Complex Subunit Assembly of Neuronal Voltage-gated K+Channels

Neurons require specific patterns of K+ channel subunit expression as well as the precise coassembly of channel subunits into heterotetrameric structures for proper integration and transmission of electrical signals. In vivo subunit coassembly was investigated by studying the pharmacological profile, distribution, and subunit composition of voltage-gated Shaker family K+(Kv1) channels in rat cerebellum that are labeled by125I-margatoxin (125I-MgTX;K d, 0.08 pm). High-resolution receptor autoradiography showed spatial receptor expression mainly in basket cell terminals (52% of all cerebellar sites) and the molecular layer (39% of sites). Sequence-directed antibodies indicated overlapping expression of Kv1.1 and Kv1.2 in basket cell terminals, whereas the molecular layer expressed Kv1.1, Kv1.2, Kv1.3, and Kv1.6 proteins. Immunoprecipitation experiments revealed that all 125I-MgTX receptors contain at least one Kv1.2 subunit and that 83% of these receptors are heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, 33% of these Kv1.1/Kv1.2-containing receptors possess either an additional Kv1.3 or Kv1.6 subunit. Only a minority of the 125I-MgTX receptors (<20%) seem to be homotetrameric Kv1.2 channels. Heterologous coexpression of Kv1.1 and Kv1.2 subunits in COS-1 cells leads to the formation of a complex that combines the pharmacological profile of both parent subunits, reconstituting the native MgTX receptor phenotype. Subunit assembly provides the structural basis for toxin binding pharmacology and can lead to the association of as many as three distinct channel subunits to form functional K+channels in vivo. Neurons require specific patterns of K+ channel subunit expression as well as the precise coassembly of channel subunits into heterotetrameric structures for proper integration and transmission of electrical signals. In vivo subunit coassembly was investigated by studying the pharmacological profile, distribution, and subunit composition of voltage-gated Shaker family K+(Kv1) channels in rat cerebellum that are labeled by125I-margatoxin (125I-MgTX;K d, 0.08 pm). High-resolution receptor autoradiography showed spatial receptor expression mainly in basket cell terminals (52% of all cerebellar sites) and the molecular layer (39% of sites). Sequence-directed antibodies indicated overlapping expression of Kv1.1 and Kv1.2 in basket cell terminals, whereas the molecular layer expressed Kv1.1, Kv1.2, Kv1.3, and Kv1.6 proteins. Immunoprecipitation experiments revealed that all 125I-MgTX receptors contain at least one Kv1.2 subunit and that 83% of these receptors are heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, 33% of these Kv1.1/Kv1.2-containing receptors possess either an additional Kv1.3 or Kv1.6 subunit. Only a minority of the 125I-MgTX receptors (<20%) seem to be homotetrameric Kv1.2 channels. Heterologous coexpression of Kv1.1 and Kv1.2 subunits in COS-1 cells leads to the formation of a complex that combines the pharmacological profile of both parent subunits, reconstituting the native MgTX receptor phenotype. Subunit assembly provides the structural basis for toxin binding pharmacology and can lead to the association of as many as three distinct channel subunits to form functional K+channels in vivo. Voltage-gated K+(Kv) 1The abbreviations used are: Kv, voltage-gated K+; MgTX, margatoxin; DTX, α-dendrotoxin; kb, kilobase. 1The abbreviations used are: Kv, voltage-gated K+; MgTX, margatoxin; DTX, α-dendrotoxin; kb, kilobase. channels serve an important function in regulating the degree of neuronal excitability. This class of channels is involved in controlling both the length of action potentials and the frequency of repetitive firing. The diversity of firing patterns displayed by individual neurons in the central nervous system is reflected by the expression of a wide variety of voltage-gated K+ channels that differ in their gating, pharmacology, and single-channel properties (1Chandy K.G. Gutman G.A. North R.A. Handbook of Receptors and Channels: Ligand and Voltage-gated Ion Channels. CRC Press, Inc., Boca Raton, FL1995: 1-71Google Scholar, 2Wei A. Jegla T. Salkoff L. Neuropharmacology. 1996; 35: 805-829Crossref PubMed Scopus (220) Google Scholar). Molecular cloning of voltage-gated K+ channels has revealed the existence of multiple members of at least eight families (2Wei A. Jegla T. Salkoff L. Neuropharmacology. 1996; 35: 805-829Crossref PubMed Scopus (220) Google Scholar, 3Pongs O. Physiol. Rev. 1992; 72: S69-S88Crossref PubMed Google Scholar). Of all the Kv channel families, the Kv1 (Shaker) class (Kv1.1–1.6) has been the most studied, due to the discovery of high-affinity blockers of these channels in the venom of snakes, scorpions, and marine organisms (4Garcia M.L. Galvez A. Garcia Calvo M. King V.F. Vazquez J. Kaczorowski G.J. J. Bioenerg. Biomembr. 1991; 23: 615-646Crossref PubMed Scopus (163) Google Scholar,5Harvey A.L. Vatanpour H. Pinkasfeld S. Vita C. Menez A. Martin Eauclaire M.F. Toxicon. 1995; 33: 425-436Crossref PubMed Scopus (29) Google Scholar). Some of these K+ channel subunits have been reported to assemble into heterotetrameric channels with distinct biophysical and pharmacological properties when expressed in vitro (6Ruppersberg J.P. Schroter K.H. Sakmann B. Stocker M. Sewing S. Pongs O. Nature. 1990; 345: 535-537Crossref PubMed Scopus (341) Google Scholar, 7Isacoff E.Y. Jan Y.N. Jan L.Y. Nature. 1990; 345: 530-534Crossref PubMed Scopus (379) Google Scholar, 8Christie M.J. North R.A. Osborne P.B. Douglass J. Adelman J.P. Neuron. 1990; 4: 405-411Abstract Full Text PDF PubMed Scopus (210) Google Scholar). In native tissue, with the combination of in situhybridization and immunocytochemical techniques, it has been possible to show that individual voltage-gated K+ channels are expressed in specific cells, occasionally even within a single neuron. In addition, these channels can be targeted to distinct subcellular compartments (9Wang H. Kunkel D.D. Martin T.M. Schwartzkroin P.A. Tempel B.L. Nature. 1993; 365: 75-79Crossref PubMed Scopus (519) Google Scholar, 10Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (292) Google Scholar, 11Wang H. Kunkel D.D. Schwartzkroin P.A. Tempel B.L. J. Neurosci. 1994; 14: 4588-4599Crossref PubMed Google Scholar, 12Sheng M. Tsaur M.L. Jan Y.N. Jan L.Y. J. Neurosci. 1994; 14: 2408-2417Crossref PubMed Google Scholar). Moreover, the assembly of distinct Kvchannel subunits (e.g. Kv1.1/Kv1.2 (9Wang H. Kunkel D.D. Martin T.M. Schwartzkroin P.A. Tempel B.L. Nature. 1993; 365: 75-79Crossref PubMed Scopus (519) Google Scholar, 11Wang H. Kunkel D.D. Schwartzkroin P.A. Tempel B.L. J. Neurosci. 1994; 14: 4588-4599Crossref PubMed Google Scholar) or Kv1.2/Kv1.4 (10Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (292) Google Scholar, 12Sheng M. Tsaur M.L. Jan Y.N. Jan L.Y. J. Neurosci. 1994; 14: 2408-2417Crossref PubMed Google Scholar)) into functional channel complexes in native brain tissue has been implied. An alternative approach to demonstrate the existence of heterotetrameric K+ channels is to use specific K+ channel ligands and define the subunit composition of the receptor. For instance, Kv1.1 and Kv1.2, but not Kv1.3 and Kv1.4, are highly sensitive to DTX (13Grissmer S. Nguyen A.N. Aiyar J. Hanson D.C. Mather R.J. Gutman G.A. Karmilowicz M.J. Auperin D.D. Chandy K.G. Mol. Pharmacol. 1994; 45: 1227-1234PubMed Google Scholar). In contrast, MgTX binds with high affinity to both Kv1.2 and Kv1.3 but displays much lower affinity for all other Kv channels (14Leonard R.J. Garcia M.L. Slaughter R.S. Reuben J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10094-10098Crossref PubMed Scopus (265) Google Scholar, 15Knaus H.G. Koch R.O. Eberhart A. Kaczorowski G.J. Garcia M.L. Slaughter R.S. Biochemistry. 1995; 34: 13627-13634Crossref PubMed Scopus (65) Google Scholar). Using125I-DTX as a marker, it has been shown that in bovine brain cortex, Kv1.2 is present in all DTX-sensitive K+ channels, whereas Kv1.1 is only present in about half of these, and that Kv1.4 and Kv1.6 subunits have a minor contribution (16Parcej D.N. Scott V.E. Dolly J.O. Biochemistry. 1992; 31: 11084-11088Crossref PubMed Scopus (120) Google Scholar, 17Scott V.E. Muniz Z.M. Sewing S. Lichtinghagen R. Parcej D.N. Pongs O. Dolly J.O. Biochemistry. 1994; 33: 1617-1623Crossref PubMed Scopus (133) Google Scholar). These results have been confirmed and extended after immunoprecipitation and Western blotting using specific antibodies for Kv1 channels (18Shamotienka O.G. Parcej D.N. Dolly J.O. Biochemistry. 1997; 36: 8195-8201Crossref PubMed Scopus (121) Google Scholar). In this study, we have quantitatively defined the subunit composition of the MgTX receptor in rat cerebellum using a combination of high-resolution autoradiography, immunocytochemistry, and immunoprecipitation experiments. The results indicate that all MgTX receptors contain at least one Kv1.2 subunit, but that less than 20% are homotetrameric channels, with the remaining composed mostly of heterotetramers of Kv1.1 and Kv1.2 subunits. Moreover, coexpression of Kv1.1 and Kv1.2 subunits in vitro reproduced the pharmacological phenotype of the cerebellum MgTX receptor. MgTX was expressed in Escherichia colias part of a fusion protein, purified (19Garcia Calvo M. Leonard R.J. Novick J. Stevens S.P. Schmalhofer W. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1993; 268: 18866-18874Abstract Full Text PDF PubMed Google Scholar), and radiolabeled as described previously (15Knaus H.G. Koch R.O. Eberhart A. Kaczorowski G.J. Garcia M.L. Slaughter R.S. Biochemistry. 1995; 34: 13627-13634Crossref PubMed Scopus (65) Google Scholar). DTX was obtained from Calbiochem. Polyethylenimine, bovine serum albumin, and ovalbumin were from Sigma, and goat serum was from Biological Industries (Kibbutz Beth Haemek, Israel). Recombinant N-glycosidase F was obtained from Genzyme. LipofectAMINE® was purchased from Life Technologies, Inc. FMOC (9-fluoroenylmethoxycarbonyl) lysine core solid-phase peptide support was from NovaBiochem (Läufelfingen, Switzerland). Hyperfilm®-βmax was from Amersham Corp., and the photo dip emulsion (NTB2®) was from Kodak. Kv1 fusion proteins were kindly provided by Dr. Olaf Pongs (Hamburg, Germany). Rat cerebellar synaptic plasma membrane vesicles were prepared as described previously (20Vazquez J. Feigenbaum P. King V.F. Kaczorowski G.J. Garcia M.L. J. Biol. Chem. 1990; 265: 15564-15571Abstract Full Text PDF PubMed Google Scholar). All technical details of the binding assay have been previously published (15Knaus H.G. Koch R.O. Eberhart A. Kaczorowski G.J. Garcia M.L. Slaughter R.S. Biochemistry. 1995; 34: 13627-13634Crossref PubMed Scopus (65) Google Scholar). Cerebellar MgTX receptors were solubilized for 30 min on ice with 2% digitonin in the presence of 500 mm KCl, and solubilized receptors were separated from particulate material as described previously (15Knaus H.G. Koch R.O. Eberhart A. Kaczorowski G.J. Garcia M.L. Slaughter R.S. Biochemistry. 1995; 34: 13627-13634Crossref PubMed Scopus (65) Google Scholar). Male Sprague-Dawley rats (250–300 g) were sacrificed by cervical dislocation, and their brains were rapidly removed and placed for 90 s in isopentane chilled to −40 °C. Thereafter, the brains were transferred for 30 min at −30 °C and stored in a sealed vial. 20-μm sections were cut on a cryostat microtome (Leitz, Germany) and thaw-mounted onto gelatin-coated slides. Slides were stored at −30 °C for up to 1 month. Sections were labeled in 20 mm Tris/HCl (pH 7.4) and 0.1% bovine serum albumin for 3 h at 22 °C at a saturating125I-MgTX concentration (5–12 pm). Nonspecific binding was determined in a series of adjacent sections by inclusion of 2 nm MgTX. Sections were then rinsed twice for 30 min in ice-cold 20 mm Tris/HCl (pH 7.4) and 150 mmNaCl, dipped in chilled distilled water, and dried rapidly in a cold stream of air. Thereafter, the sections were dipped (right after a wash with ice-cold double distilled water) in photo emulsion and stored for 3–7 days at 4 °C. Quantification of staining was done using a RGB camera system (DEI-470; Optotronics Engineering, Goleta, CA) and the MetaMorph software package (Visitron, Munich, Germany). Rabbit polyclonal sera were raised against unique carboxyl-terminal regions of the Shaker type K+ channels Kv1.1–Kv1.6 found in rat brain. Peptides were synthesized on a lysine core linked to a solid-phase peptide synthesis support. The sequences of the synthetic peptides used and their locations within the primary amino acid sequences are EEDMNNSIAHYRQANIRT (antiKv1.1(458–475)), QEGVNNSNEDFREENLKTAN (anti-Kv1.2(461–480)), QHLSSSAEELRKARSNSTL (anti-Kv1.3(456–474)), SSLGDKSEYLEMEEGVKESL (anti-Kv1.4(605–624)), KAKSNVDLRRSLYALCLDTSR (anti-Kv1.5(578–598)), and RRSSYLPTPHRAYAEKRM (anti-Kv1.6(509–526)). For the anti-Kv1.1–Kv1.4 antibodies, the amino acid numbering refers to Ref. 21Stuhmer W. Ruppersberg J.P. Schroter K.H. Sakmann B. Stocker M. Giese K.P. Perschke A. Baumann A. Pongs O. EMBO. J. 1989; 8: 3235-3244Crossref PubMed Scopus (615) Google Scholar, whereas the recognition sequences of the anti-Kv1.5 and anti-Kv1.6 antibodies are numbered according to Refs. 22Swanson R. Marshall J. Smith J.S. Williams J.B. Moyle M.B. Folander J. C. C. Neuron. 1990; 4: Full Text PDF PubMed Scopus Google and A. Schroter K.H. Ruppersberg J.P. Stocker M. T. S. Pongs O. EMBO. J. 1990; PubMed Scopus (121) Google Scholar, For we to all antibodies the All antibodies were in serum to the of The fusion proteins and and S. J. Pongs O. Neuron. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar) were purified by against mm for and with 30 of The Western blotting and immunoprecipitation have been published previously H.G. C. Koch R.O. Eberhart A. Kaczorowski G.J. H. Pongs O. Garcia M.L. J. Neurosci. 1996; PubMed Google Scholar). The of tissue sections has been described previously H.G. C. Koch R.O. Eberhart A. Kaczorowski G.J. H. Pongs O. Garcia M.L. J. Neurosci. 1996; PubMed Google Scholar). Sections were for h at the indicated with either serum or or anti-Kv1.6 were after with goat and with In was either by the antibodies with of the peptide or by the primary was using from were purchased from The Kv1.1 and Kv1.2 were obtained from Dr. Olaf The the of the Kv1.2 was and after it was into the of the expression The of the was by The of the Kv1.1 was by using a and that an and in the The was into the of the The of the Kv1.1 was by COS-1 cells were and as described previously M. Schmalhofer P. H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). were as described previously M. Schmalhofer P. H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar) using of Kv1.1 3 of Kv1.2 or a combination were to with the for h at °C. of cells were for were in mm 150 mm KCl, and 2 and plasma were as described previously M. Schmalhofer P. H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). were in mm and 20 mm (pH in and stored at °C. In purified rat cerebellar synaptic plasma membrane 125I-MgTX a single class of receptor with an of 0.08 and a of the high affinity is due to a and min at 22 (15Knaus H.G. Koch R.O. Eberhart A. Kaczorowski G.J. Garcia M.L. Slaughter R.S. Biochemistry. 1995; 34: 13627-13634Crossref PubMed Scopus (65) Google Scholar), we receptor autoradiography to the of 125I-MgTX receptors in rat the toxin binding is to individual the is in a single of receptors the molecular and cell layer and staining is found the of the molecular to cell revealed that only basket cell terminals, but not cell are labeled by whereas an of staining is found the molecular layer 2 of indicated that of toxin binding are in basket cell terminals, whereas of are found in the molecular and less than of MgTX receptors are in the cell layer and the binding in rat cryostat sections of rat cerebellum were labeled with and sections were with and dipped in photo emulsion as described molecular cell of autoradiography that most 125I-MgTX binding to terminals, whereas only are in the molecular the cerebellar expression of the MgTX receptor with a defined Kv1 subunit, we raised a of specific antibodies against individual Kv1.1–Kv1.6 channels and used these in to channel The and of these antibodies were confirmed in Western against or fusion proteins the of Kv1.1, Kv1.2, Kv1.3, Kv1.4, and Kv1.6 In all unique and the was by inclusion of 1 In fusion proteins be by the Kv1 antibodies, whereas the presence of the peptide the not the properties of the antibodies in native tissue, we Western with cerebellar 3 labeled a whereas staining of a of and and a with an of all in with previously published M. Tsaur M.L. Jan Y.N. Jan L.Y. J. Neurosci. 1994; 14: 2408-2417Crossref PubMed Google Scholar, O.G. Parcej D.N. Dolly J.O. Biochemistry. 1997; 36: 8195-8201Crossref PubMed Scopus (121) Google Scholar, Lichtinghagen R. Sewing S. Pongs O. J. Neurosci. 1995; PubMed Scopus Google Scholar). a of whereas to with a in cerebellar this a in brain not to in cerebellar the that this was of expressed in cells not the of these antibodies for their Kv1 subunits, we determined the of these subunits in cerebellum and the to obtained in important to was the of Kv1.2 and Kv1.3 subunits, these channels a high affinity for 125I-MgTX (14Leonard R.J. Garcia M.L. Slaughter R.S. Reuben J.P. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10094-10098Crossref PubMed Scopus (265) Google Scholar, 15Knaus H.G. Koch R.O. Eberhart A. Kaczorowski G.J. Garcia M.L. Slaughter R.S. Biochemistry. 1995; 34: 13627-13634Crossref PubMed Scopus (65) Google Scholar) that to the of 125I-MgTX we the with the the cell with to Kv1 channel expression and Only Kv1.1 and Kv1.2 proteins were found to in the of basket cell that the and of cell not for Kv1.1 or were only and with of 125I-MgTX binding are in the cerebellar molecular layer and This was by whereas staining were for and and In addition, high of Kv1.3 were in this All antibodies only in the cerebellar cell with the Western blotting the revealed only expression in contrast, this staining in other of the brain as the or the not in with previously published (10Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (292) Google Scholar, Lichtinghagen R. Sewing S. Pongs O. J. Neurosci. 1995; PubMed Scopus Google Scholar). was not in the rat in with Western of Kv1.1, Kv1.2, Kv1.3, and Kv1.6 in rat cryostat sections were with antibodies against Kv1.1 Kv1.2 Kv1.3 and Kv1.6 molecular cell cell in to and in F the of MgTX receptors and the cerebellar Kv1 subunits, we for heterotetrameric channel For this we cerebellar receptors to immunoprecipitation experiments using antibodies against the individual Kv1 subunits All antibodies of Moreover, inclusion of the peptide the of by not receptors and the combination of other with not the of These results indicate that in all cerebellar MgTX Kv1.2 is an of receptors be whereas and anti-Kv1.6 and of the using of both and be that these proteins are into distinct channel complexes Kv1.3 Kv1.6 subunits are with Kv1.1, we investigated the of of 125I-MgTX receptors by either or anti-Kv1.6 in the presence of the by a of with of the whereas the combination In addition, a combination of and anti-Kv1.6 not the of these indicate that Kv1.2 is the subunit of all K+ channels in rat In of the Kv1.2 is with Kv1.1, whereas in of these either Kv1.3 or Kv1.6 to be an additional of the The remaining 20% of cerebellar MgTX receptors seem to be composed of homotetrameric Kv1.2 channels. the composition of the cerebellar MgTX receptor by we expressed Kv1.1, Kv1.2, and a combination of Kv1.1/Kv1.2 subunits in COS-1 cells, and binding of 125I-MgTX or was used to the pharmacological properties of the 125I-MgTX binds to Kv1.2 and Kv1.1/Kv1.2 with of 0.08 not a to that determined with cerebellar In contrast, specific 125I-MgTX binding was for homotetrameric Kv1.1 channels. to this was used this has affinity for both Kv1.1 and Kv1.2 channels (13Grissmer S. Nguyen A.N. Aiyar J. Hanson D.C. Mather R.J. Gutman G.A. Karmilowicz M.J. Auperin D.D. Chandy K.G. Mol. Pharmacol. 1994; 45: 1227-1234PubMed Google Scholar). binds to Kv1.1, Kv1.2, and Kv1.1/Kv1.2 COS-1 with a of not The pharmacology of binding to Kv1.1, Kv1.2, and cerebellar was investigated binding to homotetrameric Kv1.1 channels with a of whereas it displays affinity for homotetrameric Kv1.2 channels pm). in Kv1.1/Kv1.2 displays a of in with the determined with cerebellar pm). These that the pharmacological profile of Kv1.1/Kv1.2 is due to the association of both subunits in a receptor complex and that this pharmacology is to that found with cerebellar the that Kv1.1 and Kv1.2 subunits are in in a complex after expression in COS-1 cells, immunoprecipitation experiments with and were For Kv1.2 or receptors can be with but not whereas the profile is receptors from Kv1.1 channels are to immunoprecipitation with these both and are to either 125I-MgTX receptors from Kv1.1/Kv1.2 In these to whereas These that Kv1.1 and Kv1.2 subunits are in a receptor complex that the of the 125I-MgTX cerebellar receptor. The of Kv channel in rat brain and their to form functional heterotetrameric channel complexes are for the diversity of K+ channel in native of that Kv1 channel subunits form heterotetramers in vitro and in vivo. of Kv1 subunits in leads to the formation of heterotetrameric Kv channels with distinct properties when with of the channels (9Wang H. Kunkel D.D. Martin T.M. Schwartzkroin P.A. Tempel B.L. Nature. 1993; 365: 75-79Crossref PubMed Scopus (519) Google Scholar, 10Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (292) Google Scholar, 11Wang H. Kunkel D.D. Schwartzkroin P.A. Tempel B.L. J. Neurosci. 1994; 14: 4588-4599Crossref PubMed Google Scholar). Moreover, in situhybridization and immunocytochemical experiments have provided the existence of overlapping Kv channel but the of these is to coassembly of subunits into this have investigated the of heterotetrameric K+ channels in vivo by immunoprecipitation of Kv1 channel by with antibodies (9Wang H. Kunkel D.D. Martin T.M. Schwartzkroin P.A. Tempel B.L. Nature. 1993; 365: 75-79Crossref PubMed Scopus (519) Google Scholar, 10Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (292) Google Scholar). this heterotetrameric channel assembly of Kv1.2/Kv1.4 subunits in (10Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (292) Google Scholar) and Kv1.1/Kv1.2 subunits in basket cell terminals and regions (9Wang H. Kunkel D.D. Martin T.M. Schwartzkroin P.A. Tempel B.L. Nature. 1993; 365: 75-79Crossref PubMed Scopus (519) Google Scholar, 11Wang H. Kunkel D.D. Schwartzkroin P.A. Tempel B.L. J. Neurosci. 1994; 14: 4588-4599Crossref PubMed Google Scholar) has been M. Tsaur M.L. Jan Y.N. Jan L.Y. Neuron. 1992; Full Text PDF PubMed Scopus Google Scholar). have extended these and to the of of defined Kv1 in bovine (18Shamotienka O.G. Parcej D.N. Dolly J.O. Biochemistry. 1997; 36: 8195-8201Crossref PubMed Scopus (121) Google Scholar). An alternative to the of channel composition is to native channels with a high-affinity and their composition by In the present study, we on the composition of K+ channels in rat In cerebellar 125I-MgTX binds to a single class of receptors with high affinity and pharmacological that are not with binding to a single Kv1 Moreover, the of Kv1 in cerebellum is in Lichtinghagen R. Sewing S. Pongs O. J. Neurosci. 1995; PubMed Scopus Google and Muniz Z.M. Dolly J.O. 1993; PubMed Scopus Google this Kv1 subunits are expressed in this brain at as Kv1.1, Kv1.2, Kv1.3, and Kv1.2 and Kv1.3 are subunits, these homotetrameric channels high-affinity receptors for 125I-MgTX J.P. Garcia M.L. Stevens Leonard R.J. Koch R. Kaczorowski G.J. Slaughter R.S. Biochemistry. 1997; 36: PubMed Scopus Google Scholar). a combination of autoradiography, immunocytochemistry, and immunoprecipitation it has been possible to the composition of the 125I-MgTX receptor in These indicate that all receptors contain at least one Kv1.2 subunit and that are heterotetramers of Kv1.1 and In addition, of these Kv1.1/Kv1.2 channels contain an additional Kv1.3 or Kv1.6 subunit. is most that all receptors found in basket cell terminals are other Kv1 subunit can be to the at the molecular Kv1.2, and Kv1.1/Kv1.2 with either Kv1.3 or Kv1.6 seem to be The unique pharmacological properties of the MgTX receptor be mostly determined by the Kv1.1/Kv1.2 channels, these are the in This has been confirmed in coexpression experiments in Kv1.1 and Kv1.2 subunits were expressed in COS-1 these subunits to a unique MgTX receptor phenotype that is to that found in cerebellar heterotetrameric channel formation can in vitro and in vivo to the and to the diversity of K+ channels in the central nervous controlling subunit assembly a important in the electrical in neuron. these to be and for technical and toxin and are for support and