Kvβ2 Inhibits the Kvβ1-mediated Inactivation of K+ Channels in Transfected Mammalian Cells
1997; Elsevier BV; Volume: 272; Issue: 18 Linguagem: Inglês
10.1074/jbc.272.18.11728
ISSN1083-351X
Autores Tópico(s)Neuroscience and Neuropharmacology Research
ResumoCloned auxiliary β-subunits (e.g.Kvβ1) modulate the kinetic properties of the pore-forming α-subunits of a subset of Shaker-like potassium channels. Coexpression of the α-subunit and Kvβ2, however, induces little change in channel properties. Since more than one β-subunit has been found in individual K+ channel complexes and expression patterns of different β-subunits overlap in vivo, it is important to test the possible physical and/or functional interaction(s) between different β-subunits. In this report, we show that both Kvβ2 and Kvβ1 recognize the same region on the pore-forming α-subunits of the Kv1 Shaker-like potassium channels. In the absence of α-subunits the Kvβ2 polypeptide interacts with additional β-subunit(s) to form either a homomultimer with Kvβ2 or a heteromultimer with Kvβ1. When coexpressing α-subunits and Kvβ1 in the presence of Kvβ2, we find that Kvβ2 is capable of inhibiting the Kvβ1-mediated inactivation. Using deletion analysis, we have localized the minimal interaction region that is sufficient for Kvβ2 to associate with both α-subunits and Kvβ1. This mapped minimal interaction region is necessary and sufficient for inhibiting the Kvβ1-mediated inactivation, consistent with the notion that the inhibitory activity of Kvβ2 results from the coassembly of Kvβ2 with compatible α-subunits and possibly with Kvβ1. Together, these results provide biochemical evidence that Kvβ2 may profoundly alter the inactivation activity of another β-subunit by either differential subunit assembly or by competing for binding sites on α-subunits, which indicates that Kvβ2 is capable of serving as an important determinant in regulating the kinetic properties of K+currents. Cloned auxiliary β-subunits (e.g.Kvβ1) modulate the kinetic properties of the pore-forming α-subunits of a subset of Shaker-like potassium channels. Coexpression of the α-subunit and Kvβ2, however, induces little change in channel properties. Since more than one β-subunit has been found in individual K+ channel complexes and expression patterns of different β-subunits overlap in vivo, it is important to test the possible physical and/or functional interaction(s) between different β-subunits. In this report, we show that both Kvβ2 and Kvβ1 recognize the same region on the pore-forming α-subunits of the Kv1 Shaker-like potassium channels. In the absence of α-subunits the Kvβ2 polypeptide interacts with additional β-subunit(s) to form either a homomultimer with Kvβ2 or a heteromultimer with Kvβ1. When coexpressing α-subunits and Kvβ1 in the presence of Kvβ2, we find that Kvβ2 is capable of inhibiting the Kvβ1-mediated inactivation. Using deletion analysis, we have localized the minimal interaction region that is sufficient for Kvβ2 to associate with both α-subunits and Kvβ1. This mapped minimal interaction region is necessary and sufficient for inhibiting the Kvβ1-mediated inactivation, consistent with the notion that the inhibitory activity of Kvβ2 results from the coassembly of Kvβ2 with compatible α-subunits and possibly with Kvβ1. Together, these results provide biochemical evidence that Kvβ2 may profoundly alter the inactivation activity of another β-subunit by either differential subunit assembly or by competing for binding sites on α-subunits, which indicates that Kvβ2 is capable of serving as an important determinant in regulating the kinetic properties of K+currents. The heterogeneity of voltage-sensitive potassium currents present in excitable and nonexcitable cells is essential for diverse biological functions (1Connor J.A. Stevens C.F. J. Physiol. ( Lond .). 1971; 213: 21-30Google Scholar, 2Byrne J.H. J. Neurophysiol. 1980; 43: 651-668Google Scholar, 3Rogawski M.A. Trends Neurosci. 1985; 8: 214-219Google Scholar, 4Hille B. Ionic Channels of Excitable Membrane. 1991; (and 99–116, Sinauer, Sunderland, MA): 58-75Google Scholar). In addition to the large number of genes encoding the channel subunits and posttranslational modulations of channel protein, the diversity of potassium channels is further enhanced by the mix-and-match assembly of different subunits (5Jan L.Y. Jan Y.N. Trends Neurosci. 1990; 13: 415-419Google Scholar, 6Salkoff L. Baker K. Butler A. Covarrubias M. Pak M.D. Wei A. Trends Neurosci. 1992; 15: 161-166Google Scholar). Within the large family of Shaker-like potassium channels, the selective subunit assembly includes heteromultimer formation of distinct pore-forming α-subunits and/or assembly of different kinds of subunits such as the α-subunits and hydrophilic cytoplasmic β-subunit(s) (7Scott 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: 1637-1641Google Scholar, 8Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Google Scholar). Together, these give rise to the vast heterogeneity of K+currents. Changes in expression of a given subunit may alter the composition of heteromultimers in vivo, which would allow a cell to tune its K+ current system(s) during development and in response to changes in the cellular environment. There are more than 60 cloned genes encoding functional Shaker-like α-subunits, which have been divided into several subfamilies. Among them, subunits in the Kv1 to Kv5 subfamily are capable of functional homomeric channels in heterologous systems, such as Xenopusoocytes (9Butler A. Wei A. Salkoff L. Nucleic Acids Res. 1990; 18: 2173-2174Google Scholar, 10Wei A. Covarrubias M. Butler A. Baker K. Pak M. Salkoff L. Science. 1990; 248: 599-603Google Scholar, 11Chandy K.G. Gutman G.A. Trends Pharmacol. Sci. 1993; 14: 434Google Scholar, 12Zhao B. Rassendren F. Kaang B.K. Furukawa Y. Kubo T. Kandel E.R. Neuron. 1994; 13: 1205-1213Google Scholar, 13Jegla T. Salkoff L. Recept. Channels. 1995; 3: 51-60Google Scholar). Four α-subunits within a given subfamily can form a functional channel either as a homotetramer or a heterotetramer (14MacKinnon R. Nature. 1991; 350: 232-235Google Scholar,15Li M. Unwin N. Stauffer K.A. Jan Y.N. Jan L.Y. Curr. Biol. 1994; 4(2): 110-115Google Scholar). In the case of auxiliary subunits in animals, four genes encoding β-subunits for Shaker-like potassium channels have been well characterized: Kvβ1, Kvβ2, Kvβ3 (which has now been suggested to be a splice variant of Kvβ1), and Hk (7Scott 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: 1637-1641Google Scholar, 8Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Google Scholar, 16Chouinard S.W. Wilson G.F. Schlimgen A.K. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6763-6767Google Scholar, 17England S.K. Uebele V.N. Shear H. Kodali J. Bennett P.B. Tamkun M.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6309-6313Google Scholar, 18Majumder K. De Biasi M. Wang Z. Wible B.A. FEBS Lett. 1995; 361: 13-16Google Scholar, 19McCormack K. McCormack T. Tanouye M. Rudy B. Stuhmer W. FEBS Lett. 1995; 370: 32-36Google Scholar, 20Morales M.J. Castellino R.C. Crews A.L. Rasmusson R.L. Strauss H.C. J. Biol. Chem. 1995; 270: 6272-6277Google Scholar). These β-subunits share at least 85% amino acid sequence identity in their COOH-terminal core regions, but differ significantly in length and sequence of the remaining NH2-terminal regions. Despite the remarkable sequence similarity among different β-subunits, their functional effects are quite different. For example, coexpression of β-subunits with certain α-subunits in Xenopus oocytes induces pronounced alterations in channel kinetic properties, most noticeably acceleration of fast inactivation by either Kvβ1 or Kvβ3 (8Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Google Scholar, 17England S.K. Uebele V.N. Shear H. Kodali J. Bennett P.B. Tamkun M.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 6309-6313Google Scholar, 18Majumder K. De Biasi M. Wang Z. Wible B.A. FEBS Lett. 1995; 361: 13-16Google Scholar, 19McCormack K. McCormack T. Tanouye M. Rudy B. Stuhmer W. FEBS Lett. 1995; 370: 32-36Google Scholar, 20Morales M.J. Castellino R.C. Crews A.L. Rasmusson R.L. Strauss H.C. J. Biol. Chem. 1995; 270: 6272-6277Google Scholar). Kvβ2, on the other hand, binds to α-subunits, such as Kv1.2 (or RCK5) (21Parcej D.N. Scott V.E. Dolly J.O. Biochemistry. 1992; 31: 11084-11088Google Scholar). However, it has little effect on inactivation of α-subunits such as Kv1.2 (RCK5) (7Scott 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: 1637-1641Google Scholar, 8Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Google Scholar, 22Scott V.E.S. Parcej D.N. Keen J.N. Findlay J.B.C. Dolly J.O. J. Biol. Chem. 1990; 265: 20094-20097Google Scholar, 23Nakahira K. Shi G. Rhodes K.J. Trimmer J.S. J. Biol. Chem. 1996; 271: 7084-7089Google Scholar). Recent data have shown that Kvβ2 is capable of increasing the surface expression of certain K+ channels in transfected cells (24Shi G. Nakahira K. Hammond S. Rhodes K. Schechter L. Trimmer J. Neuron. 1996; 16: 843-852Google Scholar). Biochemical evidence has indicated that there are more than one β-subunit present in each K+ channel complex (21Parcej D.N. Scott V.E. Dolly J.O. Biochemistry. 1992; 31: 11084-11088Google Scholar). Given that cloned β-subunits have different modulatory effects on α-subunits, it would be interesting to test whether different β-subunits can interact with each other, which could be an important mechanism to increase the diversity of potassium currents. To test this hypothesis, we have used the yeast two-hybrid system to study the interaction specificity of Kvβ2 with various α- and β-subunits. The functional consequences of heteromeric α-β and β-β interactions were evaluated by electrophysiological analyses. Plasmid vector construction was performed according to standard recombinant DNA techniques (26Sambrook J. Fristsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The vectors that express partial cDNA fragments were constructed by a high fidelity polymerase chain reaction cloning strategy according to the procedures described by Li et al.(27Li M. Jan Y.N. Jan L.Y. Science. 1992; 257: 1225-1230Google Scholar). The oligonucleotides used are listed in Table I. In yeast, the expression of different fusion proteins of α-subunits, Kvβ1, and Kvβ2 was carried out by inserting the corresponding cDNA fragments into the SmaI/NotI,SalI/NotI, or BglII/NotI sites of pPC97 and pPC86 vectors (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 29Xu J. Yu W. Jan Y.N. Jan L.Y. Li M. J. Biol. Chem. 1995; 270: 24761-24768Google Scholar, 30Fields S. Song O. Nature. 1989; 340: 245-246Google Scholar, 31Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Google Scholar). Construction of tagged Kvβ1, Kvβ2, and Kvβ2 mutants was carried out by fusing the coding fragment with a peptide which represents a heart muscle kinase recognition sequence and the 12CA5 monoclonal epitope (PYDVPDYASL), at the end of the coding sequences before the stop codon (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar). Transient expression and immunodetection of potassium channel subunits were performed according to our published protocol (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 51Li X. Xu J. Li M. J. Biol. Chem. 1997; 272: 705-708Google Scholar).Table ISequences of oligonucleotide primersOligonucleotidesSequencesaThe underlined sequence represents coding sequence or complementary to coding sequence of the indicated gene.GeneAmino acid positionML1005:GCACCATGGAGTCGACAATGTATCCGGAATCAACCACGKvβ21ML1006:CAGGCGGCCGCTTAGGATCTATAGTCCTTTTTGCKvβ2367ML1015:CAGGTCGACTGAATTCTACAGGAATCTGGGCKvβ239ML1021:CAGGAATTCAAGATCTCAATGCAAGTCTCCATAGCKvβ11ML1022:CAGAGATCTCATATAGGAATCTTGGGKvβ173ML1023:CAGAGATCTTGGTGATTCTGGGGAGCKvβ1131ML1024:CAGAGATCTACAGCAACACCCCCATGGKvβ1196ML1025:CAGGCGGCCGCTAATATTTTCCTGAAATAATTCCKvβ1289ML1026:CAGGCGGCCGCTACAGGCACCATGCCACAGCKvβ1346ML1027:CAGGCGGCCGCTAGCCTTAGGATCTATAGTCCKvβ1401ML1031:CAGGAATTCGAAGCAATGCAAGTCTCCATAGCCKvβ11ML1032:CAGGAATTCGGATCTATAGTCCTTTTTGCKvβ1401ML1044:CAGGAATTCGGCCCCATGTATCCGGAATCAACCKvβ21ML1045:CAGGAATTCGGATCTATAGTCCTTTTTGCKvβ2367ML1069:CAGGAATTCGGCCCCATGTACAGGAATCTGGGCAAATCKvβ239ML1079:CAGGAATTCGCCCTCGTTCCTCAGGKvβ2316a The underlined sequence represents coding sequence or complementary to coding sequence of the indicated gene. Open table in a new tab The procedures were performed according to our published protocol (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 29Xu J. Yu W. Jan Y.N. Jan L.Y. Li M. J. Biol. Chem. 1995; 270: 24761-24768Google Scholar) using HF7c yeast strain (MATα ura3–52 his-200 ade 2–101 lys2–801 trp1–901 leu2–3, 112 gal4–542 gal80–538 LYS2::GAL1 UAS -GAL1 TATA -HIS3 URA3::GAL4 17mer(x3) -Cycl TATA -lacZ) as host cells (25Feilotter H.E. Hannon G.J. Ruddell C.J. Beach D. Nucleic Acids Res. 1994; 22: 1502-1503Google Scholar). Whole-cell voltage clamp recordings were carried out according to the published protocol (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar,33Hamill O.P. Marty A. Nehre E. Sakmann B. Sigworth F.J. Pfluegers Arch. 1981; 391: 85-100Google Scholar). The liquid junction potential was calculated to be 7.2 mV using JPCalc software (34Barry P.H. J. Neurosci. Methods. 1994; 51: 107-116Google Scholar) and corrected from the holding potential. Typically, the cell was held at −87 mV, and the holding voltage was then jumped from this potential up to a test potential of +53 mV in 10-mV increments for 300 ms. Current data were filtered at 1 kHz, digitized at 100-μs intervals. Data analysis was done using clampfit software (pCLAMP6, Axon Instrument, Foster City, CA). A standard formula to compare two proportions was used to determine the statistical significance of different pairs of sample sets (35Pagano M. Gauvreau K. Principles of Biostatistics. Duxbury Press, Belmont, CA1993Google Scholar). We have used τ = 64 ms as the cutoff to separate populations with or without fast inactivation. In this analysis, the one-sided z test statistics were calculated using the following formula z = θ1-θ2 /{θp(1-θp)[1/n 1+ (1/n 2)]}0.5, where θ1 and θ2 are sample proportions that showed fast inactivation of each given group, θp is the weighted average of the sample proportions, andn 1 and n 2 are sample sizes. The interaction between α-subunits and Kvβ1 has been studied in more detail. In particular, the α-β complex is assembled, at least in part, by the association of the conserved core regions in Kvβ1 with NABKv1 of α-subunits, a critical assembly motif located in the hydrophilic NH2-terminal domains (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 29Xu J. Yu W. Jan Y.N. Jan L.Y. Li M. J. Biol. Chem. 1995; 270: 24761-24768Google Scholar, 36Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Google Scholar). The formation of an α-β complex presumably recruits the inactivation particle of Kvβ1 close to the "receptor" site, thereby either accelerating the rate of inactivation of α-subunits of Kv1.4 (RCK4) and ShB (or H4) or inducing inactivation of compatible α-subunits which lack intrinsic fast inactivation, such as ShBΔ(6–46) (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 36Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Google Scholar). Because the formation of the α-Kvβ1 complexes is subfamily-specific,i.e. Kvβ1 binds only to the NH2-terminal domains of Kv1 α-subunits (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar), this allows Kvβ1 to selectively modulate a subset of α-subunits. To investigate whether Kvβ2 alters the electrophysiological properties of α-subunits in transfected mammalian cells, we have constructed two plasmids that express either Kvβ1 or Kvβ2 with the 12CA5 monoclonal antibody tag fused at the COOH terminus of each coding sequence (see "Materials and Methods"). Both plasmids use the Kvβ1 5′-untranslated sequence. Thus, the two expression vectors are identical except for the amino acid coding sequence. Experiments utilizing these constructs permit better comparison of Kvβ1 and Kvβ2 expression and their ability to modulate α-subunits. By transient transfection in COS cells, we functionally expressed ShBΔ(6–46), a mutated ShB potassium channel that lacks the inactivation gate (37Hoshi T. Zagotta W.N. Aldrich R.W. Science. 1990; 250: 533-538Google Scholar, 38Zagotta W.N. Hoshi T. Aldrich R.W. Science. 1990; 250: 568-571Google Scholar). The Kvβ2 effects on this α-subunit were studied by whole-cell voltage clamp recording. Fig.1 A shows a series of traces obtained by stepping up from a holding potential of −87 mV to a final test potential of +33 mV in 20-mV increments. The recorded cells were transfected with ShBΔ(6–46) alone (upper panel), ShBΔ(6–46) in the presence of either Kvβ1 (middle panel), or Kvβ2 (bottom panel). In contrast to the ShBΔ(6–46) + Kvβ1 cotransfection in which we observed the Kvβ1-mediated inactivation (Fig. 1 A, middle panel), expression of ShBΔ(6–46) in the presence of Kvβ2 resulted in no detectable changes of the fast inactivation properties (Fig. 1 A, bottom panel). When traces were averaged within the group of ShBΔ(6–46) + Kvβ2 (n = 12) or ShBΔ(6–46) alone (n = 17), we observed little variations of inactivation properties between these two groups of recorded cells (Fig.1 B). To examine the protein expression of Kvβ2 in the experiments, total cell lysates from the transfected cells were separated by SDS-polyacrylamide gel electrophoresis. The expression of Kvβ1 and Kvβ2 was detected by immunoblot using the 12CA5 monoclonal antibody (mAb12CA5). Indeed, Kvβ2 was found to express in the transfected COS cells and exhibited higher expression as compared with that of Kvβ1 (Fig. 1 C, lanes 1 and 2). The higher expression of Kvβ2 has been reproducible in multiple transfection experiments. 1M. Bezanilla, J. Xu, and M. Li, unpublished results. The strong mAb12CA5 binding signal indicates that the failure of Kvβ2 to modulate N-type fast inactivation was not due to the lower protein expression of Kvβ2. Thus, Kvβ2 differs from Kvβ1 and by itself fails to induce the fast inactivation of the ShB α-subunit. Biochemical characterization supports direct physical interaction between Kvβ2 and Kv1.2 (RCK5) (7Scott 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: 1637-1641Google Scholar, 21Parcej D.N. Scott V.E. Dolly J.O. Biochemistry. 1992; 31: 11084-11088Google Scholar) as well as Kv1.4 (RCK4) (39Rhodes K.J. Keilbaugh S.A. Barrezueta N.X. Lopez K.L. Trimmer J.S. J. Neurosci. 1995; 15: 5360-5371Google Scholar). However, the existing results for Kvβ2 binding specificity and region(s) involved are not conclusive (Ref. 23Nakahira K. Shi G. Rhodes K.J. Trimmer J.S. J. Biol. Chem. 1996; 271: 7084-7089Google Scholar, also see "Discussion"). In addition, there is no information on whether Kvβ2 binds to ShB. To test the association between Kvβ2 and Kv1 α-subunits, we expressed various regions of α-subunits and Kvβ2 in yeast and used the yeast two-hybrid system to study the potential interaction(s) (30Fields S. Song O. Nature. 1989; 340: 245-246Google Scholar, 31Chevray P.M. Nathans D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5789-5793Google Scholar). In the case of Kvβ1, it has been found that the NH2-terminal domains of the Kv1 α-subunits are involved in the α-Kvβ1 interaction (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 36Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Google Scholar). Because Kvβ1 and Kvβ2 share considerable sequence homology, we first tested the potential interaction of Kvβ2 with the cytoplasmic regions of the Kv1.4, an α-subunit which has been found to interact with Kvβ2 (39Rhodes K.J. Keilbaugh S.A. Barrezueta N.X. Lopez K.L. Trimmer J.S. J. Neurosci. 1995; 15: 5360-5371Google Scholar). The truncated cytoplasmic fragments, i.e. the NH2-terminal domain (aa 2The abbreviation used is: aa, amino acids. 1–306) and COOH-terminal domain (aa 566–651), were expressed individually with Kvβ2 as GAL4 fusion proteins. If Kvβ2 interacts with one or both truncated Kv1.4 fragments, the resultant interaction(s) should confer the ability of the yeast transformants to grow on synthetic medium lacking histidine. Fig.2 A shows that when Kvβ2 was expressed alone either as a fusion protein of the GAL4 DNA binding domain (GAL4-DB) or that of the GAL4 transcription activation domain (GAL4-TA), the yeast transformants grew on double selection medium supplemented with histidine, indicating that they carry both plasmids (Fig.2 A, numbers 1 and 2, middle left panel). When the same number of transformants were tested to grow on the triple selection medium lacking histidine, they showed no growth (Fig. 2 A, numbers 1 and 2, lower left panel). This indicates that Kvβ2 itself does not exert any endogenous activity that permits the yeast transformants to grow on the selection medium. By contrast, the coexpression of Kvβ2 and the NH2-terminal domain (Fig. 2 A, number 3), not the COOH-terminal domain (Fig. 2 A, number 4) of Kv1.4, resulted in growth on the selection medium lacking histidine. Consistent results have been obtained using a β-galactosidase assay (data not shown). Thus, similar to Kvβ1, Kvβ2 interacts with the NH2-terminal domain of the Kv1.4 α-subunit. The ability of Kvβ2 to interact with the NH2-terminal domain of Kv1.4 suggests that the resultant association may be essential for Kvβ2 to interact with α-subunits, as seen in biochemical copurification. In the case of Kvβ1, its subfamily-specific association with the NH2-terminal domains of Kv1 α-subunits has been shown to be essential for the Kvβ1-mediated inactivation (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 36Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Google Scholar). Coimmunoprecipitation of K+ channel polypeptides in rat brain has indicated that Kvβ2 interacts with Kv1.2 and Kv1.4, but not Kv2.1 (24Shi G. Nakahira K. Hammond S. Rhodes K. Schechter L. Trimmer J. Neuron. 1996; 16: 843-852Google Scholar, 39Rhodes K.J. Keilbaugh S.A. Barrezueta N.X. Lopez K.L. Trimmer J.S. J. Neurosci. 1995; 15: 5360-5371Google Scholar). To further test the specificity of the Kvβ2, pairwise combinations of Kvβ2 and the NH2-terminal domains of eight different α-subunits were analyzed with the yeast two-hybrid system. The eight α-subunits included were: Shaker B (40Kamb A. Iverson L.E. Tanouye M.A. Cell. 1987; 50: 405-413Google Scholar, 41Tempel B.L. Papazian D.M. Schwarz T.L. Jan Y.N. Jan L.Y. Science. 1987; 237: 770-775Google Scholar, 42Pongs O. Kecskemethy N. Muller R. Krah-Jentgens I. Baumann A. Kiltz H.H. Canal I. Llamazares S. Ferrus A. EMBO J. 1988; 7: 1087-1096Google Scholar), Shabll, Shaw2, and Shal2 from Drosophila (9Butler A. Wei A. Salkoff L. Nucleic Acids Res. 1990; 18: 2173-2174Google Scholar); Kv1.4 (or RCK4) (43Stuhmer W. Ruppersberg J.P. Schroter K.H. Sakmann B. Stocker M. Giese K.P. Perschke A. Baumann A. Pongs O. EMBO J. 1989; 8: 3235-3244Google Scholar), Kv2.1 (or DRK1) (44Frech G.C. VanDongen A.M. Schuster G. Brown A.M. Joho R.H. Nature. 1989; 340: 642-645Google Scholar), Kv3.1 (or NGK2b) (45Yokoyama S. Imoto K. Kawamura T. Higashida H. Iwabe N. Miyata T. Numa S. FEBS Lett. 1989; 259: 37-42Google Scholar), and Kv4.2 (or rShal1) (46Baldwin T.J. Tsaur M.L. Lopez G.A. Jan Y.N. Jan L.Y. Neuron. 1991; 7: 471-483Google Scholar, 47Roberds S.L. Tamkun M.M. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1798-1802Google Scholar) from rat. These genes belong to the four major subfamilies, one fly gene and one rat gene for each subfamily. Among the selected NH2-terminal domains, Kvβ2 interacts only with the NH2-terminal domains of ShB and Kv1.4 (Fig.3 B, numbers 1 and 5), both of which belong to the Kv1 subfamily. Furthermore, the Kvβ2 interacting site was mapped to aa 174–306 within the NH2-terminal domain of Kv1.4 (data not shown), which coincides precisely with the domain that interacts with Kvβ1 (28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar, 36Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Google Scholar). Thus, both Kvβ1 and Kvβ2 interact subfamily-specifically with the Kv1 α-subunits and share the same binding site on the α-subunits. Based on the hydrodynamic estimates, the α-dendrotoxin acceptor (or Kv1.2) complex contains more than one Kvβ2 subunit per complex (21Parcej D.N. Scott V.E. Dolly J.O. Biochemistry. 1992; 31: 11084-11088Google Scholar). It is not clear, however, whether Kvβ2 can form an oligomeric complex in the absence of α-subunits. Additionally, since expression patterns of Kvβ1 and Kvβ2 overlapin vivo (8Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Google Scholar, 22Scott V.E.S. Parcej D.N. Keen J.N. Findlay J.B.C. Dolly J.O. J. Biol. Chem. 1990; 265: 20094-20097Google Scholar, 39Rhodes K.J. Keilbaugh S.A. Barrezueta N.X. Lopez K.L. Trimmer J.S. J. Neurosci. 1995; 15: 5360-5371Google Scholar, 52Rhodes K.J. Monaghan M.M. Barrezueta N.X. Nawoschik S. Bekele-Akcuri Z. Matos M.F. Nakahira K. Schechter L.E. Trimmer J.S. J. Neurosci. 1996; 16: 4846-4860Google Scholar), it would be interesting to examine whether different β-subunits can interact with each other to form heteromultimers. Fig. 3 shows that Kvβ2 can indeed associate to form homomultimers as the yeast transformants grow in the selection medium lacking histidine (Fig. 3, number 3). The known potassium channels in yeast have distinctive topology and belong to a subclass different from the Shaker-like potassium channels (48Ketchum K.A. Joiner W.J. Sellers A.J. Kaczmarek L.K. Goldstein S.A.N. Nature. 1995; 376: 690-695Google Scholar). Therefore, the above result supports that Kvβ2 is capable of interacting with itself in the absence of α-subunits. Both Kvβ1 and Kvβ2 are expressed in rat brain with overlapping expression patterns (8Rettig J. Heinemann S.H. Wunder F. Lorra C. Parcej D.N. Dolly J.O. Pongs O. Nature. 1994; 369: 289-294Google Scholar, 22Scott V.E.S. Parcej D.N. Keen J.N. Findlay J.B.C. Dolly J.O. J. Biol. Chem. 1990; 265: 20094-20097Google Scholar, 39Rhodes K.J. Keilbaugh S.A. Barrezueta N.X. Lopez K.L. Trimmer J.S. J. Neurosci. 1995; 15: 5360-5371Google Scholar). Because Kvβ2 forms multimers in the absence of α-subunits (Fig. 3, number 3) and has considerable overall sequence homology (73%) to Kvβ1, we tested the possible interaction between Kvβ1 and Kvβ2 and found that Kvβ1 and Kvβ2 can also interact (Fig. 3, number 4). This implies that Kvβ1 and Kvβ2 can form heteromultimers in the absence of the pore-forming α-subunits. Both Kvβ2 and Kvβ1 interact with the Kv1 α-subunits by recognizing the same region in the Kv1 α-subunits (Fig. 2 and Ref. 28Yu W.F. Xu J. Li M. Neuron. 1996; 16: 441-453Google Scholar). Additionally, Kvβ2 interacts with itself and/or Kvβ1 to form homo- and/or heteromultimers (Fig. 3). Because Kvβ1, not Kvβ2, induces the fast inactivation of the Kv1 α-subunits that lack fast inactivation (Fig.1), these data suggest that one potential function of Kvβ2 would be to alter the efficacy of the Kvβ1-mediated inactivation. One predicted outcome would be that Kvβ2 weakens the ability of Kvβ1 to inactivate, as Kvβ2 may compete with Kvβ1 for the binding site on α-subunits and/or associate with Kvβ1 to form Kvβ1-Kvβ2 heteromultimers containing fewer inactivation particles. One experiment to test this hypothesis would be to coexpress Kvβ1 and a compatible α-subunit in the presence or absence of Kvβ2 and ask whether Kvβ2 alters the ability of Kvβ1 to inactivate. We cotransfected COS cells with noninactivating ShBΔ(6–46) and Kvβ1 in a 1:6 plasmid ratio of α/Kvβ1. Fig. 4 Ashows three representative traces, one from each group, that were superimposed and normalized. These traces were recorded by stepping up the holding potential from −87 mV to a test potential of +13 mV for a duration of 300 ms. ShBΔ(6–46) alone produced a trace with fast activating kinetics lacking N-type fast inactivation. When Kvβ1 was included in the transfection, we observed a majority of transfected cells that show the Kvβ1-mediated fast inactivation (Fig.4 A). If, however, both Kvβ1 and Kvβ2 were included in a plasmid ratio of α/Kvβ1/Kvβ2 of 1:6:5, much fewer transfected cells showed fast interaction induced by Kvβ1 (see below and Fig.4 B). The lack of fast inactivation by Kvβ1 in the presence of Kvβ2 in the example shown in Fig. 4 A could have resulted from variable transfection rates of the three plasmids. To address this, we recorded 17 Shaker-positive cells for the ShBΔ(6–46) transfection, 40 cells for the ShBΔ(6–46) + Kvβ1 transfection, 44 cells for the ShBΔ(6–46) + Kvβ1 + Kvβ2 transfection. Since recorded cells were selected solely based on the presence of Shaker current and the expression of CD4 antigen that was cotransfected in all experiments, the percentage of recorded cells in a given transfection shown, fast and/or slow inactivation can then be determined. Among the recorded traces, some exhibited both fast and slow inactivation, the others showed only slow inactivation. The traces were fit by a double exponential function, and the resultant inactivation constants were plotted against the cell number in percentage (Fig. 4 B). Fig. 4 B shows plots using one inactivation constant per recorded cell, i.e. if the inactivation consists of two (fast and slow) components, only the fast inactivation constant was plotted. For the ShBΔ(6–46) transfection, we observed that all recorded cells lacked the fast inactivation and gave a slow inactivation constant (τ2) larger tha
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