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• W1975792208 abstract "Voltage-gated K+ (Kv) channels consist of α subunits complexed with cytoplasmic Kvβ subunits. Kvβ1 subunits enhance the inactivation of currents expressed by the Kv1 α subunit subfamily. Binding has been demonstrated between the C terminus of Kvβ1.1 and a conserved segment of the N terminus of Kv1.4, Kv1.5, and Shaker α subunits. Here we have examined the interaction and functional properties of two alternatively spliced human Kvβ subunits, 1.2 and 1.3, with Kvα subunits 1.1, 1.2, 1.4, and 1.5. In the yeast two-hybrid assay, we found that both Kvβ subunits interact specifically through their conserved C-terminal domains with the N termini of each Kvα subunit. In functional experiments, we found differences in modulation of Kv1α subunit currents that we attribute to the unique N-terminal domains of the two Kvβ subunits. Both Kvβ subunits act as open channel blockers at physiological membrane potentials, but hKvβ1.2 is a more potent blocker than hKvβ1.3 of Kv1.1, Kv1.2, Kv1.4, and Kv1.5. Moreover, hKvβ1.2 is sensitive to redox conditions, whereas hKvβ1.3 is not. We suggest that different Kvβ subunits extend the range over which distinct Kv1α subunits are modulated and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions. Voltage-gated K+ (Kv) channels consist of α subunits complexed with cytoplasmic Kvβ subunits. Kvβ1 subunits enhance the inactivation of currents expressed by the Kv1 α subunit subfamily. Binding has been demonstrated between the C terminus of Kvβ1.1 and a conserved segment of the N terminus of Kv1.4, Kv1.5, and Shaker α subunits. Here we have examined the interaction and functional properties of two alternatively spliced human Kvβ subunits, 1.2 and 1.3, with Kvα subunits 1.1, 1.2, 1.4, and 1.5. In the yeast two-hybrid assay, we found that both Kvβ subunits interact specifically through their conserved C-terminal domains with the N termini of each Kvα subunit. In functional experiments, we found differences in modulation of Kv1α subunit currents that we attribute to the unique N-terminal domains of the two Kvβ subunits. Both Kvβ subunits act as open channel blockers at physiological membrane potentials, but hKvβ1.2 is a more potent blocker than hKvβ1.3 of Kv1.1, Kv1.2, Kv1.4, and Kv1.5. Moreover, hKvβ1.2 is sensitive to redox conditions, whereas hKvβ1.3 is not. We suggest that different Kvβ subunits extend the range over which distinct Kv1α subunits are modulated and may provide a variable mechanism for adjusting K+ currents in response to alterations in cellular conditions. The electrical properties of excitable cells such as neurons and cardiomyocytes are strongly influenced by the K+ currents they express. A variety of voltage-gated K+ (Kv) 1The abbreviations used are: Kvvoltage-gated K+PCRpolymerase chain reactionaaamino acid(s)ntnucleotide(s)RACErapid amplification of cDNA ends. channels control the falling phase of the action potentials of excitable cells. Kv channels are also important in many nonexcitable cells, where they contribute to diverse processes such as volume regulation, hormone secretion, and activation by mitogens (1Kolb H.A. Rev. Physiol. Biochem. Pharmacol. 1990; 115: 51-91Crossref PubMed Google Scholar). Functional Kv channels assemble as tetramers of pore-forming α subunits (Kvα). Many mammalian Kvα genes have been cloned and assigned to four subclasses based on sequence similarities: Kv1, Kv2, Kv3, and Kv4 (2Pongs O. Physiol. Rev. 1992; 72: S69-S88Crossref PubMed Google Scholar). In heterologous expression systems, individual Kvα subunits confer characteristic properties of gating, selectivity, and ion conduction, but several lines of evidence suggest that native Kv channels are more complex. First, within subfamilies, Kvα subunits are able to form functionally distinct heterotetramers, which contribute to increased K+ channel diversity (3Ruppersberg J.P. Frank R. Pongs O. Stocker M. Nature. 1990; 345: 535-537Crossref PubMed Scopus (340) Google Scholar, 4Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (288) Google Scholar, 5Wang H. Kunkel D.D. Martin T.M. Schwartzkroin P.A. Tempel B.L. Nature. 1993; 365: 75-79Crossref PubMed Scopus (512) Google Scholar). Second, accessory or Kvβ subunits that modify the gating properties of coexpressed Kvα subunits have been cloned (6Scott V.E. Rettig J. Parcej D.N. Keen J.N. Findlay J.B. Pongs O. Dolly J.O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1637-1641Crossref PubMed Scopus (175) Google Scholar, 7Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Crossref PubMed Scopus (739) Google Scholar, 8Majumder K. De Biasi M. Wang Z. Wible B.A. FEBS Lett. 1995; 361: 13-16Crossref PubMed Scopus (115) Google Scholar, 9Morales M.J. Castellino R.C. Crews A.L. Rasmusson R.L. Strauss H.C. J. Biol. Chem. 1995; 270: 6272-6277Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 10England S.K. Uebele V.N. Shear H. Kodali J. Bennett P.B. Tamkun M.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6309-6313Crossref PubMed Scopus (122) Google Scholar, 11England S.K. Uebele V.N. Kodali J. Bennett P.B. Tamkun M.M. J. Biol. Chem. 1995; 270: 28531-28534Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12Heinemann S.H. Rettig J. Wunder F. Pongs O. FEBS Lett. 1995; 377: 383-389Crossref PubMed Scopus (95) Google Scholar) and found to be associated with Kvα subunits in native membranes (6Scott V.E. Rettig J. Parcej D.N. Keen J.N. Findlay J.B. Pongs O. Dolly J.O. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 1637-1641Crossref PubMed Scopus (175) Google Scholar, 13Rhodes K. Keilbaugh S.A. Barrezueta N.X. Lopez K.L. Trimmer J.S. J. Neurosci. 1995; 15: 5360-5371Crossref PubMed Google Scholar). voltage-gated K+ polymerase chain reaction amino acid(s) nucleotide(s) rapid amplification of cDNA ends. To date, five distinct mammalian Kvβ subunits have been cloned: Kvβ1.1, Kvβ1.2, Kvβ1.3, Kvβ2, and Kvβ3, according to recently proposed terminology (11England S.K. Uebele V.N. Kodali J. Bennett P.B. Tamkun M.M. J. Biol. Chem. 1995; 270: 28531-28534Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 12Heinemann S.H. Rettig J. Wunder F. Pongs O. FEBS Lett. 1995; 377: 383-389Crossref PubMed Scopus (95) Google Scholar). Kvβ subunits have been shown to alter the phenotype of a subset of Kvα subunit currents within the Kv1 subfamily (7Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Crossref PubMed Scopus (739) Google Scholar, 8Majumder K. De Biasi M. Wang Z. Wible B.A. FEBS Lett. 1995; 361: 13-16Crossref PubMed Scopus (115) Google Scholar, 9Morales M.J. Castellino R.C. Crews A.L. Rasmusson R.L. Strauss H.C. J. Biol. Chem. 1995; 270: 6272-6277Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 10England S.K. Uebele V.N. Shear H. Kodali J. Bennett P.B. Tamkun M.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6309-6313Crossref PubMed Scopus (122) Google Scholar, 12Heinemann S.H. Rettig J. Wunder F. Pongs O. FEBS Lett. 1995; 377: 383-389Crossref PubMed Scopus (95) Google Scholar, 14McCormack K. McCormack T. Tanouye M. Rudy B. Stühmer W. FEBS Lett. 1995; 370: 32-36Crossref PubMed Scopus (93) Google Scholar) primarily by introducing inactivation into noninactivating delayed rectifier channels (i.e. Kv1.1 and Kv1.5) and accelerating the intrinsic inactivation of rapidly inactivating channels (i.e. Kv1.4). The Kvβ N terminus is thought to act as a “ball peptide” to mimic the N-type inactivation characteristic of Shaker and Kv1.4 (7Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Crossref PubMed Scopus (739) Google Scholar). There are differences between Kvβ-induced and N-type inactivation, however. Whereas N-type inactivation is complete, Kvβ-induced inactivation is generally partial with significant sustained currents remaining at the end of the pulse. Recent biochemical studies have shown that Kvβ1.1 interacts with the N-terminal domains of the Kv1 subfamily α subunits, Kv1.4, Kv1.5, and Shaker, but not Kv2, Kv3, or Kv4 (15Yu W. Xu J. Li M. Neuron. 1996; 16: 441-445Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 16Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). A recently proposed model of Kvα-Kvβ interactions suggests that the inactivation conferred on Kv1 currents by Kvβ subunits is the result of two sequential interactions: 1) physical association of the two subunits through the interaction of the conserved Kvβ1 C terminus with conserved regions in the Kv1α N terminus, and 2) plugging of the Kv1α pore by functional interaction of the Kvβ1 N terminus with its corresponding receptor site on the Kv1α subunit (15Yu W. Xu J. Li M. Neuron. 1996; 16: 441-445Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 16Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). One resulting hypothesis is that all Kv1α subunits would interact with all Kvβ1 subunits but that the functional effects on a particular Kvα might differ depending upon which Kvβ1 N terminus is present. To test this hypothesis, we have studied the interaction of two human Kvβ1 subunits with a variety of Kv channels. hKvβ1.2 and hKvβ1.3 share identical C-terminal domains but have unique, nonhomologous N termini. Our results indicate that both Kvβ1 subunits interact selectively with Kv1α subunits and produce open channel block, but hKvβ1.2 is much more potent than hKvβ1.3. In addition, hKvβ1.2 is sensitive to redox potentials, while hKvβ1.3 is not. The results are consistent with our hypothesis that functional differences originate with distinct Kvβ1 N-terminal domains. 5′-RACE Ready human heart cDNA (Clontech) was used as a template in a nested PCR with antisense oligonucleotides encoding portions of the Kvβ1 subunit C terminus near the N-terminal junction point in combination with the sense oligonucleotide anchor primer supplied with the 5′-RACE Ready cDNA. The first PCR consisted of the β subunit-specific antisense oligonucleotide (R7): 5′-AGCATAGACTTCGGCAGTATC-3′ with the 5′-RACE anchor primer. A small aliquot of the first PCR product was used as template in the second reaction with an internal β subunit-specific antisense oligonucleotide (R6): 5′-TCCAAATGTCACCCATGTTCC-3′ and the anchor primer oligonucleotide. Without purification, the products from the second PCR were cloned into the pCRII vector (Invitrogen). Resulting clones were sequenced with the Sequenase kit (U. S. Biochemical Corp.), and compared to hKvβ1.2, Kvβ1.1, Kvβ2, and the Drosophila Kvβ, Hk. This protocol generated several overlapping clones, the longest of which resulted in an open reading frame of 90 amino acids (hKvβ1.3). This clone was identical to hKvβ1.2 in the region between oligonucleotide R6 and the arginine (R) residue marking the junction point between the N and C termini. Since this partial clone did not possess a putative initiating methionine residue, another 5′-RACE was performed to obtain the full N-terminal coding region. The second 5′-RACE reaction was done with the antisense β1.3-5′-RACE oligonucleotide 5′-TGAGGGACTAAGGCTGCTGTC-3′ in combination with the anchor primer, and generated a clone that contained a putative initiating methionine by virtue of two upstream in-frame stop codons. To obtain the full-length hKvβ1.3 sequence from human heart, we performed reverse transcription-PCR with human atrial total RNA using oligonucleotides spanning the proposed initiating methionine of hKvβ1.3 and the C-terminal residues plus stop codon of hKvβ1.2. A single band of approximately 1.3 kilobase pairs was obtained. Sequencing of full-length hKvβ1.3 revealed a unique N terminus identical to what had been obtained from the 5′-RACE protocol and a C terminus identical to hKvβ1.2. Fragments encoding the unique N-terminal 91 amino acids of hKvβ1.3 and 79 amino acids of hKvβ1.2 were prepared by PCR and cloned into pCRII (Invitrogen) to be used as the template to prepare β subunit-specific probes for RNase protection assays. The fragments were sequenced to confirm that no mutations were introduced by PCR, and to determine the orientation in the pCRII vector. Plasmid DNA was purified on Qiagen Midi-prep plasmid purification columns and linearized with HindIII. 32P-Labeled antisense transcripts were prepared from linearized template with T7 RNA polymerase using the MAXIscript transcription kit (Ambion) according to the manufacturer's protocol. Full-length antisense transcripts (385 bases for hKvβ1.3; 420 bases for hKvβ1.2) were gel-purified on a denaturing 5% polyacrylamide gel containing 8 M urea and eluted from the gel slices by overnight incubation at 37°C in the following buffer: 0.5 mM ammonium acetate, 1 mM EDTA, 0.2% SDS. Total RNA from human atrial appendage tissue, right atrium, right ventricle, and left ventricle was isolated using RNA-STAT-60 (Tel-Test). The ventricular and right atrial samples were obtained in accordance with Tulane University School of Medicine Institutional guidelines from the explanted heart of a 7-year-old female patient with dilated cardiomyopathy undergoing cardiac transplant. Human atrial appendage tissue was pooled from adult patients undergoing aortocoronary bypass surgery. Total RNA from whole adult human brain was purchased from Clontech (catalog no. 64020-1). The RNase protection experiments were performed using the HybSpeed RPA kit (Ambion) following the manufacturer's protocol. Ten μg of brain RNA and 50 μg of each cardiac tissue sample were incubated with 105 cpm of each β subunit probe for 20 min at 68°C. Forty μg of yeast total RNA was added to the brain RNA to keep the same total amount of RNA in each tube. As a control the probes were also incubated with 50 μg of yeast tRNA only. The hybridization mixtures were then digested for 45 min with ribonuclease T1 at 37°C. Following RNase treatment, the protected fragments were precipitated, resuspended in gel loading buffer, and electrophoresed in a 5% polyacrylamide gel containing 8 M urea. The gel was dried and exposed to Kodak X-Omat AR film for 5 days at −80°C. Protein-protein interactions were monitored with the yeast Matchmaker two-hybrid system from Clontech. EcoRI and SalI sites were incorporated into the 5′ and 3′ ends, respectively, of hKvβ1.3 and each α subunit fragment by PCR for cloning in frame to the yeast shuttle vectors, pGBT9 and pGAD424. The following putative cytoplasmic N-terminal Kvα subunit fragments were made: hKv1.4-N (aa 1-305), hKv1.4ΔN2-146 (deletion of amino acids 2-146), hKv1.5-N (aa 1-248), hKv1.2-N (aa 1-124), and hKv1.1 (aa 1-168). The C terminus of hKv1.4, hKv1.4-C (aa 562-654), was also made. The full coding sequences of hKvβ1.3 (aa 1-419) and hKvβ1.2 (aa 1-408) were prepared as yeast fusion proteins. We also subcloned the Kvβ N- and C-terminal regions and tested them for interaction with Kvα subunits separately: hKvβ1.3-N (aa 1-91), hKvβ1.2 (aa 1-79), and Kvβ1-C (the C-terminal 329 amino acids of the Kvβ1 subfamily). Protein-protein interactions were tested in two host yeast strains, SFY526 and HF7C by cotransformation with pairs of pGBT9 and pGAD424 fusion constructs according to the manufacturer's protocol. Transformations in SFY526 were plated on media lacking tryptophan (trp−) and leucine (leu−), and grown for 3-4 days at 30°C. Yeast colonies were lifted to paper filters and tested for β-galactosidase activity. Colonies turning blue within 8 h were scored as positive. Transformations in HF7C were plated on media lacking trp, leu, and histidine (his). Only yeast transformed with interacting fusion proteins should grow as a result of activation of the HIS3 gene, but β-galactosidase assays were performed for confirmation of interaction. hKvβ1.3 and hKvβ1.2 were subcloned into pSP64 (Promega) for oocyte expression. The sources of the Kvα subunit constructs and preparation of cRNA are as described previously (8Majumder K. De Biasi M. Wang Z. Wible B.A. FEBS Lett. 1995; 361: 13-16Crossref PubMed Scopus (115) Google Scholar). The hKv1.4ΔN2-146 construct was prepared by PCR using a sense oligonucleotide incorporating a NotI site, a Kozak consensus sequence for initiation and an initiating ATG linked to 15 bases of Kv1.4 sequence encoding residues 147-151, and an antisense oligonucleotide just 3′ to the internal BstEII site. After cloning into pCRII to verify the correct construction, the NotI/BstEII fragment was subcloned into hKv1.4-A+-pCRII from which the wild-type fragment had been removed. cRNA was prepared with the mMESSAGE mMACHINE kit (Ambion) using either SP6 or T7 RNA polymerase after linearization of the plasmids with EcoRI (for hKvβ1.3 and hKvβ1.2) or HindIII (for hKv1.4ΔN2-146). cRNAs were dissolved in 0.1 M KCl, stored at −80°C, and diluted immediately prior to injection. Stage V-VI Xenopus oocytes were injected with 46 nl of cRNA. Whole-cell macroscopic currents were recorded with conventional two-electrode techniques as described previously (17Kirsch G.E. Drewe J.A. Hartmann H.A. Taglialatela M. De Biasi M. Brown A.M. Joho R.H. Neuron. 1992; 8: 499-505Abstract Full Text PDF PubMed Scopus (76) Google Scholar). Electrodes filled with 3 M KCl had resistance of approximately 0.2-0.8 megohms when measured in the bath solution containing (in mM): NaCl, 100; KCl, 5; CaCl2, 0.3; MgCl2, 2; and HEPES, 10. pH was adjusted to 7.4 with Tris base. Records were digitized at 10 KHz and filtered at 3 KHz. Experiments were conducted at room temperature (20-22°C). Macropatch currents were measured in cell-attached and inside-out configuration by using pipettes made from borosilicate glass with tip openings of ∼10-15 μm. The electrodes were connected to a patch-clamp amplifier (Axopatch1-C, Axon Instrument, Foster City, CA). The pClamp suite of programs was employed for data acquisition and analysis. The bath solution contained (in mM): KCl, 100; MgCl2, 1; and HEPES, 10. The pipette (external) solution contained (in mM): NaCl, 100; KCl, 5; CaCl2, 1.5; MgCl2, 2; and HEPES, 10. pH was titrated to 7.3 with Tris base and readjusted to this value whenever additional components were added to the solution such as glutathione (GSH, 5 mM). H2O2 (0.1%) or GSH was applied to the cytoplasmic surface of the inside-out patches by changing the perfusate. Inside-out patches were positioned in the stream of a large pipette (diameter ∼2 mm) to achieve faster solution exchange (<10 s) (18Kiehn H. Wible B. Ficker E. Taglialatela M. Brown A.M. Circ. Res. 1995; 77: 1151-1155Crossref PubMed Scopus (26) Google Scholar). Data were low pass-filtered at 2 KHz before digitization (−3 dB, 4-pole Bessel filter). To minimize leak current and capacitive transient, on-line subtraction with a P-4 protocol was used in all recordings. Traces at each potential represent an average of 5-10 sweeps. Single channels were recorded in both cell-attached and inside-out patches. Fire-polished and Sylgard-coated electrodes, pulled from borosilicate glass on a horizontal Flaming-Brown micropipette puller, had a tip resistance of ∼10 megohms when filled with the external solution, which had the same composition as the bath solution described for the two-electrode experiments. Here the bath solution contained the following compositions (in mM): NaCl, 5; KCl, 100; CaCl2, 0.3; MgCl2, 2; and HEPES, 10. Data were digitized at 10 KHz and filtered with a 2-KHz cutoff frequency. Junction potentials were zeroed before formation of the membrane-pipette seal. All experiments were carried out at room temperature (∼20-22°C). Chemicals were purchased from Sigma. For whole-cell and macropatch recordings, the peak and steady-state currents were measured as the maximal current amplitude relative to the zero base-line level during a 100-ms pulse or as the current amplitude at the end of the pulse by Clampfit in pClamp. Single-channel data were analyzed with Fetchan in pClamp, and open and closed transitions were detected using a half-amplitude threshold criterion. Group data are presented as mean ± S.E. Student's t tests (paired or unpaired) were used to evaluate the statistical significance of differences between means. A two-tailed probability of 0.5% was taken to indicate statistical significance. For analysis of current kinetics, data points were fitted by Clampfit in pClamp. For other curve-fitting procedures, a nonlinear curve-fitting technique (Marquardt's procedure) was performed using Sigmaplot software (Jandel Scientific). The Kvβ1 subfamily of Kvβ subunits consists of three members, which result from alternative splicing of a single gene: Kvβ1.1 (7Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Crossref PubMed Scopus (739) Google Scholar), hKvβ1.2 (8Majumder K. De Biasi M. Wang Z. Wible B.A. FEBS Lett. 1995; 361: 13-16Crossref PubMed Scopus (115) Google Scholar), and hKvβ1.3 (11England S.K. Uebele V.N. Kodali J. Bennett P.B. Tamkun M.M. J. Biol. Chem. 1995; 270: 28531-28534Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). We originally cloned hKvβ1.2 from human atrium and found that it shared an identical C terminus to rat brain Kvβ1 (subsequently renamed Kvβ1.1). When this study was initiated, only two Kvβ subunits had been cloned: Kvβ1.1 and hKvβ1.2. Anticipating that there might be other members of this family, we searched for distinct Kvβ subunit family members with a common structural plan, i.e. a conserved C-terminal domain with a variable N terminus, by using 5′-RACE on human heart RNA. With this strategy we cloned hKvβ1.3 from human atrium, which proved to be identical to a recently published hKvβ1.3 clone from human ventricle (11England S.K. Uebele V.N. Kodali J. Bennett P.B. Tamkun M.M. J. Biol. Chem. 1995; 270: 28531-28534Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Each Kvβ1 subunit consists of an identical C-terminal domain of 329 amino acids spliced to a unique N-terminal domain. The nonhomologous N termini of the three Kvβ1 subunits are shown in Fig. 1. We examined the expression of hKvβ1.3 in human atrium, ventricle, and brain with RNase protection assays. To distinguish the expression of hKvβ1.3 from other β subunits, a radiolabeled probe covering the unique N terminus of hKvβ1.3 was hybridized to total RNA from human atrium, right ventricle, left ventricle, and brain (Fig. 2, panel A). Compared to heart RNA, 5 times less brain total RNA was used in the assay. Specific protection of a 273-nucleotide (nt) band was observed in all tissues with the strongest signals coming from left ventricle and brain. In addition, a smaller band of approximately 250 nt was observed in all tissues. The source of this second band is unknown, but could represent a second isoform of hKvβ1.3 with sequence divergence at one or the other end of the N-terminal probe. Interestingly, the relative abundance of the 250-nt band increased in atrium such that the 273- and 250-nt bands were present in at least relatively equal proportions, whereas the 250-nt band appeared to be only a minor component in ventricle and brain. For comparison, the expression of hKvβ1.2 was also examined with an N-terminal hKvβ1.2 probe (Fig. 2, panel B). hKvβ1.2 was expressed in all tissues by virtue of the protected 237-nt band seen in all lanes. Expression was highest in ventricle and brain relative to atrium, however. These data are consistent with higher levels of hKvβ1.2 expression in human ventricle compared to atrium using Northern blotting (10England S.K. Uebele V.N. Shear H. Kodali J. Bennett P.B. Tamkun M.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6309-6313Crossref PubMed Scopus (122) Google Scholar). To determine whether there is any specificity in the interaction of hKvβ1.2 and hKvβ1.3 with members of the Kv1 subfamily, we used the yeast two-hybrid system to identify the Kv1 α subunits to which hKvβ1.2 and hKvβ1.3 bind as well as to map the domains that mediate this interaction. As seen in Fig. 3, hKvβ1.3 interacts with the N terminus of hKv1.4 (aa 1-305) but not the C terminus (aa 562-654), as evidenced by the growth pattern on media lacking histidine. hKvβ1.3 also interacts with the truncated Kv1.4 N terminus, Kv1.4-ΔN2-146, indicating the binding site is within the 159 amino acids immediately preceding the putative S1 transmembrane domain. β-Galactosidase filter assays were also performed on these cotransformants and were positive for the expression of the lacZ reporter gene in those that grew on media lacking histidine (Table I).Table IYeast two-hybrid interactions of hKvβ1.3 and hKvβ1.2 with Kv1 family channel fragmentsGal4-BD hybridInteractionGal4-AD hybridGal4-BD hybridInteractionGal4-AD hybridhKvβ1.3+Kv1.4-NhKvβ1.2+Kv1.4-NKv1.4-N+hKvβ1.3Kv1.4-N+hKvβ1.2hKvβ1.3−Kv1.4-ChKvβ1.2−Kv1.4-CKv1.4-C−hKvβ1.3Kv1.4-C−hKvβ1.2hkvβ1.3+Kv1.4-ΔNhKvβ1.2+Kv1.4-ΔNKv1.5-N+hKvβ1.3Kv1.5-N+hKvβ1.2Kv1.2-N+hKvβ1.3Kv1.2-N+hKvβ1.2hKvβ1.3+Kv1.1-NhKvβ1.2+Kv1.1-NhKvβ1.3−HERG-NhKvβ1.2−HERG-NhKvβ1.3−hIRK-NhKvβ1.2−hIRK-NhKvβ1.3-N−Kv1.4-NhKvβ1.2-N−Kv1.4-NKv1.5-N−hKvβ1.3-NKv1.5-N−hKvβ1.2-NhKvβ1-C+Kv1.4-NKv1.5+hKvβ1-C Open table in a new tab The yeast two-hybrid assay was expanded to include the N-terminal portions of hKv1.5, hKv1.1, and hKv1.2. In these experiments, yeast fusion vectors were cotransformed into the SFY526 yeast strain, and the activation of the lacZ reporter gene was monitored. Table I shows that hKvβ1.3 interacts with the N-terminal sequences of not only Kv1.4, but also Kv1.5 (aa 1-248), Kv1.1 (aa 1-168), and Kv1.2 (aa 1-124). Thus, hKvβ1.3 is able to bind to all four Kv1 α subunits. hKvβ1.2 exhibits the same binding properties. As controls, we observed no interaction with either Kvβ subunit and the N terminus of the unrelated K+ channels, HERG (aa 1-396) and hIRK (aa 1-86). Table I also shows that it is the conserved C-terminal core of the Kvβ subunits which mediates their binding to the Kv1α subunit N terminus. Yeast fusion proteins were constructed with the hKvβ1.2 N terminus (aa 1-79), hKvβ1.3 N terminus (aa 1-91), and the conserved Kvβ C-terminal core region of 329 residues and tested for interaction with the N termini of Kv1.4 and Kv1.5. Only the Kvβ C-terminal core region interacts with the Kv1α N terminus. Since both Kvβ subunits are able to interact with each of the Kv1α N-terminal domains tested, we performed a detailed characterization of the functional effects of hKvβ1.3 and hKvβ1.2 by coexpression in Xenopus oocytes with a variety of Kvα subunits: Kv1.1, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv3.1. In order to compare different combinations of Kvα and β subunits, the amounts of α subunit cRNAs injected were controlled to yield steady-state currents of 4-15 μA at a potential of +60 mV when Kvβ subunits were absent. Kvβ subunits were coinjected with Kvα subunits at concentrations resulting in maximal Kvβ effects. When coinjected with hKv1.2, both β subunits introduced rapid but partial inactivation in the currents (Fig. 4A). In the presence of hKvβ1.2, Kv1.2 peak whole cell currents are reduced and inactivation is introduced at potentials above 0 mV. By contrast, at saturating concentrations of hKvβ1.3, there is a smaller reduction in peak currents and the amount of inactivation, which is only apparent at very positive potentials (>+60 mV), is less pronounced. hKvβ1.2 is a more potent modulator of Kv1.2 currents. This is further emphasized in Fig. 4B, in which I-V curves for steady-state Kv1.2 currents, plus and minus Kvβ subunits, are shown. Clearly, hKvβ1.2 reduces Kv1.2 currents over a much larger potential range than hKvβ1.3. The effects of hKvβ1.2 and hKvβ1.3 on other Kv1 channels are presented in Fig. 4C. For each Kvα-Kvβ subunit combination, the percent (%) block, defined as the difference between the peak current and the steady-state current remaining at the end of a pulse to +60 mV, is indicated. For each Kv1 channel examined (hKv1.1, hKv1.2, hKv1.4ΔN2-146, and hKv1.5), hKvβ1.2 produced significantly more block compared to hKvβ1.3. In the most dramatic example, hKvβ1.2 elicited an approximately 6-fold greater block of hKv1.2 than hKvβ1.3 did (60.7 ± 1.2% (n = 9) versus 9.8 ± 1.9% (n = 14), p < 0.01). hKvβ1.2 introduced less inactivation into Kv1.5 (52.0 ± 3.7%; n = 7), Kv1.4ΔN (48.2 ± 1.3%; n = 7), and Kv1.1 currents (31.3 ± 4.5%; n = 6). Interestingly, hKvβ1.3 produced the most marked effects on Kv1.4ΔN (% block = 32.0 ± 2.9; n = 10) and Kv1.5 (30.0 ± 4.5%; n = 7). More modest inactivation was introduced into hKv1.1 (10.6 ± 2.2%; n = 4) and hKv1.2 (see above). For Kv1.4, which exhibits intrinsic rapid inactivation, both Kvβ subunits accelerated the inactivation time course of the currents (data not shown). When intrinsic fast inactivation was removed by deletion of 146 amino acids from the N terminus (hKv1.4ΔN2-146), the channel expressed noninactivating currents that are also sensitive to Kvβ subunits. No significant changes in Kv2.1 or Kv3.1 currents were observed with either Kvβ subunit (data not shown), confirming that hKvβ1.2 and hKvβ1.3 are specific for channels of the Kv1 subfamily. To probe the mechanism by which hKvβ1.2 and hKvβ1.3 introduce inactivation and reduce peak whole-cell currents, we examined the single-channel properties of hKv1.2 with and without coexpressed Kvβ subunits. Kv1.2 currents were chosen for analysis, since the difference in the magnitude of the effects of hKvβ1.2 and hKvβ1.3 was greatest for this channel. Kv1.2 single channels recorded under different conditions from the same patches at a test potential of +70 mV, where both Kvβ subunits introduce inactivation, are presented in Fig. 5. Unitary currents from hKv1.2 in the cell-attached configuration (panel A, left) a" @default.
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• W1975792208 title "Comparison of Binding and Block Produced by Alternatively Spliced Kvβ1 Subunits" @default.
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