Elsevier

Gene

Volume 211, Issue 1, 28 April 1998, Pages 71-78
Gene

Genomic organization and expression of KCNJ8/Kir6.1, a gene encoding a subunit of an ATP-sensitive potassium channel1

https://doi.org/10.1016/S0378-1119(98)00086-9Get rights and content

Abstract

ATP-sensitive K+ (KATP) channels are implicated in the coupling of metabolic energy to membrane potential, thereby regulating many essential cell functions. Here, we demonstrate that a subunit of human KATP channel, KCNJ8/Kir6.1, is expressed preferentially in the human heart. Somatic cell-hybrid mapping and fluorescence in-situ hybridization (FISH) localize human KCNJ8 to the short arm of human chromosome 12, at 12p12. Partial characterization of the human Kir6.1 gene demonstrates that there is one large intron in the coding region and at least two additional introns in the 5′ untranslated region resulting in transcripts that have differential expression in human tissues examined. Our studies provide information on the complexity of the Kir6.1 transcript in the 5′ UTR that may be useful for future investigations on the tissue-specific regulation and function of this KATP channel gene.

Introduction

Potassium (K+) channels are essential components of cells serving important biological functions. In the heart, K+ channels are vital for the initiation and control of repolarization, for the maintenance of cellular resting potential, for regulation of the heart rate, and for cellular responses to metabolic changes [reviewed in Katz (1993)]. The diversity of mammalian K+ channels is generated by the presence of multiple gene families and alternative splicing (Kaczmarek, 1991; Perney and Kaczmarek, 1991; Pongs, 1992; Salkoff et al., 1992).

The inwardly rectifying K+ channels (Kir), are characterized by two membrane-spanning domains (M1 and M2) that flank a conserved pore (P)-region. Comparisons among the conserved sequences of inward rectifiers reveal the presence of six Kir channel subfamilies based on their degree of identity (Doupnik et al., 1995). These subfamilies are designated Kir1.x–6.x, according to the nomenclature of Chandy and Gutman (1993). ATP-sensitive potassium channels are regulated by intracellular ATP and therefore play a key role in cellular functions, such as secretion and muscle contraction, by coupling the metabolic state of the cell to the membrane potential.

KCNJ8/Kir6.1 (uKATP-1) has been identified as a member of the inward rectifiers, based on their similarity to previously reported inward rectifier K+ channel subfamilies (Inagaki et al., 1995a, Inagaki et al., 1995b, Inagaki et al., 1995c). The rat Kir6.1 cDNA encodes a 424-amino-acid residue protein that functions as an ATP-sensitive inward rectifier K+ channel. Recent results suggest that Kir6.1 might couple to another subunit, such as the sulphonylurea receptor (SUR), in order to form a functional ATP-sensitive potassium channel (Gribble et al., 1997). Although it remains to be determined which SUR subunit Kir6.1 interacts with in vivo, Kir6.1 likely encodes a subunit of an ATP-sensitive potassium channel. Expression of rat Kir6.1, as determined by Northern blot analyses, is ubiquitous. Given the physiological role that ATP-sensitive inward rectifier K+ channels play in cells, and the increasing evidence that the 5′-and 3′-untranslated regions (UTRs) of mRNAs play an important role in the regulation of gene expression (Jackson, 1993), we felt that it was of interest to examine the genomic organization of Kir6.1 and its expression in human tissues. We cloned the human Kir6.1 using a degenerate oligonucleotide-primed PCR strategy with primers derived from the pore region and a conserved region located carboxy-terminal to the M2 domain. Here, we report a quantitative tissue distribution analysis of human Kir6.1 mRNA, the genomic organization of the Kir6.1 gene and the diversity in the 5′-untranslated region (5′ UTR) of human Kir6.1 -specific transcripts. We identified three distinct exons in the 5′ UTR that exhibit tissue-selective variations in expression. Additionally, we show that the Kir6.1 gene is located on the short arm of human chromosome 12 (12p12).

Section snippets

Isolation of human Kir6.1

Degenerate oligonucleotide-primed PCR was used to identify members of the family of inward-rectifying K+ channels. Primers MABms 4 [(5′-GGCCGAAT TCGAGAC(C/G/T)(C/G)A(G/A)(G/A)(C/T)(G/A/C)ACCAT(C/A/T)GG-3′] and MABms 7 [(5′-GGCCGAATTCGGCT(C/T/G)TT(C/T/G)C(G/T)(A/C)AG(G/A)TT(G/A)(C/G)C(C/T)AC-3′], derived from the pore region and a conserved region located carboxy-terminal to the M2 domain, were used to amplify a human cardiac cDNA library (Clontech). PCR was performed as following: 94°C—5 min for

Tissue distribution of human Kir6.1

To examine the expression pattern of human Kir6.1 mRNA in several tissues, a coding-region specific probe was labeled and hybridized to a human RNA Master blot (Clontech; Fig. 1). Poly (A+) RNA samples on Master blot have been normalized to the mRNA of eight different `housekeeping' genes, allowing accurate determination of the relative expression levels of a target mRNA in different tissues. Human Kir6.1 is predominantly expressed in both fetal and adult heart. Quantitation of the Master blot (

Acknowledgements

We thank B. Kienzle for DNA Sequencing, W.A. Little for technical assistance in isolating the human homolog of Kir6.1, and F. Shalaby and J. Carman for comments on the manuscript. This work was supported by the Bristol-Myers Squibb Pharmaceutical Research Institute.

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1

Accession Nos: mRNA types A: AF015605; B: AF015606; C: AF015607.

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