©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Functional Characterization of a Novel ATP-sensitive Potassium Channel Ubiquitously Expressed in Rat Tissues, including Pancreatic Islets, Pituitary, Skeletal Muscle, and Heart (*)

(Received for publication, December 5, 1994; and in revised form, January 9, 1995)

Nobuya Inagaki (1) Yoshiyuki Tsuura (2) Noriyuki Namba (1) Kazuhiro Masuda (2) Tohru Gonoi (4) Minoru Horie (3) Yutaka Seino (2) Masanari Mizuta (1) Susumu Seino (1)(§)

From the  (1)Division of Molecular Medicine, Center for Biomedical Science, Chiba University School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan, (2)Department of Metabolism and Clinical Nutrition and (3)Third Department of Internal Medicine, Kyoto University School of Medicine, 54 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606, Japan, and (4)Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

ATP-sensitive K (K) channels play a crucial role in coupling metabolic energy to the membrane potential of cells. We have isolated a cDNA encoding a novel member (uK-1) of the inward rectifier K channel family from a rat pancreatic islet cDNA library. Rat uK-1 is a 424-amino acid residue protein (M(r) = 47,960). Electrophysiological studies of uK-1 expressed in Xenopus laevis oocytes show that uK-1 is a weak rectifier and is blocked with Ba ions. Single-channel patch clamp study of clonal human kidney epithelial cells (HEK293) transfected with uK-1 cDNA reveals that uK-1 closes in response to 1 mM ATP and has a single channel conductance of 70 ± 2 picosiemens (n = 6), indicating that uK-1 is an ATP-sensitive inward rectifier K channel. In addition, uK-1 is activated by the K channel opener, diazoxide. RNA blot analysis shows that uK-1 mRNA is expressed ubiquitously in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart, suggesting that uK-1 may play a physiological role as a link between the metabolic state and membrane K permeability of cells in almost every normal tissue. Since uK-1 shares only 43-46% amino acid identity with members of previously reported inward rectifier K channel subfamilies, including ROMK1, IRK1, GIRK1, and cK-1, uK-1 is not an isoform of these subfamilies and, therefore, represents a new subfamily of the inward rectifier K channel family having two transmembrane segments.


INTRODUCTION

The ATP-sensitive potassium (K) (^1)channel, discovered originally in cardiac muscle(1) , has a feature of regulation of channel openings by intracellular ATP or the ATP/ADP ratio(2) . Recent electrophysiological and pharmacological studies have indicated that K channels are present in pancreatic beta-cells(3, 4, 5, 6) , pituitary(7) , skeletal muscle(8) , brain(9) , and vascular smooth muscle (10) as well as in cardiac muscle. They play a key role in cellular functions such as secretion and muscle contraction by coupling metabolic status to the membrane potential of cells(2) . It has been shown that the properties of K channels vary among different tissues, suggesting molecular heterogeneity of K channels(2) .

Recently, inward rectifier K channels of a new class of K channel have been identified(11, 12) . These channels contain two putative transmembrane segments and correspond to the inner core structure of voltage-gated K channels(11, 12, 13, 14) . A cardiac K channel (cK-1) belonging to this class of K channel also has been identified(15) . Inward rectifier K channels have thus far been divided into four subfamilies, ROMK1(11) , IRK1(12) , GIRK1(16, 17) , and cK-1(15) , based on the amino acid sequence identity between subfamilies. In addition, several isoforms of IRK1 (18, 19, 20, 21, 22) and GIRK1 (23) subfamilies have also been described.

In the present study, we have identified a novel K channel (uK-1), which represents a new subfamily of the inward rectifier K channel family. Interestingly, uK-1 mRNA is expressed in all rat tissues examined, suggesting that uK-1 may play an important role in the regulation of membrane potential by metabolic energy in cells of most tissues.


MATERIALS AND METHODS

cDNA Cloning of a Novel Inward Rectifier KChannel (rIK-5)

A fragment (nucleotide +95 to +1211) of rat GIRK cDNA (16) was amplified by reverse transcriptase-polymerase chain reaction using total RNA extracted from rat heart as described previously(24) . A cDNA library has been made from rat pancreatic islets in the vector of gt22, and 7 times 10^5 plaques were screened by hybridization with a P-labeled partial rat GIRK cDNA fragment as a probe under low stringency hybridization conditions(25) . DNA sequencing was done by the dideoxynucleotide chain termination procedure after subcloning appropriate DNA fragments into M13 mp18 or mp19 (Takara). Both strands were sequenced.

Expression and Electrophysiological Analysis of rIK-5 in Xenopus laevis Oocytes

Ten µg of pGEM11Z (Promega) containing a full-length cDNA encoding rIK-5 were linearized with NotI and transcribed in vitro as described previously(24) . Xenopus oocytes were injected with 60 nl (20 ng) of transcribed cRNA. After 2-3 days, electrophysiological measurements were performed using two-electrode voltage clamp(24) ; oocytes were voltage-clamped at holding potentials near the calculated Nernst K equilibrium potential (the intracellular K concentration of oocytes was assumed to be 90 mM). Test pulse potentials ranging between -150 mV and +60 mV in steps of 15 mV and 200 ms duration were applied every 5 s. The bath solution contained a total of 90 mM KCl and NaCl (K concentrations are given in Fig. 2), 3 mM MgCl(2), and 5 mM HEPES (pH 7.4) with 0.3 mM niflumic acid to block Cl currents.


Figure 2: Whole-cell currents recorded from Xenopus oocytes expressing uK-1. A, representative traces of currents elicited by voltage steps from -150 to +60 mV in 15-mV increments (holding potential is -17 mV) in a uK-1 cRNA-injected (left) or H(2)O-injected (right) oocyte. Ba (300 µM) inhibition of the expressed currents in a cRNA-injected oocyte is shown (middle). B, current-voltage relationships in bath solutions containing 90 mM (bullet), 45 mM (circle), 20 mM (), and 4 mM (box) K ions. The holding potential was set at the zero current level in these solutions, as described under ``Materials and Methods.'' C, external K dependence of reversal potentials (E) for uK-1 currents in cRNA-injected oocytes. Each point represents measured E (mean ± S.E.) of three determinants. Measured E varies linearly with ln [K].



Expression and Single-channel Analysis of rIK-5 in HEK293 Cells

HEK293 cells were cultured in minimum essential Eagle's medium supplemented with 10% horse serum. The expression plasmid (pCMV6b) (26) that carried a full-length cDNA encoding rIK-5 was transfected into HEK293 cells using Lipofectamine and Opti-MEM I reagents (Life Technologies, Inc.) according to the manufacturer's instructions. Following transfection, cells were washed and cultured in the standard growth medium as described above for 48 h. Single-channel recordings were performed in cell-attached and inside-out membrane patch configurations, as described previously(27) . The composition of the intracellular solution was (in mM) 135 KCl, 0.1 CaCl(2), 1 EGTA, 0.001 K(2)ATP, 5 HEPES (pH 7.2, pCa 7.5), and various concentrations of MgSO(4) as indicated in Fig. 3B. The pipette solution contained 140 mM KCl, 2 mM CaCl(2), and 5 mM HEPES (pH 7.4). Continuous single channel currents were recorded through a patch clamp amplifier (Axopatch 200A, Axon Instruments) and were stored on videotape via a pulse code modulation system (PCM 501, Sony) for later analysis of channel activity, unit amplitudes, and time constants by computer (PC-98XL, NEC). The channel activity was expressed as mean patch current (n times P(o) times i) where n, P(o), and i represent the number of open channels, open probability, and unit amplitude, respectively. The electrophysiological experiments were performed at room temperature (22-25 °C). Statistical data are expressed as mean ± S.E.


Figure 3: Single channel currents of uK-1 on HEK293 cell membranes. A, representative current traces from inside-out patches at various pipette potentials in the presence (2 mM)(1, 2, 3, 4, 5, 6, 7) or absence of Mg(8) are indicated. B, a representative current-voltage relationship in the presence (2 mM) (bullet) or absence (circle) Mg. The conductance was 74 picosiemens in inward current. C, the effect of 1 mM ATP, 0.1 mM AMP-PNP, and 100 µM diazoxide on uK-1 channel activity. The channel recordings were done at -60 mV in inside-out patch. AMP-PNP and diazoxide were added in open and closed states of channels, respectively. Mean patch currents (pA) at the time indicated by arrows are: a, 0.20; b, 0; c, 1.62; d, 0.03; e, 0.20; f, 0; g, 0.24; h, 0; and i, 0.48. D, the effect of glibenclamide on uK-1 channel activity in inside-out patches. The trace was recorded at -60 mV in the presence of 1 µM ATP.



RNA Blotting Analysis

Twenty µg of total RNA from various tissues and clonal cells except for pituitary and thyroid (10 µg, each) were denatured with formaldehyde, electrophoresed on 1% agarose gel, and transferred to a nylon membrane. Hybridization was carried out under standard conditions(26) , using a P-labeled rIK-5 cDNA as a probe.


RESULTS AND DISCUSSION

Using a P-labeled GIRK cDNA fragment as a probe, a rat pancreatic islet cDNA library was screened, and five positive clones were obtained. DNA fragments isolated from a clone carrying the longest insert, designated rIK-5, were subcloned and sequenced. The sequence of 2389 base pairs contains a single open reading frame beginning with the third ATG in the cDNA sequence (there is an in-frame termination codon upstream of this ATG, and the first and second ATG are followed by termination codons), which predicts the amino acid sequence of a 424-amino acid residue protein (M(r) = 47,960) (Fig. 1). The predicted amino acid sequence of rIK-5 shows 43, 43, 44, and 46% identity with ROMK1, GIRK1, cK-1, and IRK1, respectively, each of which represents a different subfamily of the inward rectifier K channel family(11, 12, 13, 14, 15, 16, 17) . These amino acid identities are much lower than those found among various isoforms belonging to the same subfamily, where more than 60% amino acid identity is found(18, 19, 20, 21, 22, 23) . This strongly suggests that rIK-5 is not an isoform of previously described inward rectifier K channels but represents a new subfamily. Although the central region of rIK-5 protein shows a high homology with other inward rectifier K channels, the N- and C-terminal regions do not. A hydropathy plot of rIK-5 reveals two hydrophobic regions, suggesting that it has two putative transmembrane segments, a feature characteristic of inward rectifier K channels(13, 14) . There are two potential cAMP-dependent protein kinase phosphorylation sites (Thr-234 and Ser-385) and seven protein kinase C-dependent phosphorylation sites (Ser-224, Thr-345, Ser-354, Ser-379, Ser-385, Ser-391, and Ser-397) in the second intracellular region. There are also one (Thr-63) and four (Thr-234, Ser-281, Thr-329, and Ser-354) potential casein kinase II-dependent phosphorylation sites in the first and second intracellular regions, respectively. Unlike cK-1(15) , there is no potential N-linked glycosylation site in the extracellular region.


Figure 1: Comparison of the amino acid sequences of the five members of inward rectifier K channel family. Amino acids are indicated in single-letter code. The identical amino acid residues among these proteins are shown in boldface. Gaps introduced to generate this alignment are represented by dots. Predicted transmembrane (M1 and M2) and pore (H5) segments are indicated.



We also examined the electrophysiological properties of rIK-5 expressed in X. laevis oocytes. Fig. 2shows the results of the two microelectrode voltage-clamp experiments in Xenopus oocytes injected with cRNA for rIK-5 or with water. rIK-5 cRNA-injected oocytes showed inward currents at extracellular K ([K](o)) of 45 mM (Fig. 2A, left), which are blocked by external Ba (300 µM) (Fig. 2A, middle), while water-injected oocytes had negligible inward currents under the same conditions (Fig. 2A, right). The effects of various concentrations of [K](o) on rIK-5 currents are shown in Fig. 2B. As [K](o) was lowered from 90 to 4 mM, the slope conductance was decreased. A weak inward rectification was clearly observed. The reversal potential of rIK-5 currents was in good agreement with the equilibrium potential for K values predicted from the Nernst equation at various [K](o) (Fig. 2C). To further characterize the properties of rIK-5, we have performed single-channel recordings of HEK293 cells transiently transfected with the rIK-5 expression vector. Under symmetrical K conditions of 140 mM, the current-voltage relationship showed inward rectification in the presence of 2 mM Mg in the intracellular solution and a reversal potential of 0 mV (Fig. 3, A and B). The inward current was ohmic, and its channel conductance was calculated to be 70 ± 2 picosiemens (n = 6). In the case of 5 mM K in the pipette solution, the reversal potential was shifted to -79 ± 2 mV (n = 3), almost identical to that predicted by the Nernst equation (-85.6 mV at 25 °C). The single channels observed on the cell-attached and inside-out patch membranes had large fluctuations in open frequency, as shown in Fig. 3, C and D, and a flickering block was observed at -60 and -80 mV (Fig. 3A). Open time and closed time histograms of rIK-5 channel activity at -60 mV within a burst were well fitted with a single exponential, resulting in the time constants of open and closed times of 3.31 ± 0.40 and 0.91 ± 0.13 ms (n = 4), respectively. The degree of rectification was enhanced when intracellular Mg concentration was raised from 0 to 2 mM (Fig. 3, A and B). Under symmetrical K conditions of 140 mM at +80 mV, unit amplitude of rIK-5 was 3.83 ± 0.08 (n = 4), 2.73 ± 0.07 (n = 5), and 1.59 ± 0.07 (n = 3) pA at 0, 2, and 5 mM intracellular Mg concentrations, respectively. Some rectification is still observed even upon removal of intracellular Mg. Recent mutagenesis studies have suggested that the aspartate in the second transmembrane segment (amino acid residue 172 of IRK1) is a crucial determinant of rectification(28, 29) . IRK1 and GIRK1, both of which are strong rectifiers(12, 16, 17) , have aspartate at this position, while ROMK1 and cK-1, both of which are weak rectifiers(11, 15) , have asparagine. Consistent with this, uK-1, which shows weak rectification, has an asparagine at the corresponding position.

ATP sensitivity was examined using patches protected from channel run-down by 1 µM ATP. As shown in Fig. 3C, rIK-5 channel activity was completely suppressed by application of 1 mM ATP and also inhibited by 100 µM nonhydrolyzable ATP analog AMP-PNP; these effects were reversed on washout of the agents (n = 8). Diazoxide (0.1 mM), a potent opener of K channels of pancreatic beta-cells(30) , activated rIK-5 channels on inside-out and cell-attached patch membranes (Fig. 3C) (n = 3), while 200 µM pinacidil, a cyanoguanidine that activates cK channels in the presence of intracellular ATP(30, 31) , failed to reopen the rIK-5 channels inhibited by 1 mM ATP (n = 5) (data not shown), indicating a property different from that of cK-1(15) . In addition, rIK-5 channel activity was not inhibited (n = 6) by 1 µM glibenclamide, the sulfonylurea that blocks K channels (30) (Fig. 3D). These electrophysiological studies demonstrate that rIK-5 is an ATP-sensitive potassium channel, and it was designated uK-1 accordingly.

RNA blotting studies (Fig. 4) reveal that 2.7- and 1.7-kilobase transcripts are expressed in all rat tissues examined. uK-1 mRNAs are expressed at high levels in the heart, ovary, and adrenal, at moderate levels in the skeletal muscle, lung, brain, stomach, colon, testis, thyroid, and pancreatic islets, and at low levels in the kidney, liver, small intestine, and pituitary. An additional 2.9-kilobase transcript also is detected in adrenal. However, uK-1 is not expressed in any of the endocrine tissue-derived clonal cells examined, including the insulin-secreting cell lines RINm5F (rat), MIN6 (mouse), and HIT-T15 (hamster), the mouse glucagon-secreting cell line alphaTC (data not shown), the rat catecholamine-secreting cell line PC12, the rat growth hormone-secreting cell line GH3, the mouse ACTH-secreting cell line AtT-20, or in HEK293 cells. In addition, uK-1 is not expressed in vascular endothelium-derived cell lines, the calf pulmonary artery endothelial cell line CPAE, or the bovine carotid artery endothelial cell line HH. The absence of uK-1 mRNA in clonal insulin-secreting cells suggests that another K channel is expressed in these cells.


Figure 4: RNA blot analysis of uK-1 mRNA in various rat tissues, endocrine tissue- and vascular endothelium-derived clonal cells, and HEK293 cells. For autoradiography the nylon membrane was exposed to x-ray film with an intensifying screen at -80 °C for 3 days. The sizes of the hybridizing transcripts are indicated. Below the autoradiographs are the ethidium bromide-stained gels before transfer. 28 and 18 S ribosomal RNAs are shown.



Since intracellular ATP is the essential carrier of metabolic energy for all mammalian cells, it seems reasonable to assume that uK-1, expressed ubiquitously in normal tissues, may play a fundamental role in the regulation of K permeability in almost every cell by coupling metabolic energy to the membrane potential of the cell(14) . Thus, it should be interesting to examine how the activation and inactivation processes of uK-1 are regulated in altered metabolic states such as diabetes mellitus, starvation, and ischemia. In addition, since the metabolic process occurs in a glucose-dependent manner in certain cells such as pancreatic beta-cells, uK-1 may be regulated by glucose in these cells.


FOOTNOTES

*
This research was supported by scientific research grants from the Ministry of Education, Science and Culture and from the Ministry of Health and Welfare, Japan; by a grant for diabetes research from Otsuka Pharmaceutical Co., Ltd.; by a grant from Yamanouchi Foundation for Research on Metabolic Disorders; by a grant provided by Ichiro Kanehara Foundation; and by a grant from the Juvenile Diabetes Foundation International. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D42145[GenBank].

§
To whom correspondence should be addressed. Tel.: 81-43-226-2187; Fax: 81-43-221-7803.

(^1)
The abbreviations used are: K channel, ATP-sensitive K channel; AMP-PNP, adenyl-5`-yl imidodiphosphate; ACTH, corticotropin.


ACKNOWLEDGEMENTS

We thank D. L. Cook (Rain Town Biotech) and Y. Kubo (Tokyo Metropolitan Institute for Neuroscience) for critical reading of the manuscript, K. Hamaguchi (Oita Medical University) for providing us with alphaTC, and K. Yasuda and B. Chow (Kyoto University) for their help with the construction of a cDNA library. We are grateful to K. Sakurai, A. Tamamoto, and H. Aida for their expert technical assistance.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.