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Am J Physiol Heart Circ Physiol 289: H2714-H2723, 2005. First published July 29, 2005; doi:10.1152/ajpheart.00534.2005
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Differential expression of small-conductance Ca2+-activated K+ channels SK1, SK2, and SK3 in mouse atrial and ventricular myocytes

Dipika Tuteja,1 Danyan Xu,1 Valeriy Timofeyev,1 Ling Lu,1 Dipika Sharma,1 Zhao Zhang,1,5 Yanfang Xu,1,6 Liping Nie,4 Ana E Vázquez,4 J. Nilas Young,3 Kathryn A. Glatter,1 and Nipavan Chiamvimonvat1,2

1Division of Cardiovascular Medicine, University of California, Davis; 2Department of Veterans Affairs, Northern California Health Care System, Mather; 3Department of Cardiothoracic Surgery, University of California, Davis; 4Department of Otolaryngology, Center for Neuroscience, University of California, Davis, California; 5Department of Physiology, University of Zhengzhou, Zhengzhou; and 6Pharmacology Department, Hebei Medical University, Shijiazhuang, China

Submitted 20 May 2005 ; accepted in final form 20 July 2005


   ABSTRACT
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 ABSTRACT
METHODS
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 DISCUSSION
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Small-conductance Ca2+-activated K+ channels (SK channels, KCa channels) have been reported in excitable cells, where they aid in integrating changes in intracellular Ca2+ with membrane potential. We recently reported for the first time the functional existence of SK2 (KCa2.2) channels in human and mouse cardiac myocytes. Here, we report cloning of SK1 (KCa2.1) and SK3 (KCa2.3) channels from mouse atria and ventricles using RT-PCR. Full-length transcripts and their variants were detected for both SK1 and SK3 channels. Variants of mouse SK1 channel (mSK1) differ mainly in the COOH-terminal structure, affecting a portion of the sixth transmembrane segment (S6) and the calmodulin binding domain (CaMBD). Mouse SK3 channel (mSK3) differs not only in the number of polyglutamine repeats in the NH2 terminus but also in the intervening sequences between the polyglutamine repeats. Full-length cardiac mSK1 and mSK3 show 99 and 91% nucleotide identity with those of mouse colon SK1 and SK3, respectively. Quantification of SK1, SK2, and SK3 transcripts between atria and ventricles was performed using real-time quantitative RT-PCR from single, isolated cardiomyocytes. SK1 transcript was found to be more abundant in atria compared with ventricles, similar to the previously reported finding for SK2 channel. In contrast, SK3 showed similar levels of expression in atria and ventricles. Together, our data are the first to indicate the presence of the three different isoforms of SK channels in heart and the differential expression of SK1 and SK2 in mouse atria and ventricles. Because of the marked differential expression of SK channel isoforms in heart, specific ligands for Ca2+-activated K+ currents may offer a unique therapeutic opportunity to modify atrial cells without interfering with ventricular myocytes.

calcium-activated potassium current; mouse cardiac myocyte

ONE OF THE RECENTLY DISCOVERED ion channels among the six transmembrane (TM) structured channels are small-conductance Ca2+-activated K+ channels (SK channels, KCa channels) (16). SK channels are widely expressed in different tissues, including the brain, peripheral nervous system, skeletal muscle, and smooth muscle (2, 14, 16, 20–22, 25, 29, 32–35, 37). However, their presence and functional significance have not been well studied in the heart. Our laboratory was the first to report the functional contribution and cloned SK2 (KCa2.2) channel isoform from mouse and human hearts (41).

SK channels regulate neuronal excitability and hormone secretion from endocrine cells (6, 7) and have been shown to underlie slow afterhyperpolarization (sAHP) following action potential (AP) in neurons (3, 26). In contrast, in cardiac myocytes, we have found that SK channels are important in the repolarization of cardiac action potentials in mice and humans (41).

Cloning and functional expression of SK channel genes have revealed that these genes belong to a superfamily of ion channels that includes voltage-activated K+ channels (typified by Shaker K+ channel), Ca2+-activated large-conductance K+ channels (BK), and cyclic nucleotide-gated channels (4, 16, 37). SK channels are characterized by their small unitary conductance (2–20 pS), high Ca2+ sensitivity (submicromolar concentrations), very weak voltage sensitivity, and susceptibility to blockade by apamin and d-tubocurarine (10, 16, 30, 39). To date, it has been shown that SK channels are encoded by at least three genes: SK1, SK2, and SK3 (KCNN1, KCNN2, and KCNN3) (16, 33). Each subtype of SK channels consists of a pore-forming {alpha}-subunit that is ~550–730 amino acids in length with its intracellular COOH and NH2 termini. In between the termini lie six putative TM (S1–S6) regions. The region between S5 and S6 forms a pore (P region) with a "K+ channel signature sequence" (GYG) (16, 33). However, the S4 region, which is the voltage sensor in the voltage-gated K+ channels, bears a reduced number and disrupted arrangement of the positively charged amino acids in SK channels (16).

Several functional and structural studies have shown that Ca2+ gating of the SK channels is accomplished by constitutive association of calmodulin (CaM) with the intracellular COOH-terminal domain of the channel {alpha}-subunits called the CaM binding domain (CaMBD) (5, 15, 27–29, 3839, 43). By analogy to other K+ channels, SK channels are thought to exist as tetramers together with four CaMs constitutively bound to carboxyl termini at the CaMBD to form functional SK channels. Apart from gating, CaM is also required for channel assembly and trafficking (13, 19).

Another means by which the binding of CaM to SK channels could be modulated is alternative splicing. Identification of splice variants of SK channels (especially SK1 transcripts) has shed light on how deletion of key nucleotide sequences in the COOH terminus can profoundly affect either Ca2+-dependent or Ca2+-independent interaction with the CaM-SK complex (29, 43). Alternative splicing is also reported to be one of the several mechanisms responsible for generating functional, structural, and anatomical diversity of K+ channels (4).

Previous studies using cloned SK channels and SK1-SK2 dimer have demonstrated that SK subunits may form heteromeric channels, giving rise to structural and functional diversity (11). We have previously demonstrated that SK2 channels are highly expressed in the atrial myocytes compared with the ventricles (41). In the present study, we further hypothesize that the atrial and ventricular tissues may express more than one isoform of the SK channels, with differential tissue distribution of the various isoforms. The aims of the present study are to test this hypothesis directly by examining the isoform-specific expression profile of SK channels in atria and ventricles. To accomplish these goals, we first cloned the cardiac-specific SK1 and SK3 channels from the mouse atria and ventricles. Regional distribution of the different isoforms was assessed using in situ hybridization. Quantification of the transcripts was performed using real-time RT-PCR.


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 METHODS
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All animal care and procedures were approved by the University of California, Davis Institutional Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80-213, 1985).

RNA extraction and RT-PCR cloning. Total RNA was prepared from mouse and rat brain and heart using TRIzol reagent (Invitrogen, Carlsbad, CA). We used 8- to 10-wk-old male CD-1 mice (Charles River Laboratories, Wilmington, MA) and 3- to 4-mo-old male Sprague-Dawley rats (Harlan). cDNA was synthesized from total RNA samples by oligo(dT)-primed reverse transcription (Superscript II RNase H reverse transcriptase; Invitrogen). cDNA was then subjected to PCR amplification using HotStarTaq DNA polymerase (Qiagen, Valencia, CA). Full-length SK1 coding sequences were obtained from mouse atria and ventricles using the following primers: 5'-ATGAGTAGCCACAGCCACAA-3' (forward) and 5'-TCACCCACAGTCTGATCCCA-3' (reverse) according to the published sequence of mouse colonic SK1 (accession no. AF357239). Full-length SK3 coding sequences were obtained from mouse atria and ventricles using the following primers: 5'-GATGGACACTTCTGGGCACT-3' (forward) and 5'-TTAGCAACTGCTTGAACT-3' (reverse) according to the published sequence of mouse colonic SK3 (accession no. AF357241). The presence of SK1 and SK3 isoforms was detected in rat heart tissue by using PCR primers: SK1, 5'-CAGGCCCAGCAGGAGGAGTT-3' (forward) and 5'-GGCGGCTGTGGTCAGGTG-3' (reverse); and SK3, 5'-ATGAGCTCCTGCAAATACAGC-3' (reverse), 5'-GCAACTGCTTGAACTTGTGTA-3' (reverse). The absence of genomic contamination in the RNA samples was confirmed with reverse transcription-negative controls (no RT) for each experiment. Amplified products were cloned into pCRII-TOPO plasmid vector (Invitrogen) and analyzed by DNA sequencing of both forward and reverse strands.

In situ hybridization. SK1-, SK2-, and SK3-specific cDNA fragments were generated by RT-PCR and TA cloning (Invitrogen). Primers used in PCR reactions were designed from unique regions of SK1, SK2, and SK3 channels (see Fig. 3A) as follows: SK1, 5'-GGTGGTGTCAGAGCTGCAGG-3' (forward) and 5'-TCACCCACAGTCTGATCCCA-3' (reverse); SK2, 5'-CATGATTTCCGACTTAAATG-3' (forward) and 5'-CTAGCTACTCTCTGATGAAG-3' (reverse); and SK3, 5'-CTTAATCACAGAACTCAATG-3' (forward) and 5'-TTAGCAACTGCTTGAACTTG-3' (reverse). All the clones were sequenced. The sense and antisense riboprobes were synthesized in the presence of UTP-digoxigenin using the DIG RNA labeling kit (Roche). Mouse hearts procured from 8- to 10-wk-old CD-1 mice were dissected and perfused first with RNase-free phosphate-buffered saline (PBS) and later with 4% paraformaldehyde (made in PBS). Perfused hearts were fixed 24–48 h at 4°C in 4% paraformaldehyde and embedded in paraffin. Sectioning was done, and sections were laid on poly-L-lysine-coated slides (Fisher Scientific, Hampton, NH). After dewaxing and hydration, in situ hybridization (ISH) was performed with the antisense as well as the corresponding sense cRNA probes on adjacent sections. After hybridization and washes, the sections were subjected to immunological detection with anti-digoxigenin Fab fragments conjugated to alkaline phosphatase, using the DIG nucleic acid detection kit (Roche). The signals were developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Roche) added in alkaline phosphatase buffer in the presence of levamisole (Sigma) to inhibit the endogenous alkaline phosphatase. The specimens were inspected for development of purple precipitate by using bright-field microscopy (Carl Zeiss Vision). Digitized images were captured using AxioVision 4 (Zeiss).



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  		Fig. 3. RT-PCR cloning of mouse heart SK3 (mhSK3). A: a representative agarose gel of RT-PCR amplified products with an approximate size of 2,200 bp from total RNA from mouse atria (lane 2), mouse ventricles (lane 3), and mouse brain (lane 4), obtained using primers specific for the mcSK3 channel (accession no. AF357241). Lane 5 is a negative control (PCR amplified without RT). Lane 1 is lambda HindIII ladder (New England Biolabs). B: a representative agarose gel of RT-PCR amplified products with an approximate size of 1,632 bp from total RNA from rat brain (lane 1) and rat heart (lane 2), obtained using primers specific for rbSK3 channel (accession no. U69884). Lanes 3 and 4 are negative controls (PCR amplified without RT). Lane 5 is a 100-bp ladder (New England Biolabs). C: amino acid sequence alignment (ClustalW) of deduced mhSK3 protein sequence compared with that of mcSK3 and rbSK3. Six predicted TM domains (S1–S6) and P region are highlighted in yellow. Red box in P region represents K+-selective filter GYG. Symbols used are as described in Fig. 2 legend.

 
Isolation of adult mouse atrial and ventricular myocytes. To perform quantitative RT-PCR (qRT-PCR) and immunofluorescence confocal microscopy, we used single, isolated atrial and left ventricular free wall myocytes. For immunofluorescence confocal microscopy, we used freshly isolated cells. For qRT-PCR, to reduce the contamination from fibroblast, endothelial, and other cell types in the heart, we first preplated the cells for 1 h in culture, after which the cardiac myocytes were harvested and total RNA was isolated. Cell isolation was performed as previously described with minor modification (44). CD-1 mice (8–10 wk old) were anesthetized with pentobarbital sodium (100 mg/kg ip). The heart was quickly removed from the chest and retrogradely aortic perfused at constant pressure (60 mmHg) at 37°C for ~3 min with a Ca2+-free buffer containing (in mM) 120 NaCl, 5.4 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 5.6 glucose, 10 HEPES, 10 2,3-butanedione monoxime (Sigma), and 5 taurine (Sigma), pH 7.2–7.2, gassed with 100% O2. To reduce bacterial contamination, we washed the perfusion setup with 70% alcohol and then rinsed three times with sterilized distilled water before cannulation. All the solutions were filtered (0.2-µm filter). The enzymatic digestion was initiated by adding collagenase type B (0.5 mg/ml; Boehringer Manheim), collagenase type D (0.5 mg/ml; Boehringer Manheim), and protease type XIV (0.02 mg/ml; Sigma) to the perfusion solution. When the heart became swollen and hard after ~3 min of digestion, 50 µM Ca2+ was added to the enzyme solution. About 7 min later, the heart was quickly removed, separated into atria and left ventricular free wall, cut into several chunks, and further digested in a shaker (60–70 rpm) for 10 min at 37°C in the same enzyme solution. The supernatant containing the dispersed myocytes was filtered into a sterilized tube and gently centrifuged at 500 rpm for 1 min. The cell pellet was then promptly resuspended in Ca2+ solution I (125 µM Ca2+). After the myocytes were pelleted by gravity for ~10 min, the supernatant was aspirated and the myocytes were resuspended in Ca2+ solution II (250 µM Ca2+). The final cell pellet was suspended in Ca2+ solution III (500 µM Ca2+). Meanwhile, the shake-harvest procedure was repeated several times until all the chunks were digested.

Culture of adult mouse cardiac myocytes. Culture dishes were precoated for 1 h with 10 µg/ml mouse laminin (Invitrogen) in PBS (Invitrogen) with 1% penicillin-streptomycin (PS; Invitrogen) at room temperature. Freshly isolated cardiac myocytes were suspended in minimal essential medium (MEM; Invitrogen) containing 1.2 mM Ca2+, 2.5% fetal bovine serum (FBS; Invitrogen), and 1% PS (pH 7.35–7.45). After the myocytes were pelleted by gravity for ~10 min, the supernatant was aspirated and the myocytes were washed two more times using the same protocol. The myocytes were then plated at 0.5–1 x 104 cells/cm2 in MEM containing 2.5% FBS and 1% PS to allow the fibroblast to adhere to the tissue culture dishes. After 1–2 h of culture in a 5% CO2 incubator at 37°C, the myocytes were harvested and used for RNA isolation and real-time qRT-PCR.

Real-time qRT-PCR. To determine the relative abundance of SK isoforms in mouse atria vs. ventricles, we performed real-time qRT-PCR with SYBR Green PCR Master Mix (Applied Biosystems) using the ABI PRISM 7900 HT sequence detection system (Applied Biosystems). Gene-specific primers were used to analyze transcript abundance (see Fig. 4A). Mouse GAPDH transcript level was analyzed as the internal control to compensate for differences in cell numbers and/or RNA recovery and to normalize the values of transcript abundance of SK1–SK3. The following primers were used: SK1, 5'-ATCCATCAGGCTCAGAAGCTCC-3' (forward) and 5'-AGCTCTGACACCACCTCATATGC-3' (reverse); SK2, 5'-ATGGATACTCAGCTGACCAAAAGA-3' (forward) and 5'-GCTTGCAAGAATTTCCGTTGATGC-3' (reverse); SK3, 5'-CCAAGCGGATCAAGAATGCTGC-3' (forward) and 5'-GACGCTCCTCAACTGGTGGATA-3' (reverse); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5'-GCAACAGGGTGGTGGACCT-3' (forward) and 5'-GGATAGGGCCTCTCTTGCTCA-3' (reverse). The PCR reactions were performed in triplicate for each gene and were cycled 40 times by using a two-step cycle procedure (denaturation at 95°C for 15 s, annealing at 65°C for 1 min) after the initial stages (50°C for 1 s, 95°C for 10 min). To generate the standard curve, we included serially diluted ventricular cDNA in the 96-well plate along with cDNA from atria and ventricles.



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  		Fig. 4. In situ hybridization (ISH). A: nucleotide sequence alignment (ClustalW) of the COOH termini from mhSK1, mhSK2, and mhSK3 channels. Highlighted regions refer to the primers used for the PCR reactions to generate the sense and antisense riboprobes. Asterisks indicate conserved nucleotides between the three isoforms. Nucleotide sequence numbers are given at right. Photomicrographs compare the distribution of SK1 (B), SK2 (C), and SK3 (D) transcripts in mouse atria and ventricles (left). Sections obtained using the corresponding sense riboprobes are shown at right as negative controls. Arrows refer to the great vessels. Scale bars, 500 µm.

 
Immunofluorescence confocal microscopy. Immunofluorescence confocal microscopy was performed as described previously (41). Isolated myocytes were fixed with 4% paraformaldehyde in PBS for 15–20 min and then treated with 0.4% Triton X-100 in PBS for 15 min and later with antibodies (1:100 dilution, anti-SK1 or anti-SK3 antibodies; Sigma) at 4°C overnight. Immunofluorescence labeling for confocal microscopy was done by treatment with fluorescein isothiocyanate-conjugated goat anti-rabbit secondary antibody (1:400 dilution; Calbiochem). Immunofluorescence-labeled myocytes were examined using Pascal Zeiss confocal laser scanning microscopy. Control experiments were performed by preincubation of primary antibody with the respective antigenic peptide (1:1) and also by incubating myocytes with secondary antibody only, under the same experimental conditions. Identical settings were used for all specimens.

Electrophysiological recordings. Twelve-week-old male CD-1 mice were used. All chemicals were purchased from Sigma Chemical (St. Louis, MO) unless stated otherwise. Single mouse atrial and ventricular myocytes were isolated as described above. APs were recorded at room temperature using the perforated-patch technique (1, 8, 18). The patch pipettes were backfilled with amphotericin (200 µg/ml). Pipette solution contained (in mM) 120 K-glutamate, 25 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES, pH 7.4 with KOH. The external solution contained (in mM) 138 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 0.33 NaH2PO4, 10 glucose, and 10 HEPES, pH 7.4 with NaOH. All data were corrected for the liquid junction potentials as described previously (22).


   RESULTS
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Presence of a Ca2+-sensitive repolarizing current predominantly in mouse atrial compared with ventricular myocytes. We recently reported the existence of SK2 channel in human and mouse cardiac myocytes by using a combination of biochemical and physiological techniques and cloned the SK2 channel from human and mouse hearts (41). In addition, we observed that mouse atrial cardiac myocytes functionally express a higher level of SK2 current compared with ventricular tissues, using a low concentration of apamin (41). In the present study, we further examined the contribution of Ca2+-sensitive repolarizing currents in mouse atrial vs. ventricular myocytes. Perforated-patch techniques were used to record AP. Experiments were performed in the presence of niflumic acid to block Ca2+-activated Cl– current, which has been shown to be present in mouse cardiac myocytes (40). Figure 1, A and B, shows a significant prolongation of the atrial AP (left) by application of BAPTA-AM, a cell-permeant Ca2+ chelator, or nimodipine, an L-type Ca2+ channel blocker. In contrast, only a moderate effect was observed in ventricular myocytes (right). Bar graphs in insets show summary data of action potential duration at 50 and 90% repolarization (APD50 and APD90, respectively). These data suggest a differential expression of Ca2+-dependent repolarizing currents in atria compared with ventricular myocytes. Alternative possibilities included a decrease in the contribution by the forward mode of the Na+/Ca2+ exchange by BAPTA-AM or L-type Ca2+ channel blocker; however, inhibition of the Na+/Ca2+ exchange has previously been observed to abbreviate the AP (9, 31), whereas overexpression of the exchanger prolongs APD (42). To obtain data shown in Fig. 1C, we further examined the contribution from SK2 vs. SK1 or SK3 channels on the AP prolongation by BAPTA AM in atrial myocytes by using a low concentration of apamin (10 nM). SK2 channel has been shown to be highly sensitive to apamin, whereas SK1 and SK3 channels are less sensitive (11). As we have previously documented, apamin (10 nM) resulted in AP prolongation in atrial myocytes. More importantly, addition of BAPTA-AM in the presence of 10 nM apamin resulted in further prolongation of the AP in atrial myocytes, likely from additional block of the SK1 and/or SK3 channels. To directly determine the possible existence of other isoforms of the Ca2+-activated K+ channels and the possible tissue-specific differential expression of the other isoforms, we performed RT-PCR using mouse atrial and ventricular tissues.



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  		Fig. 1. Evidence for Ca2+-sensitive K+ current in mouse atrial and ventricular myocytes. A and B: examples of action potentials (AP) recorded from mouse atrial (left) and ventricular (right) myocytes using perforated patch-clamp techniques. AP traces shown were obtained in control and after application of BAPTA-AM (10 µM; A) or nimodipine (10 µM; B). Bar graphs in insets show summary data of action potential duration at 50 and 90% repolarization (APD50 and APD90, respectively) from a group of cells. *P < 0.05; n = 6 and 4 for atria and n = 4 and 6 for ventricles in A and B, respectively. C: examples of AP recorded from mouse atrial myocytes in control and after application of apamin (10 nM) compared with apamin and BAPTA-AM (10 µM).

 
Existence of SK1 and SK3 isoforms in mouse atrial and ventricular myocytes: RT-PCR amplification of SK1 and SK3 sequences from mouse heart. To directly test for these possibilities, we first performed RT-PCR cloning using previously published primers to obtain full-length coding sequences of SK1 and SK3 channels from mouse atrial and ventricular tissues as well as from rat heart tissue. Figure 2A shows RT-PCR amplified products from total RNA from mouse atria (lane 1), mouse ventricles (lane 2), and mouse brain (lane 3) obtained using primers specific for the mouse colon SK1 channel (mcSK1, accession no. AF357239). The PCR products from mouse hearts and brains show two distinct bands corresponding to 1,611 and 1,500 bp. Both amplified products were cloned into pCRII-TOPO plasmid vector and analyzed by DNA sequencing of both forward and reverse strands. The sequences were found to be those of SK1 channel with different splicing variants. Figure 2B shows RT-PCR amplified products from total RNA of rat brain (lane 2) and rat heart (lane 3) obtained using short-length (159 bp) primers specific for rat SK1 channel (accession no. AF000973). Nucleotide sequence of the PCR products was found to be similar to that of the SK1 sequence. Figure 2C shows amino acid sequence alignment of deduced SK1 protein(s) sequences from mouse atrium and ventricle compared with that of mcSK1 channel. The full-length coding nucleotide sequence of mouse heart SK1 channel (mhSK1) shows 92 and 99% identity to rat brain SK1 channel (accession no. U69882) and mcSK1 (accession no. AF357239), respectively. Variants of mouse SK1 channel (mSK1) were also detected (Fig. 2C) and differ mainly in the COOH-terminal structure affecting a portion of the sixth TM segment (S6) and CaMBD. The full-length nucleotide sequence (labeled I) was 1,611 bp, whereas the other four identified variants from the heart [variants V, VI, VII, and VIII according to nomenclature described by Shmukler et al. (29)] were 1,500, 1,494, 1,578, and 1,455 bp in length, respectively.



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  		Fig. 2. RT-PCR cloning of mouse heart SK1 (mhSK1). A: a representative agarose gel of RT-PCR amplified products with approximate sizes of 1,611 and 1,500 bp from total RNA from mouse atria (lane 1), mouse ventricles (lane 2), and mouse brain (lane 3), obtained using primers specific for the mouse colon SK1 channel (mcSK1; accession no. AF357239). Lane 4 is a negative control (PCR amplified without RT) to make certain that there is no genomic contamination of the RNA samples. Lane 5 is the Hi-Lo ladder (Bionexus). B: a representative agarose gel of RT-PCR amplified products with an approximate size of 159 bp from total RNA from rat brain (lane 2) and rat heart (lane 3), obtained using primers specific for rat brain SK1 channel (rbSK1; accession no. AF000973). Lanes 4 and 5 are negative controls (PCR amplified without RT). Lane 1 is a 100-bp ladder (New England Biolabs, Beverly, MA). C: amino acid sequence alignment (ClustalW) of deduced SK1 protein(s) sequence from mouse atrium and ventricle (mhSK1) compared with that of mcSK1. Six predicted transmembrane domains (S1–S6) and pore (P) region are highlighted in yellow. Red box in P region represents K+-selective filter GYG. ERS, endoplasmic reticulum retention signal at the NH2 terminus; CaMBD, putative calmodulin binding domain in the COOH terminus. Roman numerals denote isoform (If) nomenclature (see text for details). Residues that are identical among all of the clones are marked by asterisks (*), whereas colons (:) mark the conservative substitutions and periods (.) mark the semiconservative substitutions. Amino acid numbers for the full-length coding sequences are given at right. Dashes represent gaps in the sequence alignment. Amino acid residues are color coded (blue, acidic; magenta, basic; red, hydrophobic; green, hydrophilic).

 
Figure 3A shows the RT-PCR amplified products with an expected size of 2,200 bp from total RNA from mouse atria (lane 2), mouse ventricles (lane 3), and mouse brain (lane 4) obtained using primers specific for the mcSK3 channel (accession no. AF357240). Figure 3B shows the RT-PCR amplified products with an expected size of 1,632 bp from rat brain (lane 1) and rat heart (lane 2). The PCR products were sequenced and confirmed to be SK3 channel. Figure 3C shows amino acid sequence alignment of deduced SK3 protein sequence from mouse heart compared with that of mcSK3 and rat brain SK3 (rbSK3). Mouse SK3 channel (mSK3) variants were also detected and differ not only in the number of polyglutamine repeats (CAG repeats) in the NH2 terminus but also in the intervening sequences between the polyglutamine repeats. The nucleotide sequences of the full-length transcripts (mhSK1 and mhSK3) have been deposited in GenBank (accession nos. AY258143 and DQ140280, respectively).

In situ hybridization. SK channels may be classified pharmacologically on the basis of their sensitivity to apamin. SK2 is highly sensitive to apamin, with a half-blocking concentration of 60 pM, whereas SK1 channels are not affected by 100 nM apamin (11). SK3 channels are intermediate, with a Kd of ~1 nM. Results presented in Fig. 1 led us to further hypothesize that there may be a differential expression of SK1, SK2, and SK3 in atria and ventricles. To examine the regional distribution of the three isoforms of SK channels, we performed ISH using primers designed from mutually unique regions of the mhSK1, mhSK2, and mhSK3 channels in the COOH termini as shown in Fig. 4A to generate sense and antisense riboprobes in the presence of UTP-digoxigenin labels. Figure 4, B–D, shows photomicrographs comparing the distribution of SK1, SK2, and SK3 transcripts in mouse atria compared with ventricles. Sense riboprobes were used as a negative control from consecutive sections. All three transcripts are highly expressed in the great vessels (see arrows). In addition, SK1 and SK2 transcripts are more highly expressed in atria compared with ventricles, whereas expression of SK3 transcript appears similar in the atria and ventricles. To further confirm the differential expression of these three isoforms, we performed real-time qRT-PCR.

Comparison of expression profiles of three isoforms of SK channels between atria and ventricles. To more quantitatively analyze the expression profile between the atrial and ventricular tissues, we performed qRT-PCR using primers designed from exclusive regions of mouse cardiac SK1, SK2, and SK3 channels that we have cloned. Isolated atrial and free wall left ventricular myocytes were used. In addition, differential plating with short-term culture was used to minimize contamination from fibroblast and other cell types in the heart. Figure 5A shows the nucleotide sequence alignment from the COOH-terminal regions of the SK1, SK2, and SK3 channels and the primers used for the real-time RT-PCR analysis. Data obtained from the analysis are summarized in Fig. 5B from four to five independent experiments. Consistent with the qualitative assessment from ISH, SK1 and SK2 transcripts are significantly more abundant in atria compared with ventricles, whereas SK3 transcript was expressed at a low level and was similar in both atria and ventricles (*P < 0.05).



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  		Fig. 5. Quantification of SK1–SK3 mRNA in mouse atria vs. ventricles by real-time quantitative RT-PCR. A: nucleotide sequence alignment (ClustalW) of the COOH termini from mhSK1, mhSK2, and mhSK3 channels. Highlighted regions refer to the primers used for the PCR reactions to generate the sense and antisense riboprobes. Nucleotide sequence numbers are given at right. B: relative mRNA levels for the 3 different SK channel isoforms normalized to GAPDH, comparing atria and ventricles. *P < 0.05.

 
Immunodetection of SK1 and SK3 channels in mouse atrial and ventricular myocytes. To demonstrate the presence of SK1 and SK3 protein expression in the cardiac myocytes, we performed immunofluorescence staining using isolated mouse atrial and ventricular myocytes. Figure 6, A and B, shows specific labeling using anti-SK1 antibody in isolated mouse atrial and ventricular myocytes, respectively. In addition, both atrial and ventricular myocytes show a low level of staining using anti-SK3 antibody, consistent with a low level of expression of SK3 channels in atrial and ventricular myocytes. Preincubation of primary antibodies with the respective antigenic peptides (Fig. 6, C and F) eliminated the positive labeling, confirming that the labeling was epitope specific. In addition, staining using secondary antibody only under identical experimental conditions did not show positive labeling (data not shown).



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  		Fig. 6. Immunodetection of SK1 and SK3 channels in isolated mouse atrial and ventricular myocytes. Confocal photomicrographs show subcellular distribution of SK1 (A and B) and SK3 channels (D and E) in mouse atrial (A and D) and ventricular myocytes (B and E). Immunofluorescence labeling was done by treatment with secondary antibodies (fluorescein isothiocyanate-conjugated goat anti-rabbit antibody). In all cases, the specificity of labeling was confirmed by elimination of immunoreactivity after preincubation of antibody with the respective antigenic peptide (1:1) (C and F). Scale bars, 10 µm.

 

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 ABSTRACT
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SK channels have been shown to be expressed in a wide variety of tissues and are implicated in important physiological processes in different organ systems. For example, SK channels have been shown to underlie sAHP following AP in neurons and to regulate neuronal excitability. We recently documented that the SK2 channel is expressed and plays a prominent role in cardiac myocytes. Importantly, the channel is differentially expressed in atria compared with ventricles (41). As a direct extension of this previous study, in the present study we have shown that cardiac myocytes also express other isoforms of SK channels, namely, SK1 and SK3 channels. To investigate directly the expression profile of the three different SK channel isoforms, we first cloned the SK1 and SK3 channels from mouse hearts. Both channels show a high sequence identity to the previously described SK1 and SK3 channels from rat brain and mouse colon. We next designed isoform-specific probes to assess the expression profile in the mouse hearts by using ISH and real-time RT-PCR. Immunocytochemistry was also performed to assess expression at the protein level. Similar to the SK2 isoform, the SK1 transcript was found to be more abundant in the atria. The SK3 isoform was expressed at a similar level in both atria and ventricles.

SK channel isoforms. The different SK isoforms form channels with similar Ca2+ sensitivities and conductances. However, the intracellular NH2 and COOH termini demonstrate considerable sequence divergence, and these domains may mediate specific functions in response to intracellular signals. On the other hand, structural differences in the outer vestibule among the different SK channel isoforms determine sensitivities toward the various blockers and may reflect differential regulation by endogenous extracellular ligands (11). Pharmacologically, SK channels can be distinguished by their sensitivity toward the bee venom apamin. SK2 is highly sensitive to apamin, with a half-blocking concentration of ~60 pM, whereas SK1 channels are not affected by 100 nM apamin. SK3 channels are intermediate, with a Kd of ~1 nM. The amino acids that mediate apamin sensitivity have been determined (11). Two residues that reside on opposite sides of the deep pore, an aspartate and an asparagine, are essential for apamin sensitivity. Mutagenesis of SK1 to conform to the SK2 sequence renders SK1 channels apamin sensitive. These same residues mediate differential SK channel sensitivity to the nicotinic acetylcholine receptor antagonist d-tubocurarine and are likely to be the determinants for block by other selective SK blockers (11).

Several brain regions and peripheral tissues express more than one SK channel subtype. In addition, previous studies using cloned SK channels and SK1-SK2 dimer have demonstrated that SK subunits may form heteromeric channels, giving rise to structural and functional diversity (11). Further investigation is required to ascertain that these various isoforms can also form heteromultimeric complexes in the heart.

Roles of SK channel variants. Recently, tissue-specific truncated SK3 transcripts, SK3–1B and SK3–1C, have been identified. They are generated by alternative utilization of exons 1B or 1C instead of exon 1A, used by SK3 (17, 36). The expression pattern of the truncated forms was tissue specific. For example, the SK3–1C variant was not found in the nervous system. In the heterologous expression systems, these truncated forms lead to "dominant negative" suppression of K+ currents produced by SK1, SK2, and SK3 channels and, to a lesser extent, the intermediate-conductance Ca2+-activated K+ channels. A 30-residue domain in the COOH-terminal region shared by these truncated forms has been shown to be essential for the property of negative dominance. It was shown that this COOH-terminal domain resembles the "tetramerizing coiled-coiled" domain in the COOH termini of other K+ channels, where it contributes to tetramer stability and selectivity of multimerization (12). Therefore, it has been suggested that naturally occurring dominant negative suppression may be a widespread phenomenon through which the functional expression of multimeric proteins such as K+ channels is regulated, leading to gradations in the levels of membrane excitability (17). The presence of these truncated variants of SK channels, the regulation of the tissue-specific expression, and their functional roles remain to be investigated in the heart. Our present study documenting the expression of all three isoforms of SK channel in mouse and rat hearts opens new questions with regard to the possible existence of these naturally occurring truncated isoforms in future investigations.

Physiological significance. SK channels have been shown to play an important role in setting the tonic firing frequency of neurons (24). Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetitive AP. The increase in intracellular Ca2+ evoked by AP firing decays slowly, allowing SK channel activation to generate a long-lasting hyperpolarization, termed the sAHP. This spike-frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity. The roles of Ca2+-activated K+ channels in cardiac myocytes are not well documented. Our previous data were the first to show the important role of the SK channel in membrane repolarization, particularly in atria. Our new information on the existence of other isoforms in the heart and the differential expression profiles between atria and ventricles further suggests important roles for these specific classes of ion channels in the heart and paves the way for new investigations into the roles of these channels in health and disease. Indeed, specific ligands for the various isoforms of SK channels may offer novel selective therapeutic approaches to manipulate excitability in atria vs. ventricles.

In summary, we have documented the presence of all three SK channel isoforms in mouse and rat hearts. Furthermore, our previous data have documented the presence of SK2 isoform and its important functional roles in human atria. Potential species-specific differences in these isoforms exist. Therefore, additional experiments are required to further document the roles of the three isoforms in human hearts.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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This work was supported by a Nora Eccles Treadwell Foundation Innovative Collaboration Award; the University of California, Davis; a Department of Veterans Affairs Merit Review Grant; and National Heart, Lung, and Blood Institute Grants HL-68507 and HL-75274 (to N. Chiamvimonvat).


   ACKNOWLEDGMENTS
 
We thank Dr. Ebenezer N. Yamoah for helpful discussions and the University of California, Davis Health System Confocal Microscopy Facility.


   FOOTNOTES
 

Address for reprint requests and other correspondence: N. Chiamvimonvat, Division of Cardiovascular Medicine, Dept. of Medicine, Univ. of California, Davis, Genome and Biomedical Sciences Facility, 451 East Health Sciences Drive, Rm. 6315, Davis, CA 95616 (e-mail: nchiamvimonvat{at}ucdavis.edu)

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


   REFERENCES
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