From the Departments of Physiology and Biophysics, and
** Microbiology and Molecular Genetics, University of California Irvine,
Irvine, California 92697; Glaxo-Wellcome Research
Institute, Research Triangle Park, North Carolina 27709;
¶ Mammalian Genetics Laboratory, ABL-Basic Research Program,
NCI-Frederick Cancer Research and Development Center, Frederick,
Maryland 21702; and
Human Genome Center, Lawrence Livermore
National Laboratory, Livermore, California 94550
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ABSTRACT |
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We report the isolation of a novel mouse voltage-gated Shaker-related K+ channel gene, Kv1.7 (Kcna7/KCNA7). Unlike other known Kv1 family genes that have intronless coding regions, the protein-coding region of Kv1.7 is interrupted by a 1.9-kilobase pair intron. The Kv1.7 gene and the related Kv3.3 (Kcnc3/KCNC3) gene map to mouse chromosome 7 and human chromosome 19q13.3, a region that has been suggested to contain a diabetic susceptibility locus. The mouse Kv1.7 channel is voltage-dependent and rapidly inactivating, exhibits cumulative inactivation, and has a single channel conductance of 21 pS. It is potently blocked by noxiustoxin and stichodactylatoxin, and is insensitive to tetraethylammonium, kaliotoxin, and charybdotoxin. Northern blot analysis reveals ~3-kilobase pair Kv1.7 transcripts in mouse heart and skeletal muscle. In situ hybridization demonstrates the presence of Kv1.7 in mouse pancreatic islet cells. Kv1.7 was also isolated from mouse brain and hamster insulinoma cells by polymerase chain reaction.
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INTRODUCTION |
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Ion channels that exhibit a variety of gating patterns and ion selectivity are critical elements that transduce signals in diverse cell types (1). Voltage-gated potassium-selective (Kv)1 channels represent the largest family within this class of proteins (2), and perform many vital functions in both electrically excitable and nonexcitable cells. Following initiation of an action potential in nerve and muscle cells, Kv channels play the important role of repolarizing the cell membrane (1). Kv channels can also modulate hormone secretion, for example insulin release from pancreatic islet cells (3-6), and regulate calcium signaling during mitogen-stimulated activation of lymphocytes (7).
Kv channels in mammalian cells are encoded by an extended family of at least nineteen genes (2). The largest subfamily, Kv1, is related to the fly Shaker gene and contains six members, Kv1.1-Kv1.6 (2). The Shaker gene has 21 exons, which can be alternatively spliced to generate at least five functionally distinct transcripts (8, 9). In contrast, the protein-coding regions of each of the six known mammalian Kv1 genes and the three known Xenopus homologues are contained in a single exon (2, 10), precluding alternative splicing as a means of generating functionally different proteins. The evolutionary significance of this pattern of organization remains a puzzle.
Here we report the identification of a novel mammalian gene, Kv1.7 (Kcna7/KCNA7), that has a genomic organization distinct from the other members of the vertebrate Kv1 subfamily. We have defined the chromosomal location of this gene in the mouse and human genome, determined its tissue distribution, and characterized the biophysical and pharmacological properties of the cloned channel.
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EXPERIMENTAL PROCEDURES |
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Isolation and Characterization of mKv1.7, hKv1.7, hKv3.3, and hKv3.4 DNA Clones-- Three overlapping genomic clones (KC225, KC254, and KC256) were isolated from an AKR/J mouse genomic library screened with a mixture of mKv1.1 and rKv1.5 cDNA probes, as described previously (10), and mapped by multiple and partial restriction endonuclease digests, and by dideoxy sequencing. Kv1.7 cDNAs were amplified by the polymerase chain reaction (PCR) from mouse brain and from the hamster insulinoma cell line, HIT-T1S, using intron-flanking primers (5'-TCTCCGTACTCGTCATCCTGG-3' within S1 and 5'-AAATGGGTGTCCACCCGGTC-3' on the 3' side of S5). The resulting 588-bp PCR fragments were sequenced.
Cosmid clones encoding hKv1.7 and hKv3.3 (11) were isolated from a human chromosome 19-enriched library, Library F (12), screened with mKv1.7 and mKv3.3 probes. A 1.9-kb cDNA fragment of the Shaw family gene, hKv3.4, was isolated from a human pancreatic library (13) screened with a mixture of hKv3.1 (0.9-kb XbaI/HindIII), hKv3.3 (1.4-kb PstI/EcoRI), and mKv3.4 (0.9-kb HindII/EcoRI) probes at a final stringency of 0.5 × SSC and 0.1% SDS at 55 °C (8 × 106 phage screened). The isolated clone spans the region from S1 through the 3' end of the coding region (0.6 kb), and 1.3 kb of the 3'-noncoding region.Mice-- Pancreatic tissue sections were obtained from 9-16-week-old diabetic-prone (db/db) and healthy (db/+) C57BL/KsJ mice. Mice homozygous for the autosomal recessive diabetic susceptibility gene db, a mutated form of the leptin receptor (14, 15) on chromosome 4, develop diabetes beginning at about 6 weeks of age (16).
Mapping Mouse and Human Chromosomal Locations of Kv1.7 and Kv3.3-- Interspecific backcross progeny were generated by mating (C57BL/6J × Mus spretus)F1 females and (C57BL/6J) males, and a total of 205 N2 mice were used to map the two mouse genes, mKv1.7/Kcna7 and mKv3.3/Kcnc3, as described previously (11, 17-20). The probe for mKv1.7 was the entire 6.4-kb EcoRI fragment shown in Fig. 1, and that for mouse Kv3.3 was a 4-kb genomic HindIII fragment containing the entire 3'-exon (11). Although 155 mice were analyzed for all markers and are shown in this segregation analysis, up to 188 mice were typed for some pairs of markers. Recombination frequencies were calculated as described (11, 17-20) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to account for the allele distribution patterns. Fluorescent in situ hybridization of cosmids to metaphase human chromosomes was carried out as described previously (21, 22).
Northern Blot Assays--
A Northern blot of
poly(A)+ RNA from mouse heart, brain, spleen, lung, liver,
skeletal muscle, kidney, and testis (CLONTECH Inc.,
Palo Alto, CA) was probed with the mouse Kv1.7-specific 3'-noncoding region sequence. The PstI/SacI
Kv1.7 3'-noncoding region was labeled by the random primer
method (Boehringer Mannheim Random Primed DNA labeling kit). The RNA
blot was hybridized at 60 °C for 18 h, washed at a final
stringency of 0.2 × SSC and 0.1% SDS at 60 °C for 1 h,
and exposed to x-ray film at 80 °C with an intensifying screen for
3 days.
In Situ Hybridization--
cRNA probes labeled with
-35S-labeled UTP (1300 Ci/mmol) were alkaline-denatured
to an average size of 100 nucleotides and used for in situ
hybridization on pancreatic frozen sections (6-10 µm thick) from
db/db mice. Briefly, sections were hybridized overnight at
42 °C, RNase treated, washed five times in 0.5 × SSC at
65 °C, coated with Ilford K5 photographic emulsion, and exposed at
4 °C for varying times. After development, the sections were
counterstained with hematoxylin and eosin Y and examined with a Leitz
Aristoplan microscope equipped with reflected polarized light to
visualize silver grains in dark field. The probes used for
hybridization were as follows: insulin, 1.6-kb human insulin
gene including the 5'- and 3'-flanking sequences (ATCC no. 57399);
hKv3.4, 1.9-kb cDNA fragment spanning S1 through the 3'
end of the coding region (0.6 kb), and 1.3 kb of the 3'-noncoding
region; mKv1.7, 540-bp PstI/SacI
fragment containing 29 bp of C-terminal coding sequence and 511 bp of
3'-noncoding sequence.
Electrophysiological Studies-- To make a mKv1.7 expression construct we amplified a 588-bp fragment from mouse brain cDNA spanning the region encoded by the two Kv1.7 exons using reverse transcriptase PCR (5'-primer, 5'-TCTCCGTACTCGTCATCCTGG-3'; 3'-primer, 5'-AAATGGGTGTCCACCCGGTC-3'). Exon 1 (850-bp BspHI/BinI fragment), a 283-bp BinI/BglII fragment of our 588-bp PCR product, and exon 2 (747-bp BglII/HindIII), were ligated together at BinI and BglII sites to generate "fragment I" (1880 bp). Fragment I was blunt-ended at the 5' end and cloned into the pBluescript vector at SmaI/HindIII sites. To remove the 5'-NCR from fragment I, and for the purpose of cloning this fragment into the pBSTA expression vector, we introduced a unique BamHI site just upstream of the initiator methionine using PCR: 5'-primer, 5'-ACAAAAGCTTCATATGACTACAAGGAAAGCT-3'; and 3'-primer: 5'-AAGCGCAACCCGGCCACG-3'. The resulting PCR product (corresponding to the first 233 nucleotides of the coding region) was spliced to fragment I at the NcoI site, and the 1870-nucleotide fragment was ligated to the pBSTA vector.
mKv1.7 cRNA was transcribed in vitro (Ambion Kit, Austin, TX) and diluted in a 0.1-0.5% fluorescein-dextran (Mr 10,000, Molecular Probes, Eugene, OR) in 100 mM KCl. Rat basophilic leukemic (RBL) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Hyclone, Logan, UT) and glutamine, and were plated onto glass coverslips one day prior to use for electrophysiological experiments. RBL cells were injected with cRNA using pre-pulled injection capillaries (Femtotip) in combination with an Eppendorf microinjection system (micromanipulator 5171 and transjector 5242; Madison, WI) as described previously (23). Four to six hours later, fluorescent cRNA-injected cells were evaluated electrophysiologically. All membrane currents were recorded at room temperature (22-26 °C) with a LIST EPC-7 amplifier (Heka Elektronik, Germany). Series resistance compensation was employed if the current exceeded 2 nA, and the command input was controlled by a PDP 11/73 computer via a digital-to-analog converter. Capacitative and leak currents were subtracted using a P/8 procedure and the holding potential in all experiments was ![]() |
RESULTS |
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The Protein-coding Region of mKv1.7 Contains an Intron Unlike Its Vertebrate Homologues
A restriction map of a 6.4-kb EcoRI DNA fragment containing the entire mouse Kv1.7 coding region is shown in Fig. 1. The coding region is contained in two exons separated by a 1.9-kb intron. The upstream exon encodes the entire N terminus, S1, and part of the S1-S2 loop. The downstream exon contains the region extending from the S1-S2 loop to the C-terminal end of the protein. The intron-exon splice sites were determined by comparing the genomic sequence with cDNA sequences obtained from the hamster insulinoma cell line, HIT-T1S, and from mouse brain (Fig. 1).
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The complete coding sequence of the mKv1.7 is shown in Fig. 2. The mKv1.7 sequence is identical in the N terminus from bp 52 to 996 with the mouse EST sequence AA021711. Betsholtz et al. (24) amplified a short segment of Kv1.7 cDNA spanning the S5/S6 region from mouse (MK-6), rat (RK-6), and hamster (HaK-6) insulin-producing cells using PCR. Our sequence is identical to their MK-6 sequence, except for four nucleotide changes.
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The deduced mKv1.7 protein consists of 532 amino acids and contains six putative membrane-spanning domains, S1-S6 (Fig. 2). The hydrophobic core of this protein shares considerable sequence similarity with other Shaker family channels, while the intracellular N and C termini and the external loops between S1/S2 and S3/S4 show little conservation. The protein contains conserved sites for post-translational modifications as indicated in Fig. 2. As do all other Shaker-related channels, mKv1.7 has a potential tyrosine kinase phosphorylation site (RPSFDAVLY) in its N-terminal region (2); the proline-rich stretch within the N terminus (see residues 29-42) may be a binding site for SH3 domains of tyrosine kinases. Two protein kinase C consensus sites (Ser/Thr-X-Arg/Lys) are present in the cytoplasmic loop between S4 and S5 of mKv1.7; at least one of these sites is present in all members of the Kv1 family (2). mKv1.7, like Kv1.6, lacks an N-glycosylation site in the extracellular loop linking the S1 and S2 transmembrane segments; this consensus sequence is conserved in all other Kv1 family genes.
Fig. 3 shows a phylogenetic tree of the entire Shaker family of genes based on parsimony analysis of a nucleotide sequence alignment (generated from the amino acid sequence alignment) using the program PAUP (25). Our analysis places mKv1.7 within the Shaker family of genes. The mKv1.7 gene does not cluster with any of the known genes, and its position within the tree is not firmly established.
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Kv1.7 Is Located on Mouse Chromosome 7 and Human Chromosome 19q13.3
The mKv1.7/Kcna7 gene resides on mouse chromosome 7 (Fig. 4A), ~0.5 centimorgan telomeric to the Shaw-related K+ channel gene, mKv3.3/Kcnc3, and ~2.4 centimorgans centromeric of MyoD1 (myoblast differentiation factor). The most centromeric marker in this study was Gpi1 (glucose phosphate isomerase 1), which mapped ~6.1 centimorgans centromeric to mKv3.3/Kcnc3.
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The interval on mouse chromosome 7 containing mKv1.7/Kcna7 and mKv3.3/Kcnc3 shares a region of homology with human chromosomes 19q13 and 11p15, and the human homologues of these K+ channel genes may therefore be expected to reside on one of these chromosomes. Confirming this prediction, we mapped both genes to human 19q13.3-13.4 using fluorescent in situ hybridization. The idiogram of human chromosome 19 shown in Fig. 4B, and a more detailed map shown in Fig. 4C, indicate that hKv1.7/KCNA7 is located ~1.3 mb centromeric of hKv3.3/KCNC3. The genes for both muscle glycogen synthase (GYS1) and the histidine-rich calcium protein (HRC) also map to this region; Kv1.7/KCNA7 lies ~25 kb telomeric to GYS1 and ~200 kb centromeric to HRC (Fig. 4C). Interestingly, a putative diabetes susceptibility gene has been suggested to be present at 19q13.3 (26, 27), especially in Finnish families with associated hypertension and difficulties in insulin-stimulated glucose storage. This region has also been suggested to contain a modifier locus for cystic fibrosis (28).
Biophysical and Pharmacological Characterization of Kv1.7 Channels
We carried out a detailed characterization of mKv1.7 channels expressed in RBL cells which express no native Kv currents (29, 30). The mKv1.7 gene expressed in Xenopus oocytes produced currents (data not shown) similar to those obtained in RBL cells (Fig. 5).
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Voltage Dependence--
Patch clamp studies were performed in the
whole-cell mode. Fig. 5A shows a family of outward currents
elicited by a 200 ms depolarizing pulse from a holding potential of
80 mV in RBL cells injected with mKv1.7 cRNA; no outward
currents were detected in control cells (data not shown). The currents
activate rapidly, and from the conductance-voltage curve shown in Fig.
5B we determined that the 1/2 activation potential
(V1/2) is
20 mV.
Inactivation and Deactivation--
Inactivation of mKv1.7 channels
was rapid following a 200 ms depolarizing pulse from 80 to 40 mV
(Fig. 5A). The rate of inactivation, measured by fitting the
data to a single exponential function, yielded a time constant
(
h) of 14 ± 2.1 ms (S.E., n = 7). As
shown in Fig. 5C, the current became progressively smaller following repeated depolarizing pulses at 1-s intervals. This phenomenon, termed "cumulative inactivation," is due to the
accumulation of channels in the inactivated state which are then
unavailable for opening. The related channels, Kv1.3 (7) and Kv1.4
(31), also display this behavior.
Single-channel Conductance--
We measured single-channel
currents in an outside-out patch by applying 450-ms voltage ramps from
90 to 80 mV every second (Fig. 5E). Single channel events
were seen at potentials more positive than ~
15 mV. The
single-channel conductance of 21 pS was estimated from the slope
of the current recorded during an opening (Fig.
5E).
Pharmacology-- We determined the pharmacological sensitivity of the mKv1.7 channel using methods described previously (30, 32), IC50 values in each case being determined when block reached steady-state. The channel was blocked by several non-peptide small molecule antagonists, 4-aminopyridine (IC50 = 245 µM), capsaicin (25 µM), cromakalim (450 µM), tedisamil (18 µM), nifedipine (13 µM), diltiazem (65 µM), and resiniferatoxin (20 µM). Surprisingly, the dihydroquinoline compound, CP-339,818, that blocks rapidly inactivating Kv1 channels in the nanomolar range (30), had little effect on mKv1.7 (IC50 = 10 µM). The channel was insensitive to externally applied tetraethylammonium (C50 = 86 mM), probably because the residue at the tetraethylammonium-binding site, Ala-441 (Fig. 2), is hydrophobic.
The mKv1.7 channel is also potently blocked by a peptide (ShK toxin) obtained from sea anemone Stichodactyla helianthus (IC50 = 13 nM), and by the scorpion toxins, noxiustoxin (IC50 = 18 nM) and margatoxin (IC50 = 116 nM). The channel was resistant to charybdotoxin (IC50 >1000 nM) and kaliotoxin (IC50 >1000 nM).Expression of mKv1.7 Transcripts in Different Tissues
Northern blot assays using a mKv1.7-specific probe revealed strongly hybridizing 3-kb bands in heart and skeletal muscle; faint bands of similar size were visible in liver and lung (together with larger 7-8-kb bands), but none were seen in spleen, kidney, testis, or brain (Fig. 6) We were able to isolate mKv1.7 transcripts from mouse brain by PCR (see Fig. 1). mKv1.7 is also expressed in placenta, since the mouse EST AA021711 was derived from this tissue.
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PCR analysis demonstrated the presence of haKv1.7 mRNAs in hamster insulinoma cells (Fig. 1). We verified the presence of mKv1.7 mRNA in pancreatic islet cells obtained from 9-16-week-old diabetic db/db mice by in situ hybridization (Fig. 7C) using a mKv1.7-specific antisense probe (12-14); mKv1.7 mRNA was also present in islets from normal db/+ mice (data not shown). Scattered acinar cells outside the islets also showed mKv1.7 hybridization (Fig. 7C). In contrast, mKv3.4 mRNA was found in acinar cells surrounding islets, but not in islets, of both db/db (Fig. 7B) and db/+ mice (data not shown). As a control, insulin mRNA was detected in both normal and diabetic islets, but not in acinar cells (Fig. 7A). A Kv1.5-specific probe did not show appreciable hybridization to islets (data not shown), despite a report of Kv1.5 cDNA having been cloned from human insulinoma cells (33).
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DISCUSSION |
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Unlike all other known mammalian Shaker-related genes (Kv1.1-Kv1.6) that have intronless coding regions (2, 9), the protein-coding region of mKv1.7 is interrupted by a single 1.9-kb intron. The fly Shaker gene also contains an intron in the S1-S2 loop, raising the possibility that the intron in Kv1.7 may be ancient, predating the divergence of flies and mammals. Both the mouse Kv.1.7 and the fly Shaker intron are placed between codons, i.e. they are "phase 0" introns. While this is consistent with their having a common origin it may also be fortuitous, since there are only three possible "phases." Although we favor the idea that Kv introns were lost in the vertebrate lineage before their expansion by gene duplication (in which case the Kv1.7 intron would represent a more recent insertion), the evolutionary history of this complex gene family remains to be elucidated.
Since Kv1.7 mRNA is expressed in the mouse heart, we
searched the literature for native cardiac A-type Kv currents with
properties resembling those of Kv1.7. The Kv1.7 homotetramer shares
many properties with the rapidly inactivating transient outward
(Ito) current in cardiac Purkinje fibers, but not the
Ito current in atrial and ventricular myocytes. Kv1.7 and
the Purkinje Ito currents activate at negative potentials
(~30 to
20 mV), inactivate rapidly (
h < 25 ms),
exhibit cumulative inactivation, are blocked by micromolar
concentrations of 4-aminopyridine, and are resistant to >20
mM tetraethylammonium (34-36) (this study). In contrast, the Ito current in atrial and ventricular muscle, a product
of the Kv4.3 gene, does not exhibit cumulative inactivation
(36). These studies suggest that at least part of the Purkinje fiber Ito might be encoded by the Kv1.7 gene, although
more extensive biophysical and pharmacological studies are required to
confirm the link, and the presence of Kv1.7 mRNA
and/or protein has yet to be demonstrated in these fibers. The abundant
expression of Kv1.7 mRNA in mouse heart suggests that
this channel is also likely to be present in ventricular and/or atrial
muscle where it may co-assemble with other Kv1 family channels to form
heterotetramers.
Recent studies suggest an important role for Kv channels in regulating
islet cell function, specifically in repolarizing the membrane
potential following each action potential during the glucose-induced
bursting phase associated with insulin secretion (3-6). Despite these
interesting findings, the genes encoding Kv genes in
-cells have not been identified. Although the Kv1.5 gene
was isolated from human insulinoma cells (33), we did not detect
Kv1.5 mRNA in normal or diseased islets. We have,
however, demonstrated the presence of Kv1.7 mRNA in
these cells. Unlike the noninactivating Kv channels in pancreatic
-cells (3, 4), the Kv1.7 homotetramer exhibits rapid C-type
inactivation. Since Kv1.7 mRNA is expressed in
pancreatic islets, it is possible that heteromultimers composed of
Kv1.7 and other Kv1 subunits constitute the native Kv channels in
-cells. Enhanced levels of such Kv channels would excessively
hyperpolarize the membrane of
-cells and impair insulin secretion
(5). The mapping of the Kv1.7 gene to human chromosome
19q13.3, a region thought to contain a diabetic susceptibility gene
(26, 27), also suggests that Kv1.7 might contribute to the
pathogenesis of type II diabetes mellitus in some humans.
In conclusion, we have described a novel Kv1 family gene
with a genomic organization distinct from all the other members of the
family. The Kv1.7 channel produces a typical A-type current, and
transcripts are expressed in the heart, skeletal muscle, brain, placenta, and pancreatic -cells. This channel is biophysically and
pharmacologically similar to the Purkinje fiber Ito
current, and the gene may contribute at least one subunit to
heteromultimeric Kv channels in pancreatic
-cells.
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ACKNOWLEDGEMENTS |
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The assistance of F. Glaser, S. Plummer, B. Dethlefs, S. Hoffman, M. Christensen, T. Wymore, C. Chandy, and D. J. Gilbert is gratefully acknowledged. We are obliged to Dr. J. Aiyar for reading and improving our manuscript.
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FOOTNOTES |
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* This work was supported in part by United States Public Health Service Grants AI-24783, Shannon Award GMOD54872 (to K. G. C.), and NS14609 (to M. D. C.); by Glaxo Inc. (to K. G. C.); by United States Department of Energy Contract W-7405-Eng-48 (to H. W. and B. B.); and by the National Institutes of Health, NCI, DHHD, under Contract NO1-CO-74101 with ABL (C. M. H., N. G. C., and N. A. J.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF032099-AF032101.
§ Present address: Wyeth Ayerst Research, Cardiovascular/Metabolic Disease, CN8000, Rm. 1119A, Princeton, NJ 08543.
To whom correspondence should be addressed: Rm. 291, Joan
Irvine Simth Hall, Department of Physiology and Biophysics, Medical School, University of California Irvine, Irvine, CA 92697. Tel.: 714-824-2133; Fax: 714-824-3143; E-mail: gchandy{at}uci.edu.
1 The abbreviations use are: Kv, voltage-gated potassium selective; PCR, polymerase chain reaction; RBL, rat basophilic leukemic; bp, base pair(s); mb, millibase pair(s); kb, kilobase pair(s).
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REFERENCES |
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