Molecular characterization of human SUR2-containing KATP channels
Introduction
ATP-sensitive potassium channels (KATP) are expressed in a variety of tissues and play important roles in metabolic regulation of cellular excitability (Ashcroft and Ashcroft, 1990). KATP channels are inhibited by high levels of intracellular ATP (Noma, 1983, Trube and Hescheler, 1984) and are activated by intracellular Mg-ADP (Cook and Hales, 1988, Dunne et al., 1988, Nichols and Lederer, 1991), thereby linking the metabolic state of the cell to excitability. These channels are involved in many diverse functions such as insulin secretion from pancreatic β cells, regulation of skeletal muscle excitability, neurotransmitter release and smooth muscle relaxation (Quayle et al., 1997, Trapp and Ashcroft, 1997).
Recent cloning and expression studies have provided a molecular basis to support the heterogeneity of KATP channels in various tissues (Aguilar-Bryan et al., 1995, Chutkow et al., 1996, Inagaki et al., 1995, Inagaki et al., 1996, Isomoto et al., 1996). KATP channels have been shown to be hetero-octomeric complexes composed of four inward rectifying K+ channels belonging to the Kir 6.0 subfamily and four regulatory proteins, the sulfonylurea receptor (SUR) (Clement et al., 1997). The SUR subunit belongs to the ATP-binding cassette (ABC) superfamily and has two members, SUR1 and SUR2, that are encoded by two genes containing 39 and 38 exons, respectively (Aguilar-Bryan et al., 1998, Bryan and Aguilar-Bryan, 1999). In addition, the SUR2 gene has been shown to undergo alternative splicing at exon 38, generating two splice variants SUR2A (exon 38a) and SUR2B (exon 38b) (Isomoto et al., 1996). Expression of these recombinant KATP channel subunits has revealed distinct KATP channels with biophysical and pharmacological properties similar to those described in pancreas, cardiac and smooth muscle tissues (Nelson and Quayle, 1995). For example, expression of SUR2B-Kir 6.2 subunits in HEK 293 cells forms channels that are activated by diazoxide, pinacidil and cromakalim, like those expressed in various smooth muscles (Hambrock et al., 1999, Isomoto et al., 1996, Schwanstecher et al., 1998). In contrast, KATP channels composed of SUR2A-Kir 6.2 are insensitive to diazoxide, but are activated by pinacidil and cromakalim similar to cardiac KATP channels (Inagaki et al., 1996, Okuyama et al., 1998).
The expression of KATP channels containing SUR2 has been investigated extensively at a molecular level using reverse transcriptase-polymerase chain reaction (RT-PCR) in rodents. These studies have shown widespread distribution of the SUR2B subunit in mouse tissues (Isomoto et al., 1996), whereas mRNA for SUR2A was shown to be predominantly expressed in skeletal muscle, heart and bladder. Additional splicing of the mouse SUR2 gene arising from deletions of exon 14 has also been reported (Chutkow et al., 1996). This exon 14− splice variant is restricted to cardiac tissues and, unlike the other splice variants of the SUR2 gene, this isoform does not generate functional channels when expressed in combination with Kir 6.2 in COS-7 cells (Chutkow et al., 1999). More recently, another SUR2 splice variant that lacks exon 17 (SUR2 exon 17−) has been identified in several mouse tissues, showing high mRNA levels in heart, skeletal muscle, bladder and gut. KATP channels formed by co-expression of this splice variant with Kir 6.2 exhibit differential sensitivity to ATP compared with the exon 17+ counterpart (Chutkow et al., 1999).
Despite earlier evidence of diverse SUR2 splice variants and their possible relevance to KATP channel function, efforts to characterize potential splice variants in human tissues have been lacking. In the present study, we have examined the distribution of SUR2 splice variants in human tissues using RT-PCR. We have extended this study to evaluate quantitatively the expression levels of SUR2 exon 17+ and 17− splice variants using real-time Taqman PCR techniques, and further demonstrate that these splice variants can form functional KATP channels in mammalian cells.
Section snippets
Reverse transcription-polymerase chain reaction analysis
First strand cDNA was synthesized from human total and poly A+ RNA (Analytical Biological Services Inc. and Clontech) using Superscript II (Life Technologies) according to the manufacturer's instructions. Briefly, the RNA (2 μg total/1 μg poly A+) was primed with 200 ng of random hexamers or a SUR2 gene-specific primer (5′- GAATACATGGCCCGTCGGAG-3′) and incubated at 70°C for 10 min. PCR buffer (20 mM Tris–HCl pH 8.4, 50 mM KCl), 2.5 mM MgCl2, 1 mM dNTP and 10 mM DTT were added at 25°C and incubated for 5
Expression patterns of the SUR2 gene
The SUR2A and SUR2B transcripts arise due to alternative splicing at the extreme 3′ end of the SUR2 gene (Isomoto et al., 1996). Since the exon that encodes the SUR2A variant (exon 38a) is found immediately upstream of the exon encoding the SUR2B (exon 38b) variant, specific oligonucleotide primers were designed such that the forward primer is to a region that is conserved in both SUR2A and SUR2B, upstream of this splicing site (exon 36), and the reverse primer is located within the sequence of
Discussion
This study provides a detailed examination of the mRNA expression patterns of SUR2 splice variants across a variety of human tissues. Our analysis reveals a widespread distribution of SUR2B across skeletal, smooth and cardiac muscle preparations. In contrast, SUR2A was found to be more discretely expressed in the heart and skeletal muscle, and at low levels in brain and prostate. An additional splice variant of SUR2 that lacked exon 17 with a differential pattern of expression was also
Acknowledgments
We would like to thank Dr. Joe Bryan and Dr. Lydia Aguilar-Bryan for SUR2A-pECE and SUR2B-pECE constructs that were used in this study.
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2012, Matrix BiologyCitation Excerpt :There are few practical methods for the routine quantitative analysis of alternative mRNA splice forms. Most commonly, PCR-based, co-amplification approaches, generating multiple PCR products have been used, with primers that flank alternative exon–exon junctions (Kafert et al., 1999; Dalski et al., 2000; Davis-Taber et al., 2000; Favy et al., 2000; Brown et al., 2004; Connell et al., 2005; Meidan et al., 2005; Yoong et al., 2005; Nygard et al., 2010; Long et al., 2011; Sylvestersen et al., 2011). Although convenient, the co-amplification approach, is semi-quantitative at best, and may under- or over-estimate relative quantities of alternative splice forms.
Gain-of-function mutation S422L in the KCNJ8-encoded cardiac K <inf>ATP</inf> channel Kir6.1 as a pathogenic substrate for J-wave syndromes
2010, Heart RhythmCitation Excerpt :KCNJ8-S422L is highly conserved across species and has not been observed in nearly 2,000 reference alleles based on previously reported data. Heterologous expression of KCNJ8-S422L in COS1 cells evidenced a marked gain of function in the KATP current associated with Kir6.1-S422L channels when coexpressed in the setting of SUR2A, the predominant SUR isoform in cardiac ventricular tissue.36 This gain of function coupled with the preferential epicardial distribution of Kir6.1 could explain the J-wave syndrome phenotype observed in the three unrelated patients known to host this specific missense mutation.
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