New Modulatory α Subunits for Mammalian ShabK+ Channels
1997; Elsevier BV; Volume: 272; Issue: 39 Linguagem: Inglês
10.1074/jbc.272.39.24371
ISSN1083-351X
AutoresMiguel Salinas, Fabrice Duprat, Catherine Heurteaux, Jean‐Philippe Hugnot, Michel Lazdunski,
Tópico(s)Receptor Mechanisms and Signaling
ResumoTwo novel K+ channel α subunits, named Kv9.1 and Kv9.2, have been cloned. The Kv9.2 gene is situated in the 8q22 region of the chromosome. mRNAs for these two subunits are highly and selectively expressed in the nervous system. High levels of expressions are found in the olfactory bulb, cerebral cortex, hippocampal formation, habenula, basolateral amygdaloid nuclei, and cerebellum. Interestingly Kv9.1 and Kv9.2 colocalized with Kv2.1 and/or Kv2.2 α subunits in several regions of the brain. Neither Kv9.1 nor Kv9.2 have K+ channel activity by themselves, but both modulate the activity of Kv2.1 and Kv2.2 channels by changing kinetics and levels of expression and by shifting the half-inactivation potential to more polarized values. This report also analyzes the changes in electrophysiological properties of Kv2 subunits induced by Kv5.1 and Kv6.1, two other modulatory subunits. Each modulatory subunit has its own specific properties of regulation of the functional Kv2 subunits, and they can lead to extensive inhibitions, to large changes in kinetics, and/or to large shifts in the voltage dependencies of the inactivation process. The increasing number of modulatory subunits for Kv2.1 and Kv2.2 provides an amazingly new capacity of functional diversity. Two novel K+ channel α subunits, named Kv9.1 and Kv9.2, have been cloned. The Kv9.2 gene is situated in the 8q22 region of the chromosome. mRNAs for these two subunits are highly and selectively expressed in the nervous system. High levels of expressions are found in the olfactory bulb, cerebral cortex, hippocampal formation, habenula, basolateral amygdaloid nuclei, and cerebellum. Interestingly Kv9.1 and Kv9.2 colocalized with Kv2.1 and/or Kv2.2 α subunits in several regions of the brain. Neither Kv9.1 nor Kv9.2 have K+ channel activity by themselves, but both modulate the activity of Kv2.1 and Kv2.2 channels by changing kinetics and levels of expression and by shifting the half-inactivation potential to more polarized values. This report also analyzes the changes in electrophysiological properties of Kv2 subunits induced by Kv5.1 and Kv6.1, two other modulatory subunits. Each modulatory subunit has its own specific properties of regulation of the functional Kv2 subunits, and they can lead to extensive inhibitions, to large changes in kinetics, and/or to large shifts in the voltage dependencies of the inactivation process. The increasing number of modulatory subunits for Kv2.1 and Kv2.2 provides an amazingly new capacity of functional diversity. Voltage-gated potassium channels (Kv) 1The abbreviations used are: Kv, voltage-gated potassium channel; EST, expressed sequence tag; ORF, open reading frame; PBS, phosphate buffered saline; FaNaCh, FMRF-activated Na+ channel; kb, kilobase pair(s). form the largest and most diversified class of ion channels. These proteins are present in both excitable and nonexcitable cells. Their main functions are associated with the regulation of the resting membrane potential and the control of the shape and frequency of action potentials (1Rudy B. Neuroscience. 1988; 25: 729-749Crossref PubMed Scopus (1074) Google Scholar, 2Hille B. Catterall W.A. Siegel G.J. Agranoff B.W. Albers R.W. Molinoff P.B. Basic Neurochemistry: Molecular, Cellular, and Medical Aspects. 5th Ed. Raven Press, New York1994: 75-95Google Scholar). K+ channel functions are included in very diverse processes such as neuronal integration, cardiac pacemaking, muscle contraction, and hormone secretion in excitable cells (3Hille B. 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Sequence similarities between members of the Kv family were initially used to define the different subfamilies of α subunits. The different members within a given subfamily share only a percentage of 30–50% with members of others subfamilies. To date 20 functional voltage-gated potassium channels α subunits have been described. They belong to six subfamilies designated Kv1 (Shaker), Kv2 (Shab), Kv3 (Shaw), Kv4 (Shal), KvLQT, and EAG. Another family (Kv7) has a single member which has been cloned fromAplysia (9Zhao B. Rassendren F. Kaang B.K. Furukawa Y. Kubo T. Kandel E.R. Neuron. 1994; 13: 1205-1213Abstract Full Text PDF PubMed Scopus (47) Google Scholar). The diversity of potassium channel functions comes from the diversity of potassium channel genes and is increased by alternate splicing (10Luneau C.J. Williams J.B. Marshall J. Levitan E.S. Oliva C. Smith J.S. Antanavage J. Folander K. Stein R.B. Swanson R. Kaczmarek L.K. Buhrow S.A. Proc. Natl. Acad. Sci. U. S. 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These α subunits, although they cannot generate K+ channel activity by themselves, can modulate in a specific way the function of Kv2.1 and Kv2.2 subunits (22Salinas M. de Weille J. Guillemare E. Lazdunski M. Hugnot J.-P. J. Biol. Chem. 1997; 272: 8774-8780Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 23Post M.A. Kirsch G.E. Brown A.M. FEBS Lett. 1996; 399: 177-182Crossref PubMed Scopus (82) Google Scholar). They inhibit the Kv2.1 and Kv2.2 channels when expressed at high concentrations (21Hugnot J.P. Salinas M. Lesage F. Guillemare E. Weille J. Heurteaux C. Mattéi M.G. Lazdunski M. EMBO J. 1996; 15: 3322-3331Crossref PubMed Scopus (108) Google Scholar, 23Post M.A. Kirsch G.E. Brown A.M. FEBS Lett. 1996; 399: 177-182Crossref PubMed Scopus (82) Google Scholar). The Kv4 family now also has a modulatory α subunit (jShalγ1) (24Jegla T. Salkoff L. J. Neurosci. 1997; 17: 32-44Crossref PubMed Google Scholar) of the same type. This sort of regulation by heteromultimerization with homologous subunits, which do not form functional channels by themselves, was also previously described for nucleotide-gated channels (25Chen T.Y. Peng Y.-W. Dhallan R.S. Ahamed B. Reed R.R. Yau K.-W. Nature. 1993; 362: 764-767Crossref PubMed Scopus (274) Google Scholar, 26Liman E.R. Buck L.B. Neuron. 1994; 13: 611-621Abstract Full Text PDF PubMed Scopus (215) Google Scholar, 27Bradley J. Li J. Davidson N. Lester H.A. Zinn K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8890-8894Crossref PubMed Scopus (210) Google Scholar, 28Biel M. Zong X. Ludwig A. Sautter A. Hofmann F. J. Biol. Chem. 1996; 271: 6349-6355Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). This study reports the cloning and brain localization of two new neuronal modulatory α subunits that define a new structural family designated as Kv9. Kv9.1 and Kv9.2 are new modulators of Kv2.α subunits but have no functional activity by themselves. The study also shows that Kv5.1 and Kv6.1 are also potent regulators of channels of the Kv2 family. The functional properties of Kv9.1/Kv2.α and Kv9.2/Kv2.α heteromultimers are compared with those of Kv5.1/Kv2.α and Kv6.1/Kv2.α assemblies. A BLAST search of the expressed sequence tag (EST) data base, using the query peptide sequence of Kv8.1, reveals two groups of matching EST coding for two new homologues of Kv channels. Sense and antisense oligonucleotides corresponding to the extremities of these two EST groups were synthesized (Genosys) and used in two polymerase chain reaction amplifications on mouse brain cDNA. The sequencing of cloned polymerase chain reaction products allowed the isolation of Kv9.1 and Kv9.2 probes that were used to screen 5 × 105 clones of a mouse brain cDNA library constructed with λZAP II vector (Stratagene). Probes ([α-32P]dCTP-labeled Kv9.1 and Kv9.2 fragments) were produced with a random primers kit from Amersham Corp. Filters were hybridized in 50% formamide, 5 × SSC, 4 × Denhardt's solution, 0.1% SDS, and 100 μg/ml denatured salmon sperm DNA at 45 °C overnight and washed to a final stringency of 0.2 × SSC, 0.1% SDS at 55 °C. Three independent clones for Kv9.1 and one for Kv9.2 were isolated. The cDNA inserts were characterized by restriction enzyme analysis and by partial or complete DNA sequencing on both strands by the dideoxy nucleotide method using an automatic sequencer (Applied Biosystems model 373A). Two Kv9.1 clones (2.2–2.3 kb long) and the Kv9.2 insert (5 kb long) were shown to contain a full-length ORF. The third Kv9.1 clone was identical but incomplete in its 5′-coding sequence. A mouse multiple tissue Northern blot (CLONTECH) was probed with the32P-labeled inserts of pBS Kv9.1 or pBS Kv9.2 according to the manufacturer's protocol. Experiments were performed on adult Swiss mice using standard procedures (29Heurteaux C. Messier C. Destrade C. Lazdunski M. Mol. Brain Res. 1993; 18: 17-22Crossref PubMed Scopus (67) Google Scholar). The dissected organs (brains, spinal cords, and retina) were fixed in ice-cold 4% (w/v) paraformaldehyde, 0.1 m sodium phosphate-buffered solution (PBS, pH 7.4) for 8 h and then immersed overnight at 4 °C in a 20% sucrose, PBS solution. Frozen sections (10 μm) were cut on a Cryostat (Leica) at −25 °C, collected on 3-aminopropylethoxysilane-coated slides, and stored at −20 °C until use. Specific antisense cRNA probes were generated with T7-RNA polymerase (Boehringer Mannheim) by in vitro transcription using d-[33P]UTP (3000 Ci/mmol, ICN Radiochemicals), from EcoRI-linearized plasmid containing a 900-base pair fragment of Kv9.1 cDNA or a 560-base pair fragment of Kv9.2 cDNA in the 3′-untranslated sequence and in the cytoplasmic C-terminal coding sequence, inserted into pBluescript SK−. Sections were treated consecutively with 0.1 m glycine in PBS for 10 min, PBS for 3 min, 5 μg/ml proteinase K diluted in 0.1m Tris, 50 mm EDTA (pH 8.0) for 15 min at 37 °C, 4% paraformaldehyde, and PBS (pH 7.2) for 5 min. Slides were then rinsed for 10 min in PBS, acetylated for 10 min in 0.25% acetic anhydride in 0.1 m triethanolamine and dehydrated. Hybridization was carried out overnight at 55 °C in hybridization buffer (50% deionized formamide, 10% dextran sulfate, 500 μg/ml denatured salmon sperm DNA, 1% Denhardt's solution, 5% Sarcosyl, 250 mg/ml yeast tRNA, 20 mm dithiothreitol, 20 mmNaPO4 in 2 × SSC, and the radiolabeled probe (at 0.2 ng/ml with specific activities of 8 × 108 dpm/μg). Following hybridization, sections were washed in 4 × SSC for 15 min and then twice in 1 × SSC for 30 min at 60 °C, treated with RNase A (5 μg/ml) in 2 × SSC for 15 min at 37 °C, and washed twice with 1 × SSC for 30 min, followed by two 15-min washes in 0.1 × SSC at 30 °C. Specimens were then dehydrated, air-dried, and exposed to Amersham β-max Hyperfilm for 5 days at 4 °C. Selected slides were dipped in Amersham LM1 photographic emulsion and exposed for 3 weeks at 4 °C and then developed in Kodak D-19 for 4 min. All slides were counterstained with hematoxylin/eosin. For control experiments, adjacent sections were hybridized with corresponding sense riboprobes or digested with RNase before hybridization. The coding sequences of Kv9.1, Kv9.2, Kv5.1 (IK8) and Kv6.1 (K13) were amplified using a low error rate DNA polymerase (PWO DNA polymerase, Boehringer Mannheim) and subcloned into pEXO vector (30Lingueglia E. Renard S. Voilley N. Waldmann R. Chassande O. Lazdunski M. Barbry P. Eur. J. Biochem. 1993; 216: 679-687Crossref PubMed Scopus (45) Google Scholar). Capped cRNAs were synthesized in vitrofrom linearized plasmid by using the T7, T3, or SP6 RNA polymerase (Promega). Cloning of cDNA and synthesis of complementary RNA have been previously described for Kv1.1 (31Tempel B.L. Jan Y.N.J. Jan L.Y. Nature. 1988; 332: 837-839Crossref PubMed Scopus (231) Google Scholar), Kv1.2 (32Guillemare E. Honore E. Pradier L. Lesage F. Schweitz H. Attali B. Barhanin J. Lazdunski M. Biochemistry. 1992; 31: 12463-12468Crossref PubMed Scopus (57) Google Scholar), Kv1.3 (33Attali B. Romey G. Honoré E. Schmid-Alliana A. Mattéi M.-G. Lesage F. Ricard P. Barhanin J. Lazdunski M. J. Biol. Chem. 1992; 267: 8650-8657Abstract Full Text PDF PubMed Google Scholar), Kv1.4 (34Tseng-Crank J.C.L. Tseng G.N. Schwartz A. Tanouye M.A. FEBS Lett. 1990; 268: 63-68Crossref PubMed Scopus (128) Google Scholar), Kv1.5 (11Attali B. Lesage F. Ziliani P. Guillemare E. Honore E. Waldmann R. Hugnot J.-P. Mattéi M.-G. Lazdunski M. Barhanin J. J. Biol. Chem. 1993; 268: 24283-24289Abstract Full Text PDF PubMed Google Scholar), Kv2.1 (DRK1) (35Frech G.C. Van Dongen A.M.J. Schuster G. Brown A.M. Joho R.H. Nature. 1989; 340: 642-645Crossref PubMed Scopus (359) Google Scholar), Kv2.2 (CDRK) (36Hwang P.M. Glatt C.E. Bredt D.S. Yellen G. Snyder S.H. Neuron. 1992; 8: 473-481Abstract Full Text PDF PubMed Scopus (103) Google Scholar), Kv3.3 (37Vega-Saenz de Miera E. Moreno H. Fruhling D. Kentros C. Rudy B. Proc. R. Soc. Lond. B Biol. Sci. 1992; 248: 9-18Crossref PubMed Scopus (71) Google Scholar), Kv3.4 (38Schroter K.H. Ruppersberg J.P. Wunder F. Rettig J. Stocker M. Pongs O. FEBS Lett. 1991; 278: 211-216Crossref PubMed Scopus (115) Google Scholar), Kv4.1 (39Pak M.D. Covarrubias M. Ratcliffe A. Salkoff L. J. Neurosci. 1991; 11: 869-880Crossref PubMed Google Scholar), Kv5.1 (IK8), and Kv6.1 (K13) (20Drewe J.A. Verma S. Frech G. Joho R.H. J. Neurosci. 1992; 12: 538-548Crossref PubMed Google Scholar). Xenopus laevis were purchased from C.R.B.M. (Montpellier, France). Pieces of the ovary were surgically removed, and individual oocytes were dissected away in a saline solution (ND96) containing 96 mm NaCl, 2 mm KCl, 1.8 mmCaCl2, 2 mm MgCl2, and 5 mm HEPES at pH 7.4 with NaOH. Stage V and VI oocytes were treated for 2 h with collagenase (1 mg/ml, type Ia, Sigma) in ND96 to discard follicular cells. cRNA solutions were injected (50 nl/oocyte) using a pressure microinjector (Inject+Matic, Switzerland). We have previously shown that a high level of expression of some cloned K+ channels could lead to high magnitude K+currents with major kinetic and voltage-dependence modifications when compared with currents of lower intensity (32Guillemare E. Honore E. Pradier L. Lesage F. Schweitz H. Attali B. Barhanin J. Lazdunski M. Biochemistry. 1992; 31: 12463-12468Crossref PubMed Scopus (57) Google Scholar, 40Honoré E. Attali B. Romey G. Lesage F. Barhanin J. Lazdunski M. EMBO J. 1992; 11: 2465-2471Crossref PubMed Scopus (56) Google Scholar). Therefore, particular attention was paid to compare currents of similar and relatively low intensities (under 5 μA for a test pulse at +50 mV). cRNAs were injected at 0.5 ng/oocyte for Kv2.1 and 10 ng/oocyte for Kv2.2. The oocytes were kept at 19 °C in the ND96 saline solution supplemented with gentamycin (5 μg/ml). Oocytes were studied within 2–4 days following injection of cRNA. In a 0.3-ml perfusion chamber, a single oocyte was impaled with two standard glass microelectrodes (1–2.5-megohm resistance) filled with 3 m KCl and maintained under a voltage clamp using a Dagan TEV 200 amplifier. Stimulation of the preparation, data acquisition, and analysis were performed using pClamp software (Axon Instruments, Burlingame, CA). All experiments were performed at room temperature (21–22 °C) in ND96 solutions. The activation phase of currents elicited by voltage steps to +30 mV, 1 s in duration, were fitted with a single exponential and the time constant τact was determined. Inactivation curves (see Figs. 5,E and F, and 7, E and F) were fitted with a Boltzman distribution of the form I = 1/1 + exp(V 1/2 inact/k inact).V 1/2 inact is the potential of half-inactivation and k inact is the slope factor. The tail currents were recorded at −40 mV after a 500-ms long prepulse to +50 mV, in standard ND96 solution. These currents were fitted with a single exponential characterized by a time constant τtail. The coding sequence of Kv9.1 was subcloned into the Flag-pRc/CMV vector as described previously for N-terminal-tagged Kv2.2 protein (21Hugnot J.P. Salinas M. Lesage F. Guillemare E. Weille J. Heurteaux C. Mattéi M.G. Lazdunski M. EMBO J. 1996; 15: 3322-3331Crossref PubMed Scopus (108) Google Scholar). However, to link the T7 tag epitope (MASMTGGQQMG) to the N terminus of Kv9.2, the double-stranded oligonucleotide 5′-GGGCTAGCTGATCAGAGGCCTCACCATGGCTAGTATGACTGGAGGACAGCAAATGGGATATCGCGACCAGCTGACTCGAGAATTCG-3′, which contains the restriction sites NheI andEcoRI in its extremities, was subcloned into the pCI vector (Promega) at the corresponding sites. This construction designated T7.tag-pCI contains EcoRV, NruI,PvuII, and XhoI cloning sites. The PCR fragment of Kv9.2 (see above under "cRNA Synthesis, Injection, and Electrophysiological Measurement in Xenopus Oocyts") was digested in 5′ by XhoI and inserted inEcoRV-XhoI-linearized T7.tag-pCI vector to create a tagged-Kv9.2-expressing vector. COSm6 cells were grown in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum and antibiotics (60 μg/ml penicillin, 50 μg/ml streptomycin). Two days before transfection 15 × 103 cells were plated on cover glasses onto 15-mm Petri dishes. The cells were transfected by a modification of the DEAE-dextran/chloroquine method (41Lopata M.A. Cleveland D.W. Sollner-Webb B. Nucleic Acids Res. 1984; 12: 5707-5717Crossref PubMed Scopus (518) Google Scholar) using 1 or 2 μg of supercoiled DNA. Two days after transfection the cells grown on glass coverslips were fixed for 15 min with 4% (v/v) paraformaldehyde in PBS. After rinsing twice with PBS, cells were permeabilized by incubation for 10 min with 0.1% Triton X-100. The sites of nonspecific binding were blocked by 2-h incubation with 5% goat serum, 2% bovine serum albumin in PBS at room temperature. The cells were then incubated for 2 h with a mixture of anti-FLAG M2 monoclonal antibody (1/150 dilution, Kodak) or T7.Tag monoclonal antibody (1/250, NOVAGEN) with a BS solution (2% bovine serum albumin in PBS), followed by washing with PBS and incubation for 1 h with fluorescein isothiocyanate-conjugated goat anti-mouse Ig (1/200, Sigma) in BS. After washing in PBS then in 10 mm Tris-HCl, pH 7.5, cells were mounted in Vectashield Medium (Vector Laboratories, Inc.) and observed with a Leitz Aristoplan microscope (Wild Leitz) using an interference blue (fluorescein isothiocyanate) filter and a 40× lens. Computer analyses were performed using Genetics Computer Group software. The phylogenetic tree (see Fig. 1 C) and the percentage of identity (Table I) were generated using the PILEUP multiple sequence analysis program with the conserved region corresponding to the amino acids from the position 24–435 of Kv9.1 (starting from residues NVGG to Y). The members of subfamilies used and their accession numbers are as follows: mKv1.1, Y00305; hKv1.3, M85217; rKv2.1, X16476; rKv2.2, M77482; rKv3.3, M84211; rKv3.4, X62841; rKv4.1,S64320; rKv5.1, M81783; rKv6.1, M81784; aKv7.1, L35766; and Kv8.1,U62810.Table IAmino acid identity between voltage-gated potassium channels of different subfamiliesKv5.1Kv6.1Kv8.1Kv9.1Kv9.2%3736373740Kv1.13937373738Kv1.34546494245Kv2.14547494245Kv2.24036373535Kv3.33935363334Kv3.43735383536Kv4.13730353334Kv7.110038393740Kv5.1100414039Kv6.11004449Kv8.110058Kv9.1100Kv9.2Percentages of identical positions were calculated as described under "Experimental Procedures." The percentages for K+ channels of the same subfamily are shown in boldface. Open table in a new tab Percentages of identical positions were calculated as described under "Experimental Procedures." The percentages for K+ channels of the same subfamily are shown in boldface. The Kv8.1 subunit is a new K+ channel subunit which is inactive by itself and which has recently been described to regulate both active subunits of the Kv2 potassium channel subfamily (21Hugnot J.P. Salinas M. Lesage F. Guillemare E. Weille J. Heurteaux C. Mattéi M.G. Lazdunski M. EMBO J. 1996; 15: 3322-3331Crossref PubMed Scopus (108) Google Scholar, 22Salinas M. de Weille J. Guillemare E. Lazdunski M. Hugnot J.-P. J. Biol. Chem. 1997; 272: 8774-8780Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). This result has suggested the existence of a new class of regulatory α subunits. To identify potential genes coding for parent sequences, the peptide sequence of the Kv8.1 subunit was used to search related sequences in GenBankTM data base using the BLAST sequence alignment program (42Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (70758) Google Scholar). We identified two groups of human EST encoding a portion of a new α subunit (named Kv9.1) from the S3 to the S6 transmembrane domain for the EST group corresponding to the accession numbers H18119, H43697, H49525, H42586,H49759, H2O365, H18164, and H42586, and another new α subunit (named Kv9.2) from the S2 to S4 transmembrane domains for the EST groupR19352, H19204, and R34920. It was then postulated that these two groups of EST were partial copies of mRNAs coding for two new subunit homologues of the Kv8.1 K+ channel. Two DNA probes derived from these two groups of sequences were used to screen a mouse cDNA brain library. Three clones for Kv9.1 and one clone for Kv9.2 were obtained. Two of the three independent Kv9.1 clones found were full-length and presented the same expected open reading frame (ORF). Nevertheless, these two clones differed in their 5′-noncoding sequence upstream of the nucleotide −21. The 5.0-kb cDNA insert of the Kv9.2 clone also comprises the expected ORF. The methionine initiation codon for the longest ORF is preceded by several in-frame termination triplets. In addition, nucleotide sequences surrounding the initiation codon of both Kv9.1 and Kv9.2 clones correspond to the Kozak consensus sequence (43Kozak M. Nucleic Acids Res. 1987; 15: 8125-8148Crossref PubMed Scopus (4168) Google Scholar). The ORFs of Kv9.1 and Kv9.2 encode proteins of 497 and 477 amino acids, respectively, with a calculated molecular mass of 54.9 and 54.3 kDa (Fig. 1 A). Protein sequences reveal that all the structural characteristics of outward rectifier voltage-gated K+ channel α subunits (Kv) are conserved in Kv9.1 and Kv9.2, i.e. six putative transmembrane segments (S1 to S6), a transmembrane region (S4) showing five positively charged amino acids and a conserved pore-forming region (named H5 or P domain). As indicated in Fig. 1 A, Kv9.1 and Kv9.2 subunits contain several putative phosphorylation sites located in the cytoplasmic regions for protein kinase C, cAMP-dependent protein kinase, Ca2+-calmodulin kinase II, casein kinase II, and tyrosine kinase. No N-glycosylation sequence was detected. The identity between these two new α subunits is about 58% and they have only 33–49% identity with other Kv channel subunits (TableI). Thus, they belong to the same subfamily (44Chandy K.G. Gutman G.A. Voltage-gated K+ Channels. CRC, Boca Raton, FL1995Google Scholar). The phylogenetic tree presented in Fig. 1 Cindicates that the Kv9 α subunits have a common ancestor with other "nonfunctional" α subunits such as Kv5.1, Kv6.1, and Kv8.1, as well as with the Kv2 family. Northern blot analysis presented in Fig.2 shows that Kv9.1 and Kv9.2 mRNAs are expressed only in the brain. Specific probes detected two transcripts for Kv9.1 with an estimated size of 2.2 and 2.7 kb and one for Kv9.2 of approximately 5.3 kb. No expression was observed in heart, spleen, lung, liver, skeletal muscle, kidney, or testis. The two Kv9.1 transcripts probably represent alternatively spliced variants in the 5′-noncoding region as shown by sequencing of the different clones. The regional distribution of Kv9.1 and Kv9.2 mRNA was studied from analysis of x-ray film images and emulsion-coated sections of adult mouse brains cut in sagittal and coronal planes and detected byin situ hybridization. Specific cRNA probes revealed a wide and heterogeneous expression pattern in adult mouse brain (Fig.3 A). The heterogeneous distribution of the hybridization signal and the observation of emulsion-dipped sections suggested a neuronal localization of the transcripts. Sense probes did not show significant hybridization (data not shown). Examination of adjacent sections hybridized with Kv9.1 and Kv9.2 cRNA probes indicated that the distribution of Kv9.1 transcripts was strikingly similar to that observed for Kv9.2, with highest expression levels in the main olfactory bulb, cerebral cortex, hippocampal formation, habenula, basolateral amygdaloid nuclei, and cerebellum. In the olfactory system (Fig. 3 B), cells expressing Kv9.1 and Kv9.2 mRNA were localized in the glomerular cell layer, the densely packed internal granular layer, and the thin mitral and internal plexiform cell layers. Within the glomerular layer, transcripts were restricted to the periglomerular cells whose processes extend into the glomerula. In contrast, light diffuse labeling was observed in the external plexiform layer and the olfactory nerve layer. In addition to the main olfactory bulb, Kv9.1 and Kv9.2 expression was also prominent in the anterior olfactory nuclei and the piriform (primary olfactory) cortex. In the hippocampus (Fig. 3 C), Kv9.1 and Kv9.2 mRNAs were strongly expressed in dentate granule cells and hilar neurons as well as in CA1–CA3 pyramidal cells. In addition, intense hybridization signals were observed in large interneurons located in stratum oriens and radiatum of all subfields. Many cells in the subiculum and enthorinal cortex of the hippocampal formation were also found to be positive. In the neocortex (Fig.3 D), Kv9.1 and Kv9.2 gene expression was observed in all cortical areas with a labeling pattern reflecting the laminar structure of cell distribution. These two transcripts were strongly expressed in the large cortical pyramidal neurons as compared with the small cell bodies throughout the cortical layers, which may represent nonpyramidal neurons, oligo-, astro-, or microglia cells and which failed to show hybridization signals. Kv9.1 and Kv9.2 mRNAs were also densely distributed throughout most of the amygdala, including the cortical, lateral and basolateral nuclei. Within the thalamus, the medial and lateral habenula showed particularly high levels of Kv9.1 and Kv9.2. The ventromedial hypothalamic nucleus displayed an intense hybridization signal. A distinct laminar expression pattern was observed in the cerebellum (Fig. 3 E). The most intense labeling was detected in the Purkinje and granule cell layers. The molecular layer was weakly labeled except for a few strongly positive cells that are scattered in the cerebellar molecular layer and may be stellate cells and/or basket cells. Large neurons in all deep cerebellar nuclei expressed moderate to high levels of Kv9.1 and Kv9.2 mRNAs. A diffuse expression was observed over most other regions including striatum, globus pallidus of the basal ganglia, substantia nigra, midbrain, and brainstem (pons and medulla). Kv9.1 and Kv9.2 transcripts were also intensely expressed in the rat retina with a distinct stratification pattern. High labeling was apparent in the inner nuclear layer (INL), composed of nuclei of Müller cells and amacrine, bipolar, and horizontal neurons, in the retinal ganglion cell layer (RGC) and in the photoreceptor inner segments (IS) (Fig. 3 F). The spinal cord showed an intense hybridization signal in several neuronal types. This hybridization pattern was similar from the upper cervical to the sacral region. The majority of spinal cord neurons, includi
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