUR2 mutant mouse was previously generated by disrupting the first nucleotide-binding domain (NBD1), where a glibenclamide action site was located. In the mutant ventricular myocytes, a non-conventional glibenclamide-insensitive (10 μM), ATP-sensitive current (IKATPn) was detected in 33% of single-channel recordings with an average amplitude of 12.3±5.4 pA per patch, an IC50 to ATP inhibition at 10 μM, and a mean burst duration at 20.6±1.8 ms. Newly designed SUR2-isoform or variant-specific antibodies identified novel SUR2 short forms in the sizes of 28 and 68 kDa in addition to a 150-kDa long form in the sarcolemmal membrane of wild-type (WT) heart. We hypothesized that channels constituted by these short forms that lack NBD1, confer IKATPn. The absence of the long form in the mutant corresponded to loss of the conventional glibenclamide-sensitive KATP currents (IKATP) in isolated cardiomyocytes and vascular smooth muscle cells but the SUR2 short forms remained intact. Nested exonic RT-PCR in the mutant indicated that the short forms lacked NBD1 but contained NBD2. The SUR2 short forms co-immunoprecipitated with Kir6.1 or Kir6.2 suggesting that the short forms may function as hemi-transporters reported in other eukaryotic ABC transporter subgroups. Our results indicate that different KATP compositions may co-exist in cardiac sarcolemmal membrane.
KATP channels are widely distributed in various tissues and play important physiological roles in regulating insulin secretion in pancreatic β-cells , providing ischemic protections to the heart  and modulating vascular tone in smooth muscles . It is generally agreed that the sacrolemmal KATP is a hetero-octamer that consists of a Kir6.0 pore and a sulfonylurea receptor (SUR) . SUR1 (~177 kDa) is a high-affinity SUR that is primarily present in pancreatic β-cells  while SUR2 (~174 kDa) is a low-affinity SUR that is mainly detected in the heart . Multiple alternative splice variants have been found in each SUR gene . The SUR in cardiac and vascular smooth muscle is encoded by two splice variants of SUR2 (ABCC9), SUR2A and SUR2B, which differ in the alternative use of the last exon . These SUR isoforms and splice variants have increased the diversity of KATP.
SURs belong to the ATP-binding cassette (ABC) transporter superfamily. A typical eukaryotic ABC transporter  contains two symmetric transmembrane domains (TMD1 and TMD2) and two nucleotide-binding domains (NBD1 and NBD2). However, SURs diverge from other ABC transporters  by having an additional TMD0 and two asymmetric NBDs, with NBD1 as the initial ATP-binding site while NBD2 is more likely involved in ATP hydrolyzation . The role of SURs has evolved from other ABC transporters by regulating the KATP complex instead of transporting a specific substrate itself. ABC transporters also exist as hemi-transporters, either in homodimeric or heterodimeric forms, employing the only TMDs to receive regulatory signals from their single NBDs .
In SUR1, glibenclamide acts on an N-end component between NBD1/transmembrane helix (TM) 12 and a C-end component located between TM15−16 . Mutating either component abolishes the drug effect. A previously generated SUR2 mutant mouse has a disruption cassette inserted between exons 10−16 to disrupt NBD1 of SUR2 . The glibenclamide effect is expected to be altered in the mutant. Earlier characterizations at the nucleic acid level suggest that the disruption cassette is present in SUR2, which is confirmed by the loss of the conventional glibenclamide-sensitive KATP currents (IKATP) in isolated cardiomyocytes  and vascular smooth muscle cells . This mouse was thought to be a SUR2 null mouse based on lines of functional data. In the present work, however, a non-conventional glibenclamide-insensitive, ATP-sensitive current (IKATPn) was found in the mutant. Using a panel of newly developed SUR2 isoform- or variant-specific antibodies that were unavailable in earlier study , novel SUR2 short forms in the sizes of 28−68 kDa were discovered in the mutant. Evidence at both mRNA and protein levels indicated that the short forms lacked NBD1 but had NBD2, and they could interact with Kir6.1 or Kir6.2, which may account for IKATPn. Our observations suggested a greater diversity for KATP structure as previously proposed based on pharmacological and physiological studies.
Detailed protocols and additional supporting information are provided in an on-line Supplement
The SUR2 (ABCC9) cDNA clones were from mouse  while the SUR1 (ABCC8) cDNA clone was from rat . The mouse KIR6.2  or KIR6.1 [17, 18] gene was cloned from a mouse heart cDNA library (BD BioSciences, San Jose, CA) by PCR. The KIR6.2 gene was amplified by using primers, P1a: 5'-GGAGCCA TGCTGTCCCGAAAGGGC-3' and P2a: 5'-ACAAGTGAGTGGGGGCCTGAGG-3' while the mouse KIR6.1 gene was amplified by primers, P3a: 5'-ATGCTGGCCAGGAAGAGCAT-3' and P4a: 5'-GTCATCGGGACTCAGTGAG-3'. PCR conditions were performed as previously described  and the KIR6.2 or KIR6.1 PCR products were then subcloned into pcDNA3 (Invitrogen, Carlsbad, CA) as pYB2 or pYB1. Primers used in the nested exonic RT-PCR experiment to amplify possible transcripts encoding the SUR2 short forms were P1: 5'-ATGAATCCCCAGAAAGTGAAGCCT-3'; P2: 5'-ATGTTTCCAGAGACACTCTCATCAAA-3'; P3: 5'-ATGATTGTAGGCCAAGTGGGTTGTGG-3'; P4: 5'-ATGCAGACCCTGGAAGGAAAAGTTTA-3'; P5: 5'-ATGTATTCAAGAGAGGCCAAGGCAC-3'; P6: 5'-ATGGGCCTCACAGCCGCCAAGAAC-3'. P7: 5'-GGCCATGTCGATGGAAGCAG TGGC-3'
The SUR2 mutant mouse  was previously described and generated in C57BL-6J/129SV/J mixed background (Jackson Laboratories, Bar Harbor, Maine). It was on this mixed background that mice were previously characterized. The SUR2 targeted allele was then bred heterologously through six generations onto the FVB background . Heterologous mice were then interbred and genotyped to obtain homozygous mutants. Homozygotes were maintained for experiments described in this work. Mouse protocols were performed following the guidelines of NIH at the University Wisconsin Animal Core Facility.
Cell culture and transfection were performed as described previously . Single colonies were screened and confirmed by RT-PCR using the SuperScript II Kit (Invitrogen) and Western blot analysis. The RT-PCR primers for KIR6.2, P5a: 5'-AGAATATCGTCGGGCTGATG-3' and P6a: 5'-GTTTC TACCACGCCTTCCAA-3' and for KIR6.1, P7a: 5'-CTATCATGTGGTGGCTGGTG-3' and P8a: 5'-CGTGGTTTTCTTGACCACCT-3'.
T1 was raised against amino acids 268−287 while BNJ-2 was raised against amino acids 636−649 of SUR2. BNJ-39 was raised against the C-terminus of SUR2A (1525−1546) while BNJ-40 was raised against the C-terminus of SUR2B (1523−1536). Epitopes were synthesized by KLH-conjugation at its N-terminus and affinity-purified (Invitrogen) against the peptide. The isoform or variant specificity of each antibody was tested in the Kir6.2 stable cell line by introducing SUR2 or SUR1. Anti-Kir6.2 (sc-11224) or anti-Kir6.1 (sc-11228) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Nav1.5 (06−811) was obtained from Upstate (Charlottesville, VA) and Anti-HCN4 (APC-052) was from Alomone Labs (Jerusalem, Israel). Anti-Na/K ATPase (ab-7671), anti-VDAC1 (ab-3434) or anti-COXIV (ab-14744) was obtained from Abcam (Cambridge, MA).
Ventricular myocytes were isolated from mouse hearts by enzymatic treatment as previously described . Immunostaining was performed as previously described . BNJ-39 (1:500) or BNJ-40 (1:250) was the primary antibody while the anti-rabbit Alexa Fluor-488 IgG (1:250) was the secondary antibody (Invitrogen). All confocal recordings were performed at room temperature and the images were analyzed by Confocal Assistant software.
Protein isolation  was handled on ice or at 4 °C to prevent proteolysis and Westerns were performed as described . Concentration was determined by the Lowry method using a DC Protein Assay Kit (Bio-Rad, Hercules, CA). Secondary antibodies were from Invitrogen and Amersham (Piscataway, NJ) and added at 1:10,000−1:12,500. Blots were scanned by the BioSpectrum Imaging System (UVP, Upland, CA).
Cardiac membrane fractions of WT and SUR2 mutant mouse heart tissues were prepared as previously described . Tissues from 8 mice were pooled for isolation. Hearts (the atria were removed) were rapidly removed and put in ice-cold extraction buffer. The crude extracts were separated by a discontinuous gradient containing 21%, 31%, 40% and 55% sucrose at 141,000 ×g for 2 h resulting in three distinct interfaces. The very top fraction at 21% sucrose was used for surface membrane protein isolation. Contamination of mitochondria was determined by Western blots using anti-VDAC1 (1:250), anti-COXIV (1:5000) and anti-Na/K ATPase (1:1000). Protein quality was determined by the detections of Nav1.5 (1:200) and HCN4 (1:200).
Co-IP experiments were carried out using Dynabeads Protein A or G (Invitrogen) by following manufacturer's recommended procedures. 5 μg of T1 or control IgG was used to IP ~100 μg purified membrane proteins isolated from WT hearts in the forward IP experiments followed by a Western blot using BNJ-39 (1:2000). In the reverse IP experiments by BNJ-39, T1 was the Western blot antibody (1:2000). In the experiments to study associations of the SUR2 short forms with Kir6.1 or Kir6.2, 5 μg of BNJ-39, BNJ-40 or control IgG was used to IP ~100 μg purified membrane proteins isolated from the SUR2 mutant hearts in the forward IP experiments. Anti-Kir6.1 (1:200) and anti-Kir6.2 (1:200) were used as the Western antibodies. In the reverse IP experiments by anti-Kir6.1, BNJ-39 (1:2000) was the Western antibody.
IKATP was recorded by single-channel recordings using inside-out configuration at room temperature as previously described . Ventricular myocytes were isolated from mice that were >12-wks-old. In the glibenclamide experiment, macroscopic currents were recorded from whole cells as described . Experimental data was represented as mean±SEM. Data analysis and presentation were carried out as described in . A t-test was used in any comparison of two groups while a χ square test was used in any comparison between two variables. p<0.05 was considered a significant difference.
In the SUR2 mutant mouse with a disrupted NBD1, the conventional glibenclamide-sensitive IKATP was previously reported to be absent in both ventricular myocytes  and vascular smooth muscle cells . Single channel recordings in the ventricular myocytes isolated from the SUR2 mutant hearts, however, recorded a non-conventional K+ current (designated as IKATPn , Fig. 1A) that significantly differed from the WT conventional K+ current (IKATP, Fig. 1B). IKATPn was sensitive to cytoplasmic ATP and completely inhibited at 1 mM concentration (Fig. 1C). In addition, IKATPn was present at a much lower density with fewer patches showing currents (Fig. 2A), and with smaller amplitude per patch (Fig. 2B). KATPn was four times more sensitive to ATP than KATP (Fig. 2C) with a 10.0 μM half-maximal inhibition value (IC50). Open probability (Po) for KATPn in the mutant cells was ~10% higher than that for KATP in WT cells (Fig. 3A) while single channel conductance (pS) for the mutant cells was quite similar to WT cells at a membrane potential of 0 mV (Fig. 3B). The mean burst duration for KATPn was half of that for KATP (Fig. 3C). KATPn showed a significantly shorter time constant for closed intervals within (τ1) or between bursts (τ2) than those recorded in KATP in the open and closed time histogram (Fig. 3D). Resting potential for the mutant or WT cells was −76±0.58 mV and −75.5±0.56 mV (Fig. 4A), respectively. In contrast to IKATP recorded in WT cells (Fig. 4B), IKATPn in the SUR2 mutant cells was insensitive to 10 μM glibenclamide (Fig. 4C). Single channel activity consistent with IKATPn (sensitive to ATP, insensitive to 10 μM glibenclamide) was also detected in WT cells. These results suggested that IKATPn had distinct ATP sensitivity, kinetics and drug response compared to IKATP.
Several types of KATP channels may account for IKATPn in the SUR2 mutant cells. A Kir6.2 with a truncated C-terminus (Kir6.2ΔC35) that is able to travel to cell surface without an SUR subunit  could be a candidate. However, such a recombinant channel expressed in COS1 cells (n=6) showed a mean burst duration of 4.6±1.0 ms, a Po of 0.19±0.03 mV and a single channel conductance value of 25.9±0.9 at a membrane potential of 0 mV (p<0.05 compared to KATPn) in our study (data not shown) suggesting it did not underlie IKATPn. A Kir6.2/SUR1 (glibenclamide-sensitive) channel mainly functions in β-cells [4-5] but not in cardiac cells and displays distinct kinetics from a SUR2-based channel. These observations led us to develop a new panel of SUR2 antibodies (Fig. 5A) to identify whether any SUR2 forms remained in the mutant, which may confer IKATPn. COS1 cell lines stably expressing KIR6.2 or KIR6.1 were generated to facilitate specificity tests for the new SUR2 antibodies. Positive candidates from the stable cell line selection were confirmed by RT-PCR (Fig. 5B) and Western blot analysis (Fig. 5C). One Kir6.2 and two Kir6.1 stable lines were obtained. The specificity of each antibody was then tested by expressing each SUR isoform or splice variant into the Kir6.2 stable cell line. T1 or BNJ-2, recognized a 170-kDa SUR2 band only in the cells expressing KIR6.2 and SUR2A cDNA suggesting they are SUR2 isoform-specific antibodies (Fig. 5D).
BNJ-39 and BNJ-40 were designed to distinguish the two SUR2 splice variants, with BNJ-39 specific to the C-terminus of SUR2A and BNJ-40 specific to the C-terminus of SUR2B. Because SUR2A differs from SUR2B only in the last exon (contains 42 amino acids) and the 42 amino acids in each variant share ~30% homology, a relatively long epitope was designed to achieve better antigenicity for BNJ-39. It was noted that the last 42 amino acids at the C-terminus of SUR2B shares ~70% homology with that of SUR1. We excluded the last 10 of the 42 amino acids in SUR2B to improve the specificity for BNJ-40. These designs set our antibodies apart from other reported SUR2 antibodies. BNJ-39 detected a 170-kDa SUR2A band only in the cells containing SUR2A and KIR6.2 (Fig. 5E). A trace amount of the 170-kDa band was found in the untransfected COS1 cells indicating that endogenous SUR2A was present in our COS1 line. On the other hand, two bands in the sizes of 170-kDa and 100-kDa were detected by BNJ-40 in the cells when KIR6.2 and SUR2B were co-expressed (Fig. 5F). The 100-kDa band was present in the untransfected COS1 cells and the cells containing SUR2A and KIR6.2 but it was present much more abundantly in those cells containing SUR2B and KIR6.2. This suggested that our COS1 line had the endogenous 100-kDa SUR2B, which could be a subtype splice variant for SUR2B. Thus, SUR2A or SUR2B was only recognized by the antibody generated based on the corresponding variant.
A cardiac membrane fraction from WT mouse hearts was fractionated as previously described  and proteins isolated from the very top fraction showed no detectable mitochondrial contamination in gels based on a Western blot analysis (Fig. 6A, left panel) of two mitochondria markers, VDAC1 (outer membrane) and COXIV (inner membrane), while a plasma membrane marker, Na/K ATPase was abundantly present. To assess whether large molecular-weight ion channel proteins (>150-kDa) were intact in this fraction, the α subunit of the voltage-gated sodium channel, Nav1.5 and the pacemaker channel isoform 4, HCN4, were used as quality controls (Fig. 6A, right panel). Both channels appeared intact in the fraction, suggesting that the protein isolation process did not cause proteolysis in these proteins.
When the T1 or BNJ-2 antibody was used to cross-react with the WT cardiac membrane fraction, a 150-kDa band was found (Fig. 6B, Lanes 1&2). The band detected by T1 appeared to be prominent, which was unexpected. We found later that a theoretical secondary glycosylation site was included in this relatively long epitope. Differentiated glycosylation has been reported in SUR proteins , therefore, T1 may detect a group of bands in various sizes as observed in Fig. 6B (Lane 1). BNJ-2, however, was raised against a different region of SUR2, did not detect such a pattern. The C-terminal antibody, BNJ-39, only detected two short forms (Fig. 6B, Lane 5) in the sizes of 68-kDa (designated as 68A) and 28-kDa (designated as 28A) from the membrane fraction. The presence of these short forms was confirmed in purified membrane proteins isolated from individual ventricular myocytes (data not shown). SUR2B is thought to be a major splice variant present in smooth muscle and it is not expected to be found in ventricular tissues. BNJ-40, detected a faint 150-kDa band in a whole heart lysate (Fig. 6B, Lane 4), which could be contaminated by some smooth muscle. In the cardiac membrane fraction, BNJ-40 (Fig. 6B, Lane 6) only detected a 28-kDa band (designated as 28B). These results suggested that the SUR2-specific antibodies detected SUR2 in various sizes in mouse cardiac membrane but they detected full-length SUR2 when each variant was co-expressed with Kir6.2 in COS1 cells (Figs 5D-F).
To further understand why BNJ-39 could not detect a 150-kDa band that was detected by the internal antibodies, T1 and BNJ-2, co-immunoprecipitation experiments were carried out. The cardiac membrane fraction was immunoprecipitated with T1 in the forward IP experiments followed by a Western blot using BNJ-39 (Fig. 6C, Lane 1). Two bands in 150-kDa and 28-kDa were detected by BNJ-39 after IP, but the 68-kDa band was not. These results were confirmed in the reverse IP experiments (Fig. 6C, Lane 3) suggesting 28A had an association with a 150-kDa form in our preparations. In the reverse IP experiments, two additional bands in the sizes of 100-kDa and 55-kDa were found, which could be from the T1 antibody because a faint 55-kDa band was observed in the control (Fig. 6C, Lane 4). Because these two bands were not detected in the forward IP, they were probably not part of the 150/28A complex. Immunostaining was performed by using BNJ-39 and BNJ40 in ventricular myocytes isolated from the WT hearts. Both antibodies detected green fluorescence signals in WT ventricular myocytes (Fig. 6D, Panels II). In WT controls where no primary antibodies were added, green fluorescence was not detected (Fig. 6D, Panels IV). These results confirmed the Western blot results in Fig. 6B.
The SUR2 mutant mouse was generated with a disruption cassette inserted between exons 10 and 16 of SUR2 (Fig. 7A) to disrupt NBD1 . The SUR2 antibodies were then used to cross-react with a cardiac membrane fraction isolated from the SUR2 mutant. Neither T1 nor BNJ-2 detected a 150-kDa SUR2 band suggesting that it was disrupted as expected (Fig. 7B, Lanes 1&2). This data was consistent with previous observations that glibenclamide-sensitive IKATP was absent in both mutant ventricular myocytes  and vascular smooth muscle cells . However, BNJ-39 and BN-40 detected the SUR2 short forms in the mutant heart tissues (Fig. 7B, Lanes 3&4) as observed in WT. Immunostaining was performed to confirm the Western blot results in the mutant. Both C-terminal antibodies were used to detect SUR2 signals in ventricular myocytes isolated from the mutant hearts. BNJ-39 and BNJ-40 detected green fluorescence signals (Fig. 7C, Panels II) suggesting that the SUR2 short forms were intact. In mutant controls where no primary antibodies were added, no green fluorescence was detected (Fig. 7C, Panels IV).
To study the possible locations of SUR2 transcripts encoding the short forms in the SUR2 mutant, six forward primers (see Fig. 7A) were designed for a nested exonic RT-PCR experiment based on regions before exon 10 (P1, P2), within exons 10−16 (P3, P4) and after exon 16 (P5, P6). Each forward primer was paired with a reverse P7 primer that was designed based on exon 36 of SUR2 to amplify the expected regions using SUR2A cDNA  as the templates. PCR products in expected sizes were amplified successfully by all primer pairs (Fig. 7D, top panel). The six primer pairs were then used to perform nested exonic RT-PCR using mRNA isolated from WT or mutant heart tissues. Expected RT-PCR products were amplified from WT (Fig. 7D, middle panel) but RT-PCR products were only amplified by primer pairs 5/7 and 6/7 from the mutant (Fig. 7D, bottom panel). Cloning and sequencing of each RT-PCR band amplified from the mutant suggested that they encode partial SUR2 sequences that lacked NBD1 but contained NBD2. The 1.7-Kb and 1.3-Kb products were overlapped as expected, which could be the partial transcript encoding 68A while the 0.6-Kb product could be a partial transcript encoding 28 forms. These observations provided initial evidence that transcripts encoding the remaining short forms could be generated by alternative splicing and they were located after exon 16.
A protein level approach was attempted to identify the starting amino acid sequences for the SUR2A-based short forms. BNJ-39 was used to immnuoprecipitate a mutant cardiac membrane fraction and the sample was separated by 2-dimensional gels (data not shown). A candidate spot at ~68-kDa (for 68A) was detected and confirmed by Western blot analysis using BNJ-39 in one blot. The 68-kDa spot was excised from the other blot and processed for N-terminal protein sequencing to determine the starting amino acids for this candidate. However, this experiment did not yield any information because the N-terminal end of the 68-kDa protein was acetylated, which is a common modification scheme in certain eukaryotic proteins.
A previous report in a Kir6.2/SUR1 channel study demonstrated that the C-terminus of SUR1 was essential for channel complex trafficking . Kir6.2 was further shown to interact with either TMD0 (transmembrane domain 0) or TMD2 but not TMD1 . By sequence homology between SUR1 and SUR2, TMD1 may not be as critical as TMD0 or TMD2 for binding of SUR2 to the pore. A more recent study in a Kir6.2/SUR2A channel suggested that Kir6.2 interacted with SUR2A at a region of amino acid position 1294−1358, which is located within NBD2 . To investigate whether each SUR2 short form had an association with Kir6.1 or Kir6.2, Co-IP experiments were performed using the purified mutant cardiac membrane fraction. Anti-Kir6.2 detected a 43-kDa Kir6.2 band in the samples immunoprecipitated by either BNJ-39 or BNJ-40 (Fig. 8A). In the reverse IP experiments, BNJ-39 detected both 68A and 28A while BNJ-40 detected 28B. Thus, each SUR2 short form could interact with Kir6.2. By contrast, anti-Kir6.1 detected a 47-kDa Kir6.1 band only in the sample immunoprecipitated by BNJ-39 (Fig. 8B, Lane 2). Therefore, BNJ-39 was tested in the reverse IP, and it detected 68A and 28A (Fig. 8B, Lane 5). This indicated that the SUR2A-based short forms had an association with Kir6.1 as well. These observations and data from Fig. 6C may lead to KATP channels with different compositions, regulation and pharmacology.
A novel glibenclamide-insensitive, ATP-sensitive current (IKATPn) was recorded in a SUR2 mutant mouse. Due to the significantly lower current density, smaller amplitude of the current, and insensitivity to glibenclamide, IKATPn was relatively difficult to detect in WT cells. However, the mutant provided a background in which the 150-kDa SUR2 long form was disrupted allowing for IKATPn detection. The kinetics, regulation and drug response of IKATPn were distinctively different from those reported for the conventional IKATP. The constituents possibly accounting for this current were then further investigated in the SUR2 mutant mice. Because the kinetics of IKATPn differed significantly from that of a recombinant Kir6.2Δ35 channel , Kir6.2Δ35 was probably not responsible for conferring IKATPn. The unavailability of commercial SUR2 isoform- or SUR2 splice variant-specific antibodies made characterization at the protein level difficult in earlier studies [14,15]. To search for possible SUR2 forms in the mutant, we designed new antibodies to distinguish SUR2 isoforms and splice variants. These SUR2 antibodies detected novel SUR2 short forms in the sizes of 28 and 68 kDa from WT hearts. Nested exonic RT-PCR results suggested that the short forms could be generated by alternative splicing, therefore, they were “unaffected” by the disruption. Such splicing events have been reported earlier in other eukaryotic genes . The complete sequences for these SUR2 variants, however, remain to be identified.
Two SUR2 isoform-specific antibodies, T1 and BNJ-2, detected a 150-kDa SUR2 band but the C-terminal BNJ-39 antibody was able to detect it only after an IP. SUR2 has an estimated size of 174-kDa but we and others [33,34] detected ~140−150-kDa in WT native cells. These observations let us hypothesize that two 150-kDa forms may exist in WT cardiac membrane, one lacks a N-terminal domain while the other lacks a C-terminal domain in the size of ~25−35 kDa. Both forms are expected to be recognized by T1 but BNJ-39 can only detect the first form in a Western blot. This form is thought to be glibenclamide-sensitive (Fig. 8C, I) while the second form is not due to the truncation in the C-terminus, which is required for glibenclamide-sensitivity . The detection of a 150-kDa band by BNJ-39 after an IP indicates that the first form may be present scarcely in the cell. Moreover, the second form can not be detected by BNJ-39 in the Co-IP experiments unless an SUR2 short form is associated with it. The detection of a 28A band in the Co-IP experiments suggested that it could interact with the second 150-kDa form as a complex (Fig. 8C, II). We further hypothesize that the 150/28A complex is glibenclamide-sensitive and it may underlie IKATP in WT cells. In the SUR2 mutant mice, however, both 150-kDa forms are expected to be disrupted leading to glibenclamide-insensitivity. The exact nature and functional/pharmacological consequences of these 150-kDa forms requires further study.
The Co-IP results indicated that the 68A short form probably had no association with the 150-kDa forms. Because 68A could interact with Kir6.1 or Kir6.2, these short form channels may co-exist with the long-form based channels in cardiac membrane (Fig. 8C, III). The 28B short form also had an association with Kir6.2 suggesting Kir6.2/28B channels may be present as well (Fig. 8C, IV). Previous photo-affinity labeling in β-cells identified three SUR1 proteins in the sizes of 140-kDa, 65-kDa and 33-kDa . [3H]Glibenclamide was predominantly incorporated into the 140-kDa and 33-kDa forms simultaneously but not into the 65-kDa form . Instead, the 65-kDa form was primarily labeled by [3H]glimepiride, a sulfonylurea with different receptor binding kinetics. The [3H]glibenclamide labeling of two other SUR1 forms inhibited the labeling of the 65-kDa form by [3H]glimepiride. These results implied that SUR1 proteins exist in different sizes and configurations (a 140/33 kDa complex and a 65-kDa form). The sizes of the detected SUR1 forms by photo-affinity labeling are in close proximity to the SUR2 forms detected by our antibody approach. The biological significance and regulation of the SUR short forms have not been extensively elucidated. This report represents the initial effort of a long-term study to address the composition and function of the SUR2 short form-based channels.
The intended SUR2 mutant mouse was designed to disrupt NBD1, where action sites for several sulfonylurea drugs are located , i. e. glibenclamide . Results from the nested exonic RT-PCR experiment in the mutant suggested that the SUR2 short forms lacked NBD1 but contained NBD2. Channels formed by these short forms were therefore expected to be insensitive to glibenclamide. Pharmacological study of IKATPn showed that it was indeed insensitive to 10 μM glibenclamide. Our study was extended to identify the pore-forming subunit that would interact with these SUR2 short forms. Co-IP results showed that each SUR2 short form had an association with Kir6.2 but only the SUR2A-based short forms could interact with Kir6.1. These results suggested that the SUR2 short forms may act as regulatory subunits for KATPn possibly by dimerization as hemi-ABC transporters. They could use NBD2 to receive regulatory signals from a TMD as reported previously for other hemi ABC transporters . Obtaining the complete sequences for SUR2 short forms is expected to allow us to further characterize these channels in recombinant systems. We are aware that we can not completely exclude the involvement of SUR1 short forms [35,36] in underlying IKATPn because several SUR1 splice variants have been reported earlier [38-40], and SUR1 signals have been detected by antibodies in heart  probably from a short splice variant .
KATP also exists in mitochondrial inner membrane  and may be important in conferring ischemic protection to the heart [43,44]. A long form of SUR2 in the size of 140-kDa  and a 28-kDa short form  have been detected by SUR2 antibodies in heart mitochondria. The identities and sequences of these proteins, however, are unclear. Although the exact molecular composition for mitochondrial KATP remains to be elucidated, the novel SUR2 short forms from this study could be SUR2 candidates that are targeted to heart mitochondria. Future study of the molecular mechanisms that generate the novel SUR2 short forms can expand our knowledge on sacrolemmal and mitochondrial hemi-ABC transporters, and the roles for the two types of KATP channels in ischemic preconditioning. In conclusion, we have identified an ATP-sensitive current with unique kinetics and pharmacology in a SUR2 mutant mouse that contains novel SUR2 short forms. These short forms may act as hemi-transporters similar to reported cases in other eukaryotic ABC transporter subgroups. More study is required to assess the significance of these findings in WT hearts, but our results suggest that KATP channels with diverse structures, regulatory function and pharmacology may play roles under normal physiological and stress conditions.
This study was supported by The American Heart Association National Center (0630268N to N.Q.S), (0435030N to B.Y) and by The National Institute of Health (2R01HL-57414 to J.C.M.).
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