Cloning and expression of the delayed-rectifier LsK channel from neonatal rat heart and diethylstilbestrol-primed rat uterus ( voltage clamp / Xenopus oocytes / antisense inhibition / polymerase chain reaction )

cDNAs encoding a delayed-rectifier-type K+ channel were cloned from both neonatal rat heart and ovariectomized, diethylstilbestrol-primed rat uterus by using the polymerase chain reaction. Both clones have nucleotide sequences identical to that encoding the rat kidney LK channel [Takumi, T., Ohkubo, H. & Nakanishi, S. (1988) Science 242, 1042-1045] and encode a putative protein of 130 amino acids. Iiuection ofRNA transcripts ofthecDNAs intoXenopus oocytes resulted in the expression of a slowly activating, voltagedependent K+ current. An antisense oligonucleotide, derived from the sequence of the clone, specifically inhibited the expression of the slow, outward current observed in cells injected with mRNAs isolated from the parent tissues (i.e., kidney, heart, and uterus), indicating that the cloned gene underlies the major K+ current expressed from RNA isolated from these tissues. K+ channels form a diverse group of membrane-spanning proteins that have been classified largely according to their electrophysiological and pharmacological properties. Despite extensive characterization of their function and regulation, knowledge of channel structure is only beginning to emerge. Recent advances in the molecular biology of these proteins-specifically, the cloning and expression of K+ channels from the Shaker, Shab, and Shaw loci ofDrosophila (1-6) and of homologues from rodent brain (7, 8)-have greatly facilitated studies of their molecular structure. These Shaker-type K+ channels all share structural similarities that link them to the voltage-dependent Na+ and Ca2l channel families (9)-i.e., a motif of six or seven putative transmembrane segments including an amphipathic region thought to be involved in sensing changes in membrane potential. Recently, a gene for a structurally distinct K+ channel was cloned by expression from rat kidney (10). This channel, the IsK protein (11), is structurally unrelated to any ion channel previously described. When expressed by microinjection of RNA into Xenopus oocytes, it induces a slow, voltagedependent K+ current that closely resembles the delayedrectifier K+ current expressed from uterine poly(A)+ mRNA isolated from ovariectomized, diethylstilbestrol (DES)treated rats (12, 13). Both of these currents also bear a strong resemblance to the classical delayed outward rectifier (IV,), the main repolarizing K+ conductance in the cardiac ventricle (14, 15). To our knowledge, the nature of the proteins underlying these delayed rectifier K+ currents in heart and uterus has not been determined previously. In this report, we describe the cloning and expression of the IsK-type K+ channel from heart and DES-primed uterus of the rat. Expression of the clones in Xenopus oocytes induces a slow, outward K+-selective current. We further demonstrate, by hybrid arrest of expression with antisense oligonucleotides (16, 17), that this protein is responsible for the slow K+ current observed in oocytes injected with poly(A)+ mRNA from kidney, neonatal heart, and DES-primed uterus. Therefore, the lsK protein underlies the major K+ current expressed from mRNAs isolated from these tissues. MATERIALS AND METHODS Materials and General Methods. Avian myeloblastosis virus reverse transcriptase, poly(A) polymerase, pUC13, and the Klenow fragment of DNA polymerase were purchased from Pharmacia, and restriction enzymes were purchased from New England Biolabs. Transformation-competent Escherichia coli XL1-Blue and all reagents for in vitro transcription were from Stratagene. Reagents for the polymerase chain reaction (PCR) were purchased from Cetus; collagenase (type II), from Worthington; and DNA sequencing reagents, from United States Biochemical. Oligo(dT)cellulose (type III) was from Collaborative Research and Zeta-Probe membranes were from Bio-Rad. Female Xenopus laevis were from Nasco (Fort Atkinson, WI), and ovariectomized rats (200-250 g) were from Taconic Farms. Standard molecular biological procedures [e.g., RNA isolation, RNA (Northern) and DNA (Southern) blotting, and fragment isolation and subcloning] were performed as described (18, 19). Oligonucleotides (20-mers to 60-mers) were synthesized on an Applied Biosystems model 380B DNA synthesizer with /3-cyanoethylphosphoramidite chemistry. Cloning of K+ Channel cDNAs. PCR was used to clone cDNAs from the poly(A)+ fraction of mRNAs isolated from neonatal rat heart, ovariectomized and DES-primed rat uterus, and adult rat kidney (20). The oligonucleotide used to prime the first-strand cDNA synthesis was derived from the kidney lsK cDNA sequence (10) and was complementary to the sense strand, 18-42 bases downstream of the protein coding region (nucleotides 408-431 in Fig. 2). The first-strand cDNA was then used directly in the PCR. An upstream oligonucleotide primer was constructed that contained a phage T7 promoter (5'-TAATACGACTCACTATAGGGAGA-3'; ref. 21) followed by a sequence located upstream of the coding region in the IsK cDNA (from nucleotides -40 to -19 in Fig. 2). The PCR was performed for 40-50 cycles of 1-min strand separation at 94°C, 2-min hybridization at 56°C, and 3-min extension at 72°C. Reaction products of the expected size [-450 base pairs (bp)] were isolated by preparative gel electrophoresis and cloned into pUC13 by standard techniques. Nucleotide sequences of the clones were determined by the chain-termination method (22) with a Abbreviations: DES, diethylstilbestrol; PCR, polymerase chain reaction. *To whom reprint requests should be addressed at: W26-200B, Merck Sharp & Dohme Research Laboratories, West Point, PA 19486. 2975 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2976 Neurobiology: Folander et al. modified T7 DNA polymerase (Sequenase). Three independently amplified cDNAs from each poly(A)+ mRNA pool were sequenced to eliminate potential artifacts resulting from the high error frequency of the Thermus aquaticus DNA polymerase used in the PCR (23). Expression and Assay ofK+ Channels. RNAwas synthesized both from the bulk PCR reaction products (using the T7 promoter incorporated into the upstream primer) and from individual cDNAs after their subcloning into pUC13. T7 transcripts were prepared in vitro under conditions described for transcription with phage SP6 RNA polymerase (24). Transcripts were capped at the 5' end by including diguanosine triphosphate in the transcription reaction (25) and were polyadenylylated at the 3' end (to >30 mol of adenosine/mol of RNA) by reaction with ATP and poly(A) polymerase (26). Oocytes were obtained from female Xenopus laevis, defolliculated by collagenase digestion, and injected with RNA (50 nl; 2 mg/ml) as described (27). Injected cells were incubated for 2-3 days at room temperature in 96 mM NaCl/2 mM KCl/1.8 mM CaCl2/1 mM MgCl2/5 mM Hepes-NaOH, pH 7.6, supplemented with penicillin (100 units/ml), streptomycin (100 ,ug/ml), and gentamicin (10 ,&g/ml) and then were screened electrophysiologically for the expression ofK+ channels with a standard two-microelectrode voltage clamp. Currents were measured in 82.5 mM NaCl/2.5 mM KCl/1 mM MgCl2/5 mM HepesNaOH, pH 7.6, at room temperature (22-24°C). Microelectrodes were filled with 3 M KCl and had tip resistances from 0.5 to 2.0 MQ. The current was sampled at 2 kHz, low-pass-filtered at 30 Hz, and the data were stored for analysis. Leakage currents were removed from most recordings by pulsing to hyperpolarized potentials and then subtracting a linearly scaled, inverted pulse from the depolarization-induced current records. In antisense inhibition experiments, 2.5 1,u of poly(A)+ mRNA (1-3 mg/ml) was mixed with 0.5 ,ul of antisense (or control) oligonucleotide at 30 or 300 ,ug/ml in 500 mM KCl, denatured at 65°C for 2 min, and annealed by cooling to room temperature. Injection and recording were then carried out as described above.