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• W2092509098 abstract "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 K. Luneau C.J. Antanavage J. Oliva C. Buhrow S.A. Bennett C. Stein R.B. Kaczmarek L.K. Neuron. 1990; 4: 929-939Abstract Full Text PDF PubMed Scopus (262) Google Scholar and 23Grupe A. Schroter K.H. Ruppersberg J.P. Stocker M. Drewes T. Beckh S. Pongs O. EMBO. J. 1990; 9: 1749-1756Crossref PubMed Scopus (121) Google Scholar, respectively. For simplicity, we subsequently refer to all antibodies without the corresponding numbering. All sequence-directed antibodies were diluted in nonimmune serum to keep the overall amount of IgG/tube approximately constant. The fusion proteins glutathioneS-transferase-Kv1.1 and β-galactosidase-Kv1.2, -Kv1.3, -Kv1.4, and -Kv1.6 (24Sewing S. Roeper J. Pongs O. Neuron. 1996; 16: 455-463Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) were purified by SDS-polyacrylamide gel electrophoresis, electroeluted, dialyzed against 50 mm NaHCO3 for 24 h, and reacted with 30 μCi of 125I-labeled Bolton-Hunter reagent. The respective Western blotting and immunoprecipitation protocols have been published previously (25Knaus H.G. Schwarzer C. Koch R.O. Eberhart A. Kaczorowski G.J. Glossmann H. Wunder F. Pongs O. Garcia M.L. Sperk G. J. Neurosci. 1996; 16: 955-963Crossref PubMed Google Scholar). The preparation of tissue sections has been described previously (25Knaus H.G. Schwarzer C. Koch R.O. Eberhart A. Kaczorowski G.J. Glossmann H. Wunder F. Pongs O. Garcia M.L. Sperk G. J. Neurosci. 1996; 16: 955-963Crossref PubMed Google Scholar). Sections were incubated for 48–96 h at the indicated dilutions with either crude serum (anti-Kv1.1, 1:5000; anti-Kv1.2, 1:2000) or affinity-purified anti-Kv1.3 (1:1500), anti-Kv1.4 (1:1000), or anti-Kv1.6 (1:1000). Immunoreaction products were visualized after coupling with rabbit peroxidase-antiperoxidase via goat anti-rabbit IgG and color reaction with 3,3′-diaminobenzidine. In control sections, nonspecific immunoreactivity was assessed either by preadsorbing the antibodies with 10 μm of the respective peptide or by incubation without the primary antibody. Polymerase chain reaction amplification was carried out using Pfu DNA polymerase from Stratagene. Restriction endonucleases were purchased from Promega. The Kv1.1 and Kv1.2 genes were obtained from Dr. Olaf Pongs. The 2.2-kbHindIII/XbaI fragment containing the open reading frame of the Kv1.2 gene was excised, and after generating blunt ends, it was subcloned into the SmaI site of the mammalian expression vector pCI-neo (Promega). The orientation of the insert was checked by restriction analysis. The open reading frame of the Kv1.1 gene was amplified by polymerase chain reaction using a forward (5′-AAGAATTCCGCCACCATGACGGTGATGTCTGGGGAG-3′) and reverse (5′-TTATTCTAGATTTAAACATCGGTCAGTAGCTTGCTC-3′) primer that introduced an EcoRI and XbaI site in the flanking region. The polymerase chain reaction fragment was subcloned into the corresponding sides of the pCI-neo vector. The integrity of the entire amplified Kv1.1 cDNA was verified by nucleotide sequencing. COS-1 cells were grown and maintained as described previously (26Hanner M. Schmalhofer W.A. Munujos P. Knaus H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2853-2858Crossref PubMed Scopus (82) Google Scholar). Transfections were carried out as described previously (26Hanner M. Schmalhofer W.A. Munujos P. Knaus H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2853-2858Crossref PubMed Scopus (82) Google Scholar) using 12 μg of Kv1.1 DNA, 3 μg of Kv1.2 DNA, or a combination thereof per T-225 flask being transfected. Cells were allowed to incubate with the transfection medium for 5 h under 10% CO2, 100% humidity, at 37 °C. After removal of transfection medium, cells were grown for 72 h. Cell pellets were resuspended in lysis buffer (5 mm EGTA/NH4OH, pH 10.6, 150 mm KCl, and 2 mmMgCl2), and plasma membranes were isolated as described previously (26Hanner M. Schmalhofer W.A. Munujos P. Knaus H.G. Kaczorowski G.J. Garcia M.L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2853-2858Crossref PubMed Scopus (82) Google Scholar). Membranes were resuspended in 100 mm NaCl and 20 mm Hepes-Tris (pH 7.4), quickly frozen in liquid N2, and stored at −80 °C. In purified rat cerebellar synaptic plasma membrane vesicles, 125I-MgTX labels a single class of receptor sites with an equilibrium dissociation constant of 0.08 pm and a maximum density of 0.9 pmol/mg protein (Fig.1). Given the very high affinity of125I-MgTX, which is due to a very slow and monophasic radioligand dissociation (t ½ = 126 min at 22 °C) (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 performed receptor autoradiography to investigate the distribution of 125I-MgTX receptors in rat cerebellum. At the macroscopic level, toxin binding is spatially restricted to two individual compartments: (a) the highest site density is detected in a single sheet of receptors located between the molecular and granule cell layer (Fig.2 A); and (b) strong autoradiographic staining is found throughout the entire thickness of the molecular layer. Higher magnification (after dye counterstaining to identify cell bodies) revealed that only basket cell terminals, but not Purkinje cell bodies, are heavily labeled by 125I-MgTX, whereas an intermediate density of autoradiographic staining is found throughout the entire molecular layer (Fig. 2 B). Counting of silver grains indicated that 52% of toxin binding sites are clustered in basket cell terminals, whereas 39% of sites are found in the molecular layer, and less than 10% of MgTX receptors are located in the granule cell layer and the folial white matter.Figure 2Autoradiographic localization of125I-MgTX binding sites in rat cerebellum. A, sagittal cryostat sections (15 μm) of rat cerebellum were labeled with 11 pm125I-MgTX, and slide-mounted sections were counterstained with cresyl violet and dipped in photo emulsion as described under “Experimental Procedures.”ML, molecular layer; GL, granule cell layer;PT, pinceau terminals. Bar, 200 μm.B, representative image of high-resolution125I-MgTX autoradiography (80-fold magnification). Note that most 125I-MgTX binding occurs to pinceau terminals, whereas only modest levels are observed in the molecular layer.Bar, 50 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To correlate the cerebellar expression pattern of the MgTX receptor with a defined Kv1 subunit, we raised a panel of specific antibodies against individual Kv1.1–Kv1.6 channels and used these in immunohistochemical studies to elucidate channel distribution. The specificity and reactivity of these antibodies were confirmed in Western blots against glutathioneS-transferase or β-galactosidase fusion proteins containing the carboxyl terminus of Kv1.1, Kv1.2, Kv1.3, Kv1.4, and Kv1.6 (Fig. 3,A–D). In all cases, each antibody exclusively recognized its unique antigen, and the immunostaining signal was blocked by inclusion of 1 μm antigenic peptide. In addition,125I-labeled fusion proteins could be immunoprecipitated by the respective Kv1 antibodies, whereas the presence of the antigenic peptide blocked the reaction (data not shown). To investigate the properties of the antibodies in native tissue, we carried out Western blots with cerebellar membranes (Fig. 3 E). Anti-Kv1.1 specifically labeled a polypeptide ofM r 78,000, whereas anti-Kv1.2 yielded very diffuse staining of a polypeptide of aboutM r 80,000, and anti-Kv1.3 faintly and diffusely stained a polypeptide with an apparentM r of 85,000, all in agreement with previously published data (12Sheng M. Tsaur M.L. Jan Y.N. Jan L.Y. J. Neurosci. 1994; 14: 2408-2417Crossref PubMed Google Scholar, 18Shamotienka O.G. Parcej D.N. Dolly J.O. Biochemistry. 1997; 36: 8195-8201Crossref PubMed Scopus (121) Google Scholar, 27Veh R.W. Lichtinghagen R. Sewing S. Wunder F. Grumbach I.M. Pongs O. Eur. J. Neurosci. 1995; 7: 2189-2205Crossref PubMed Scopus (286) Google Scholar). Anti-Kv1.6 stained a protein of M r 56,000, whereas anti-Kv1.4 failed to react specifically with a polypeptide in cerebellar membranes, although this antibody clearly recognized a polypeptide ofM r 95,000 in whole brain membranes (data not shown). Anti-Kv1.5 failed to give any signal in cerebellar membranes, despite the fact that this antiserum was clearly capable of immunostaining Kv1.5 expressed in HEK-293 cells (data not shown). Given the specificity of these antibodies for their respective Kv1 subunits, we determined the distribution of these subunits in cerebellum and compared the data to those obtained in autoradiographies. Particularly important to us was the distribution of Kv1.2 and Kv1.3 subunits, because these two channels exhibit 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 makes them likely candidates to match the observed autoradiographic distribution of 125I-MgTX binding. First, we inspected the region with the highest density of125I-MgTX receptors, the Purkinje cell layer, with respect to Kv1 channel expression (Fig.4, A and B). Only Kv1.1 and Kv1.2 proteins were found to reside in the terminal field of basket cell axons that wrap around the base and initial axon segments of Purkinje cells; Purkinje cell somata did not express any immunoreactivity for Kv1.1 or were only very faintly and irregularly stained with anti-Kv1.2. As mentioned above, significant levels of 125I-MgTX binding are also observed in the cerebellar molecular layer (Fig. 2,A and B). This region was intensely immunostained by anti-Kv1.1, whereas moderate staining levels were observed for anti-Kv1.2 and anti-Kv1.6, respectively (Fig. 4, A, B, E, and F). In addition, reasonably high levels of Kv1.3 immunoreactivity were also detected in this region (Fig. 4, C andD). All antibodies gave only faint immunostaining signals in the cerebellar granule cell layer. Consistent with the Western blotting data, the anti-Kv1.4 antibody revealed only very low expression levels; in contrast, this antiserum yielded strong staining in other parts of the brain such as the substantia nigra pars reticulata or the ventral pallidum (data not shown), in agreement with previously published data (10Sheng M. Liao Y.J. Jan Y.N. Jan L.Y. Nature. 1993; 365: 72-75Crossref PubMed Scopus (292) Google Scholar, 27Veh R.W. Lichtinghagen R. Sewing S. Wunder F. Grumbach I.M. Pongs O. Eur. J. Neurosci. 1995; 7: 2189-2205Crossref PubMed Scopus (286) Google Scholar). Kv1.5 immunoreactivity was not detected in the rat cerebellum, in agreement with Western blot data.Figure 4Distribution of Kv1.1, Kv1.2, Kv1.3, and Kv1.6 immunoreactivity in rat cerebellum. 40-μm sagittal cryostat sections were stained with antibodies against Kv1.1 (A), Kv1.2 (B), Kv1.3 (C, D), and Kv1.6 (E andF). ML, molecular layer; PL, Purkinje cell layer; GL, granule cell layer; PC, Purkinje cell; PT, pinceau terminals. Bar in E(50 μm) also applies to A, B, and C;bar in F (200 μm) also applies toD.View Large Image Figure ViewerDownload Hi-res image Download (PPT) After having established the distribution of MgTX receptors and the cerebellar Kv1 subunits, we sought direct biochemical evidence for heterotetrameric channel assembly. For this purpose, we subjected125I-MgTX prelabeled cerebellar receptors to immunoprecipitation experiments using antibodies against the individual Kv1 subunits (Fig.5 A). All antibodies yielded saturable levels of precipitation. Moreover, inclusion of the competing peptide always decreased the level of precipitation by >90% (data not shown). Anti-Kv1.2 antibody precipitated >95% of125I-MgTX receptors (n = 48), and the combination of any other anti-Kv1 antibody with anti-Kv1.2 did not increase the amount of immunoprecipitated material. These results indicate that in virtually all cerebellar MgTX receptors, Kv1.2 is an essential component. Using solely anti-Kv1.1, 83 ± 5% of receptors (n = 32) could be precipitated, whereas anti-Kv1.3 and anti-Kv1.6 recognized 21 ± 5 (n = 16) and 16 ± 4% (n = 11) of the sites, respectively. By using saturating concentrations of both anti-Kv1.3 and anti-Kv1.6, additive precipitation levels (33 ± 7%; n = 13) could be achieved, indicating that these two proteins are segregated into distinct MgTX-sensitive channel complexes (see “Discussion”). To determine whether Kv1.3 and/or Kv1.6 subunits are coassembled with Kv1.1, we investigated the extent of precipitation of 125I-MgTX receptors by either anti-Kv1.3 or anti-Kv1.6 in the presence of anti-Kv1.1. Neither antibody significantly increased the levels achieved by anti-Kv1.1 alone (83 ± 5%, see above). Thus, a saturating amount of anti-Kv1.1 together with anti-Kv1.3 precipitated 87 ± 4% (n = 20) of the receptors, whereas the anti-Kv1.1/anti-Kv1.6 combination yielded 86 ± 5% (n = 20) precipitation. In addition, a combination of anti-Kv1.1, anti-Kv1.3, and anti-Kv1.6 did not further increase the amount of precipitation. Taken together, these data indicate that Kv1.2 is the dominant subunit of all MgTX-sensitive K+ channels in rat cerebellum. In 80% of the receptors, Kv1.2 is assembled with Kv1.1, whereas in one-third of these receptors, either Kv1.3 or Kv1.6 seems to be an additional integral component of the complex. The remaining 20% of cerebellar MgTX receptors seem to be composed of homotetrameric Kv1.2 channels. To confirm the composition of the cerebellar MgTX receptor by independent means, we heterologously expressed Kv1.1, Kv1.2, and a combination of Kv1.1/Kv1.2 subunits transiently in COS-1 cells, and binding of 125I-MgTX or 125I-DTX was used to determine the pharmacological properties of the resulting complex. 125I-MgTX binds to Kv1.2 and Kv1.1/Kv1.2 membranes with aK d value of 0.08 pm (data not shown), a value identical to that determined with cerebellar membranes (see Fig.1). In contrast, no specific 125I-MgTX binding signal was observed for homotetrameric Kv1.1 channels. Due to this fact, 125I-DTX was used instead, because this ligand has equal 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). 125I-DTX binds to Kv1.1, Kv1.2, and Kv1.1/Kv1.2 COS-1 membranes with a dissociation constant of 0.2 pm (data not shown). The pharmacology of 125I-DTX binding to Kv1.1, Kv1.2, Kv1.1/Kv1.2, and cerebellar membranes was investigated next (Fig. 5 C). Charybdotoxin inhibits125I-DTX binding to homotetrameric Kv1.1 channels with a K i value of ∼4000 pm, whereas it displays higher affinity for homotetrameric Kv1.2 channels (K i, ∼3 pm). However, in Kv1.1/Kv1.2 membranes, charybdotoxin displays a K i of 21 pm, in close agreement with the K i value determined with cerebellar membranes (18 pm). These data suggest that the observed pharmacological profile of Kv1.1/Kv1.2 membranes is due to the association of both subunits in a receptor complex and that this pharmacology is similar to that found with cerebellar membranes. To further validate the idea that Kv1.1 and Kv1.2 subunits are in fact associated in a complex after transient expression in COS-1 cells, immunoprecipitation experiments with anti-Kv1.1 and anti-Kv1.2 were performed (Fig. 5 B). For homomultimeric Kv1.2 channels,125I-MgTX or 125I-DTX receptors can be fully precipitated with anti-Kv1.2 but not anti-Kv1.1, whereas the opposite profile is observed when125I-DTX receptors from homomultimeric Kv1.1 channels are subjected to immunoprecipitation with these antibodies. However, both anti-Kv1.1 and anti-Kv1.2 are able to immunoprecipitate either 125I-MgTX or125I-DTX receptors from Kv1.1/Kv1.2 membranes (Fig. 5 B). In these experiments, anti-Kv1.2 precipitated close to 100% of125I-MgTX receptors, whereas anti-Kv1.1 precipitated ∼80%. These data imply that Kv1.1 and Kv1.2 subunits are indeed functionally associated in a receptor complex that very closely resembles the major constituent of the 125I-MgTX cerebellar receptor. The multiplicity of Kv channel genes in rat brain and their ability to form functional heterotetrameric channel complexes are two potential mechanisms for generating the enormous diversity of K+ channel currents observed in native neurons. Several lines of evidence suggest that Kv1 channel subunits form heterotetramers in vitro and in vivo. Coexpression of Kv1 subunits in Xenopus oocytes leads to the formation of heterotetrameric Kv channels with distinct properties when compared with those of the corresponding homomultimeric 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 evidence concerning the existence of overlapping Kv channel expression, but the resolution of these techniques is too low to directly detect coassembly of different subunits into tetrameric structures. To address this issue, some studies have investigated the occurrence of heterotetrameric K+ channels in vivo by immunoprecipitation of Kv1 channel complexes, followed by cross-blotting with subtype-selective anti-Kv1 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). Using this approach, heterotetrameric channel assembly of Kv1.2/Kv1.4 subunits in hippocampus (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 juxtaparanodal 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 demonstrated (28Sheng M. Tsaur M.L. Jan Y.N. Jan L.Y. Neuron. 1992; 9: 271-284Abstract Full Text PDF PubMed Scopus (400) Google Scholar). More recent studies have extended these findings and led to the identification of several subpopulations of defined Kv1 oligomers in bovine cerebral cortex (18Shamotienka O.G. Parcej D.N. Dolly J.O. Biochemistry. 1997; 36: 8195-8201Crossref PubMed Scopus (121) Google Scholar). An alternative way to address the issue of channel composition is to label native channels with a high-affinity ligand and establish their composition by quantitative immunoprecipitation. In the present study, we focused exclusively on the composition of MgTX-sensitive K+ channels in rat cerebellum. In cerebellar membranes 125I-MgTX binds to a single class of receptors with very high affinity (K d < 0.1 pm), slow ligand dissociation kinetics, and pharmacological characteristics that are not consistent with binding to a single homomultimeric Kv1 channel. Moreover, the pattern of Kv1 protein distribution in cerebellum is understood in some detail (Refs.27Veh R.W. Lichtinghagen R. Sewing S. Wunder F. Grumbach I.M. Pongs O. Eur. J. Neurosci. 1995; 7: 2189-2205Crossref PubMed Scopus (286) Google Scholar and 29McNamara N.M. Muniz Z.M. Wilkin G.P. Dolly J.O. Neuroscience. 1993; 57: 1039-1045Crossref PubMed Scopus (52) Google Scholar; this report). Several Kv1 subunits are expressed in this brain region at significant levels, such as Kv1.1, Kv1.2, Kv1.3, and Kv1.6. Kv1.2 and Kv1.3 are particularly interesting subunits, because these homotetrameric channels represent very high-affinity receptors for 125I-MgTX (30Helms L.M.H. Felix J.P. Bugianesi R.M. Garcia M.L. Stevens F. Leonard R.J. Knaus H.-G. Koch R. Wanner S.G. Kaczorowski G.J. Slaughter R.S. Biochemistry. 1997; 36: 3737-3744Crossref PubMed Scopus (50) Google Scholar). By a combination of autoradiography, immunocytochemistry, and immunoprecipitation studies, it has been possible to determine the composition of the 125I-MgTX receptor in cerebellum. These studies indicate that all receptors contain at least one Kv1.2 subunit and that ∼80% are heterotetramers of Kv1.1 and Kv1.2. In addition, some of these Kv1.1/Kv1.2 channels also contain an additional Kv1.3 or Kv1.6 subunit. It is most likely that all receptors found in basket cell terminals are Kv1.1/Kv1.2, because no other Kv1 subunit can be mapped to the compartment. However, at the molecular layer, homomultimeric Kv1.2, Kv1.1/Kv1.2, and Kv1.1/Kv1.2 with either Kv1.3 or Kv1.6 seem to be present. The unique pharmacological properties of the MgTX receptor should be mostly determined by the Kv1.1/Kv1.2 channels, because these are the major constituents in cerebellum. This idea has been confirmed in coexpression experiments in which Kv1.1 and Kv1.2 subunits were transiently expressed in COS-1 cells. Interestingly, these two subunits coassemble to yield a unique MgTX receptor phenotype that is very similar to that found in cerebellar membranes. Thus, heterotetrameric channel formation can occur in vitro and in vivo to the same extent and contributes to the diversity of K+ channels in the central nervous system. Those mechanisms controlling subunit assembly must play a very important role in determining the overall electrical activity in any given neuron. However, these processes remain to be characterized. We thank Maria Trieb, Emanuel Emberger, William Schmalhofer, and Jerry DiSalvo for technical contributions and recombinant toxin synthesis. Drs. Hartmut Glossmann, Jörg Striessnig, and Günther Sperk are gratefully acknowledged for continuous support and discussion." @default.
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