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• W2000483745 abstract "The human KCNQ gene family encodes potassium channels linked to several genetic syndromes including neonatal epilepsy, cardiac arrhythmia, and progressive deafness. KCNQ channels form M-type potassium channels, which are critical regulators of neuronal excitability that mediate autonomic responses, pain, and higher brain function. Fundamental mechanisms of the normal and abnormal cellular roles for these channels may be gained from their study in simple model organisms. Here we report that a multigene family of KCNQ-like channels is present in the nematode, Caenorhabditis elegans. We show that many aspects of the functional properties, tissue expression pattern, and modulation of these C. elegans channels are conserved, including suppression by the M1 muscarinic receptor. We also describe a conserved mechanism of modulation by diacylglycerol for a subset of C. elegans and vertebrate KCNQ/KQT channels, which is dependent upon the carboxyl-terminal domains of channel subunits and activated protein kinase C. The human KCNQ gene family encodes potassium channels linked to several genetic syndromes including neonatal epilepsy, cardiac arrhythmia, and progressive deafness. KCNQ channels form M-type potassium channels, which are critical regulators of neuronal excitability that mediate autonomic responses, pain, and higher brain function. Fundamental mechanisms of the normal and abnormal cellular roles for these channels may be gained from their study in simple model organisms. Here we report that a multigene family of KCNQ-like channels is present in the nematode, Caenorhabditis elegans. We show that many aspects of the functional properties, tissue expression pattern, and modulation of these C. elegans channels are conserved, including suppression by the M1 muscarinic receptor. We also describe a conserved mechanism of modulation by diacylglycerol for a subset of C. elegans and vertebrate KCNQ/KQT channels, which is dependent upon the carboxyl-terminal domains of channel subunits and activated protein kinase C. The human genome contains five KCNQ genes that encode a family of K+ channel α-subunits possessing six transmembrane domains and a single pore loop. Functional channels are assembled from four α subunits, and may be either homo- or heterotetramers, depending on cell type. These channels serve a wide range of physiological roles. In the heart, KCNQ1 (originally designated KvLTQ1) is co-assembled with the product of the KCNE1 gene (variously designated minK, IsK, and MiRP) to form the cardiac IKs delayed rectifier-like K+ current (1Barhanin J. Lesage F. Guillemare E. Fink M. Lazdunski M. Romey G. Nature. 1996; 384: 78-80Crossref PubMed Scopus (1368) Google Scholar, 2Sanguinetti M.C. Curran M.E. Zou A. Shen J. Spector P.S. Atkinson D.L. Keating M.T. Nature. 1996; 384: 80-83Crossref PubMed Scopus (1492) Google Scholar). Mutations in either KCNQ1 or KCNE1 cause inherited long QT syndrome (LQT1 or LQT5, respectively), a condition leading to arrhythmia (Romano-Ward syndrome), as well as an associated form of deafness (Jervell and Lange-Nielsen syndrome) (3Wang Q. Curran M.E. Splawski I. Burn T.C. Millholland J.M. Van-Raay T.J. Shen J. Timothy K.W. Vincent G.M. de Jager T. Schwartz P.J. Toubin J.A. Moss A.J. Atkinson D.L. Landes G.M. Connors T.D. Keating M.T. Nat. Genet. 1996; 12: 17-23Crossref PubMed Scopus (1472) Google Scholar, 4Neyroud N. Tesson F. Denjoy I. Leibovici M. Donger C. Barhanin J. Faure S. Gary F. Coumel P. Petit C. Schwartz K. Guicheney P. Nat. Genet. 1997; 15: 186-189Crossref PubMed Scopus (733) Google Scholar, 5Chouabe C. Neyroud N. Guicheney P. Lazdunski M. Romey G. Barhanin J. EMBO J. 1997; 16: 5472-5479Crossref PubMed Scopus (237) Google Scholar, 6Splawski I. Tristani-Firouzi M. Lehmann M.H. Sanguinetti M.C. Keating M.T. Nat. Genet. 1997; 17: 338-340Crossref PubMed Scopus (664) Google Scholar, 7Schulze-Bahr E. Wang Q. Wedekind H. Haverkamp W. Chen Q. Sun Y. Rubie C. Hordt M. Towbin J.A. Borggrefe M. Assmann G. Qu X. Somberg J.C. Breithardt G. Oberti C. Funke H. Nat. Genet. 1997; 17: 267-268Crossref PubMed Scopus (364) Google Scholar). KCNQ1 can also co-assemble with KCNE3, and may form the channel carrying the basolateral cAMP-regulated K+ current present in colonic crypt cells (8Schroeder B.C. Kubisch C. Stein V. Jentsch T.J. Nature. 1998; 396: 687-690Crossref PubMed Scopus (432) Google Scholar, 9Schroeder B.C. Waldegger S. Fehr S. Bleich M. Warth R. Greger R. Jentsch T.J. Nature. 2000; 403: 196-199Crossref PubMed Scopus (416) Google Scholar). KCNQ2/KCNQ3 heteromultimers are thought to underlie the prototypic M-current. Mutations in either of these genes cause an inherited neonatal epilepsy (Benign Familial Neonatal Convulsion, BfnC) (10Charlier C. Singh N.A. Ryan S.G. Lewis T.B. Reus B.E. Leach R.J. Leppert M. Nat. Genet. 1998; 18: 53-55Crossref PubMed Scopus (812) Google Scholar, 11Singh N.A. Westenskow P. Charlier C. Pappas C. Leslie J. Dillon J. Anderson V.E. Sanguinetti M.C. Leppert M.F. Brain. 2003; 126: 2726-2737Crossref PubMed Scopus (231) Google Scholar, 12Biervert C. Schroeder B.C. Kubisch C. Berkovic S.F. Propping P. Jentsch T.J. Steinlein O.K. Science. 1998; 279: 403-406Crossref PubMed Scopus (921) Google Scholar, 13Yang W.P. Levesque P.C. Little W.A. Conder M.L. Ramakrishnan P. Neubauer M.G. Blanar M.A. J. Biol. Chem. 1998; 273: 19419-19423Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 14Wang H.S. Pan Z. Shi W. Brown B.S. Wymore R.S. Cohen I.S. Dixon J.E. McKinnon D. Science. 1998; 282: 1890-1893Crossref PubMed Scopus (1008) Google Scholar). The KCNQ4 gene may encode the molecular correlate of I(K,n) in outer hair cells of the cochlea, and I(K,L) in Type I hair cells of the vestibular apparatus, mutations that lead to a form of inherited progressive adult deafness (Autosomal Dominant Non-syndromic Deafness, DfnA2) (15Kubisch C. Schroeder B.C. Friedrich T. Lutjohann B. El-Amraoui A. Marlin S. Petit C. Jentsch T.J. Cell. 1999; 96: 437-446Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar). The recently identified KCNQ5 gene is expressed in brain and skeletal muscle and can co-assemble with KCNQ3, suggesting it may contribute to M-current heterogeneity (16Lerche C. Scherer C.R. Seebohm G. Derst C. Wei A.D. Busch A.E. Steinmeyer K. J. Biol. Chem. 2000; 275: 22395-22400Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 17Schroeder B.C. Hechenberger M. Weinreich F. Kubisch C. Jentsch T.J. J. Biol. Chem. 2000; 275: 24089-24095Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), although no linkage to a hereditary disease has yet been reported. Thus, mutations in four of the five human KCNQ genes are associated with hereditary diseases, suggesting a uniquely important role for this class of channels in a variety of physiological functions. In this article we show that a three-gene family of KCNQ-like channels is also present in the nematode worm, Caenorhabditis elegans, which we call KQT channels. Many aspects of their functional properties, tissue distribution, and modulation have striking parallels with their mammalian orthologues. For example, C. elegans pharyngeal muscles possess many cardiac-like properties including electrical coupling and rhythmic myogenic contractions generated by prolonged action potentials (18Avery L. Thomas J.H. Riddle D. Blumenthal T. Meyer B.J. Preiss J.R. C. elegans II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 679-716Google Scholar). We show that kqt-1 is expressed in pharyngeal muscles, where it may serve a role analogous to IKs in mammalian cardiac muscle. Additional kqt expression was found in nematode mechanosensory neurons, chemosensory neurons, and intestinal cells that may mediate cellular functions analogous to those in vertebrate species. We also show, that like all mammalian KCNQ channels, C. elegans KQT channels are modulated by the M1 muscarinic receptor that utilizes a Gαq signaling pathway. We address one potential signaling pathway of receptor-stimulated inhibition of M-currents using kqt and KCNQ genes from both C. elegans and vertebrates. We find that a subset of these kqt/KCNQ genes encode channels that are potently suppressed by submicromolar concentrations of the water soluble diacylglycerol analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), implicating DAG as a signaling intermediary. By analysis of chimeric subunit constructs, we find that this OAG 1The abbreviations used are: OAG, 1-oleoyl-2-acetyl-sn-glycerol; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; PKC, protein kinase C; GFP, green fluorescent protein; PIP2, phosphatidylinositol 4,5- bisphosphate.1The abbreviations used are: OAG, 1-oleoyl-2-acetyl-sn-glycerol; DAG, diacylglycerol; PMA, phorbol 12-myristate 13-acetate; DIDS, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid; PKC, protein kinase C; GFP, green fluorescent protein; PIP2, phosphatidylinositol 4,5- bisphosphate. suppression is mediated through the carboxyl-terminal “tail” domains of OAG-sensitive channel subunits. We observe that OAG-mediated suppression is mimicked by phorbol 12-myristate 13-acetate (PMA), a potent pharmacological activator of protein kinase C (PKC). Pretreatment with staurosporine, a PKC-specific inhibitor effectively blocks OAG inhibition. These results suggest that DAG-stimulated PKC may mediate receptor-coupled inhibition of a subset of M-currents through a mechanism involving the carboxyl-terminal tails of OAG-sensitive channel subunits. This mechanism is conserved for a subset of KCNQ/KQT channels in both C. elegans and vertebrates. Molecular Biology and C. elegans Transformed Strains—Full-length cDNAs of kqt-1, kqt-2, and kqt-3 were generated by a combination of hydridization screens with 32P-labeled DNA probes, PCR from a commercial C. elegans cDNA library (number 937007, Stratagene, San Diego, CA), and reverse transcriptase-PCR from mRNA extracted from mixed staged wild-type animals, using oligonucleotide primer sequences based on predicted cDNAs from cosmid sequences C25B8 (kqt-1; GenBank™ accession number U41556), M60 (kqt-2; GenBank accession number U39995), and YAC sequence Y54G9a (kqt-3; GenBank accession number AL032648). PCR-generated cDNAs were sequenced and compared with GenBank entries, as well as independently sequenced wild-type genomic fragments in some instances, to resolve sequence discrepancies with GenBank predictions. These cDNA sequences can be accessed with GenBank accession numbers AY572974 (kqt-1) and AY572975 (kqt-3). A full-length kqt-2 cDNA was contained within an EST (yk25b9) kindly provided by Yuji Kohara (National Institutes of Genetics, Mishima, Japan). C. elegans kqt cDNAs were modified by introduction of a Kozak initiation consensus sequence to each initiation methionine, and deletion of all 5′ non-coding sequences. Full-length KCNQ4 was reconstructed from a partial cDNA (AK074957, obtained from the National Institute of Technology and Evaluation, Department of Biotechnology, Biological Resource Center, Chiba, Japan), with additional 5′ fragments generated by PCR from cDNAs derived from human brain RNA and genomic DNA (Clontech, Palo Alto, CA), using primers based on GenBank accession number AF105202. Additional cDNAs used in this study were generously provided by Jacques Barhanin (KCNQ1; Institut de Pharmacologie Moleculaire et Cellulaire, Valbonne, France), David McKinnon (KCNQ2, rat KCNQ3; State University of New York at Stony Brook), Klaus Steinmeyer, Christian Lerche, Guiscard Seebohm and Andreas Busch (KCNQ5; Aventis, Frankfurt, Germany), and Narasimhan Gautam (human M1 muscarinc receptor; Washington University School of Medicine). Chimeric KQT/KCNQ constructs were generated by the overlap-extension PCR technique (19Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2614) Google Scholar). All cDNAs were subcloned into pOX (20Wei A. Solaro C. Lingle C. Salkoff L. Neuron. 1994; 13: 671-681Abstract Full Text PDF PubMed Scopus (227) Google Scholar) for Xenopus oocyte expression studies. To promote efficient heterologous expression in Xenopus oocytes, pOX incorporates 5′ and 3′ untranslated sequences from the Xenopus β-globin gene into transcribed cRNAs. All constructs were completely verified by sequencing. C. elegans translational GFP fusion constructs for each kqt gene were generated using subclones of genomic cosmid (C25B8, M60) and YAC (Y54G9a) clones provided by the C. elegans Sequencing Consortium (Genome Sequencing Center, Washington University School of Medicine, and Sanger Institute, Hinxton, UK). Subclones were designed to encompass most or all exons for each kqt gene, and substantial lengths of 5′ and 3′ non-coding sequences. The kqt-1::GFP construct was created by first subcloning an 11.6-kb NruI fragment of C25B8, which includes the entire kqt-1 gene and ∼9.0 kb of 5′ untranslated and ∼2.1 kb of 3′ untranslated sequences, into pBluescriptII KS+ (Stratagene, San Diego). This genomic subclone was then modified by the insertion of a GFP cDNA sequence in-frame at a unique SpeI site within exon 16 (immediately 3′ of the codon corresponding to T672), preserving the native 3′ splice site of exon 16. The kqt-2::GFP construct was created by subcloning a 11.9-kb NsiI fragment from Cosmid M-60, encompassing the first 12 exons of kqt-2 and ∼9.0 kb of 5′ non-coding sequence, into pPD95.81 (gift of Andy Fire, Carnegie Institution of Washington), linearized with PstI. This construct thus produces a 3′ translational fusion of KQT-2 at Ala-605 with GFP, lacking the last 70 predicted carboxyl-terminal amino acids encoded in exon 13. The kqt-3::GFP construct was created by subcloning a ∼5.0-kb NsiI/NheI fragment amplified from Y54G9a by PCR, into pPD95.75 (gift of Andy Fire, Carnegie Institution of Washington). This construct encompasses all kqt-3 exons and ∼1.5 kb of the non-coding sequence 5′ of the initiation methionine, and creates a translational fusion of GFP with the C terminus of KQT-3. Two additional overlapping PCR-generated genomic fragments were made, encompassing sequences ∼6.2 kb further 5′ of the initial subcloned kqt-3 fragment. Coinjection of these PCR-generated fragments with the 5′ linearized kqt-3::GFP construct yielded lines with stable labeling of chemosensory neurons, because of transgenes presumably generated by efficient in vivo recombination (21Yuan A. Dourado M. Butler A. Walton N. Wei A. Salkoff L. Nat. Neurosci. 2000; 3: 771-779Crossref PubMed Scopus (95) Google Scholar). Transformed C. elegans strains were created by the standard germline injection technique (22Mello C.C. Kramer J.M. Stinchcomb D. Ambros V. EMBO J. 1991; 10: 3959-3970Crossref PubMed Scopus (2392) Google Scholar), using ∼20 –50 ng/μl of each construct and rol-6(+) as a selectable marker. GFP-positive chemosensory neurons were identified in L1 staged larvae, assisted by co-labeling amphid sensory neurons (ASK, ADL, ASI, AWB, ASH, and ASJ) with DiI (Molecular Probes, Eugene, OR), as landmark cells (23Hedgecock E.M. Culotti J.G. Thomson J.N. Perkins L.A. Dev. Biol. 1985; 111: 158-170Crossref PubMed Scopus (336) Google Scholar). Electrophysiology—Two-electrode voltage-clamp and patch clamp recordings were made from Xenopus oocytes injected with cRNAs, as previously described (20Wei A. Solaro C. Lingle C. Salkoff L. Neuron. 1994; 13: 671-681Abstract Full Text PDF PubMed Scopus (227) Google Scholar). Dose-response series were obtained with a recording chamber with a volume of ∼150 μl, and solution changes with ∼3 times the recording chamber volume, applied manually without plastic tubing to minimize potential error because of retention of lipophilic reagents to tubing. Two-electrode voltage-clamp measurements were made at steady-state, typically 2–3 min after drug application. Between drug series, all recording surfaces were flushed with 70% ethanol to remove residual drug samples, then with ND96 recording solution. Stock solutions of drugs were dissolved in Me2SO, stored at –20 °C, and diluted in ND96 for each experimental series. In all instances, final Me2SO concentrations never exceeded 1% (v/v). Control experiments showed that 1% Me2SO had no effect on either endogenous or heterologously expressed currents in Xenopus oocytes. All measurements were made with 1.0 mm DIDS (Sigma) to block endogenous Ca2+-activated Cl– currents present in Xenopus oocytes. Composition of ND96 (in mm) was: 96 NaCl, 2.0 KCl, 1.8 CaCl2, 1.0 MgCl2, 5.0 HEPES (pH 7.2). Excised inside-out patch-clamp recordings were made under symmetric 160 mm K+ recording conditions, with zero Ca2+ and nearly Cl– free solutions. Recordings were obtained and low-pass filtered at 2.0 KHz, with either an Axopatch 200A (Molecular Devices, Sunnyvale, CA) or a model 2400 (A-M Systems, Carlsborg, WA) patch-clamp amplifier. Composition of pipette solution (in mm) was: 160 K-gluconate, 2.0 MgCl2, 5.0 HEPES (pH 7.2). Composition of cytoplasmic bath/perfusion solution (in mm) was: 160 K-gluconate, 5.0 EGTA, 5.0 HEPES (pH 7.2). Linopirdine was a gift from Barry Brown (DuPont Pharmaceuticals, Wilmington, DE). OAG, phorbol 12-mystristate 13-acetate (PMA), and poly-lysine HCl (15–30,000 Mr) were purchased from Sigma. Staurosporine and oxotremorine were purchased from Tocris Cookson (Ellisville, MO). A KCNQ-like Multigene Family in C. elegans—As in humans, KCNQ-like genes are represented in C. elegans as a multigene family. The C. elegans genome encodes 71 potassium channel genes (24Wei A. Jegla T. Salkoff L. Neuropharmacology. 1996; 35: 805-829Crossref PubMed Scopus (218) Google Scholar), of which three have similarity to KCNQ genes, forming a family of genes we call kqt for “K+ channel related to QT interval.” The three members of this family are kqt-1 (C25B8.1, Wormbase notation), kqt-2 (M60.2), and kqt-3 (Y54G9a.3). Primary sequence alignments revealed two domains of high conservation (∼70% identity), defining the “core” transmembrane segments S1–S6, and an additional region of ∼115 residues within the putative cytoplasmic carboxyl-terminal tail, implicated in subunit multimerization (25Maljevic S. Lerche C. Seebohm G. Alekov A.K. Busch A.E. Lerche H. J. Physiol. 2003; 548: 353-360PubMed Google Scholar, 26Schwake M. Jentsch T.J. Friedrich T. EMBO Rep. 2003; 4: 76-81Crossref PubMed Scopus (118) Google Scholar) (Supplemental Materials Fig. S1). Phylogenetic comparison of all C. elegans KQT and human KCNQ primary sequences identified two conserved subfamilies: C. elegans KQT-1 defining one subfamily with KCNQ2–5, whereas C. elegans KQT-3 defined a second subfamily with KCNQ1. KQT-2 did not group with either subfamily, nor did it possess a human ortholog. However, BLAST similarity searches clearly showed higher KQT-2 similarity in the vertebrate KCNQ gene family than to any other vertebrate potassium channel gene family (Fig. 1A). Translational fusions of kqt genes with GFP revealed prominent expression in a variety of C. elegans tissues including pharyngeal muscles (kqt-1), intestinal cells (kqt-2, kqt-3), mechanosensory neurons (kqt-1, kqt-3), chemosensory neurons (kqt-3), and other head neurons (kqt-1, kqt-3) (Fig. 1, B–D). Some individual GFP-positive neurons were identified based on unique morphology and location, or double labeling with the lipophilic tracer DiI, which reproducibly labels a set of chemosensory amphid and phasmid neurons (23Hedgecock E.M. Culotti J.G. Thomson J.N. Perkins L.A. Dev. Biol. 1985; 111: 158-170Crossref PubMed Scopus (336) Google Scholar). The mechanosensitive touch neurons PLM and ALM were clearly labeled by both kqt-1::GFP and kqt-3::GFP (Fig. 1E). In addition, a large subset of DiI-positive chemosensory amphid and phasmid neurons consistently labeled with kqt-3::GFP. These identified kqt-3::GFP positive neurons included the amphid neurons ADL, ASI, AWB, and ASH located in the head, and the phasmid neurons PHA and PHB in the tail (Fig. 1F). An additional DiI-negative neuron in the head labeled consistently with kqt-3::GFP, which we tentatively identified as the chemosensory neuron AWC. Additional unidentified head neurons were labeled by kqt-1::GFP and kqt-3::GFP, although expression was not reliably observed in neurons of the ventral or dorsal cord. Conserved Functional Properties and Muscarinic Modulation of C. elegans KQT Channels—Potassium currents with functional properties resembling vertebrate M-currents were expressed by full-length kqt-1 or kqt-3 cDNAs in Xenopus oocytes. Functional expression of kqt-2 was not observed. KQT-1 and KQT-3 potassium currents displayed unusually slow activation and deactivation kinetics, under two-electrode voltage-clamp (Fig. 2, A and B). Normalized conductance-voltage plots revealed voltages for half-maximal conductance similar to vertebrate KCNQ orthologs (Fig. 2C) (KQT-1, V50 = –16 mV; KQT-3, V50 = –13 mV, versus –19 to –8 mV reported for vertebrate KCNQ channels (27Selyanko A.A. Hadley J.K. Wood I.C. Abogadie F.C. Jentsch T.J. Brown D.A. J. Physiol. 2000; 522: 349-355Crossref PubMed Scopus (142) Google Scholar); but others report –40 to –20 mV (28Jentsch T.J. Nat. Rev. Neurosci. 2000; 1: 21-30Crossref PubMed Scopus (675) Google Scholar)). Both KQT-1 and KQT-3 potassium currents were reversibly blocked by the KCNQ-specific compound linopridine (KQT-1 IC50 = 44 μm; KQT-3, IC50 = 79 μm), with ∼4–8-fold lower sensitivity compared with vertebrate KCNQ channels (Fig. 2D). All vertebrate KCNQ channels can be modulated through the M1 muscarinic receptor (17Schroeder B.C. Hechenberger M. Weinreich F. Kubisch C. Jentsch T.J. J. Biol. Chem. 2000; 275: 24089-24095Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 27Selyanko A.A. Hadley J.K. Wood I.C. Abogadie F.C. Jentsch T.J. Brown D.A. J. Physiol. 2000; 522: 349-355Crossref PubMed Scopus (142) Google Scholar). Significantly, we observed that this mode of regulation was conserved with C. elegans subunits, by coexpressing KQT-1 or KQT-3 with the human M1 muscarinic receptor in Xenopus oocytes. KQT-1 or KQT-3 currents expressed in these experiments were rapidly suppressed by bath application of the muscarinic agonist, oxotremorine (10 μm) (Fig. 2, E and F). M1 mediated suppression was more effective for KQT-1 (∼90% inhibition) than for KQT-3 (∼16% inhibition) (Fig. 2G). These experiments demonstrate that C. elegans KQT channels encode M-currents, which are functionally similar to M-currents encoded by vertebrate KCNQ channels. A Subset of C. elegans KQT and Vertebrate KCNQ Channels Are Suppressed by Submicromolar OAG—The lipid metabolite diacylglycerol generated by phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) has been described as a potential signaling intermediary for receptor-coupled inhibition of native M-currents in gastric smooth muscles (29Sims S.M. Singer J.J. Walsh Jr., J.V. Science. 1988; 239: 190-193Crossref PubMed Scopus (39) Google Scholar) and perhaps other tissues (30Stemkowski P.L. Tse F.W. Peuckmann V. Ford C.P. Colmers W.F. Smith P.A. J. Neurophysiol. 2002; 88: 277-288Crossref PubMed Scopus (24) Google Scholar). To test if DAG may mediate inhibition of cloned KQT channels, we applied the water soluble DAG analog, OAG, to oocytes expressing KQT-1 or KQT-3 channels. Potent suppression of KQT-1 currents was observed with submicromolar concentrations of OAG (IC50 = 0.2 μm), resulting in ∼80% maximal inhibition at saturating concentrations, within 2 min (Fig. 3, A and B). KQT-3 currents were also suppressed by OAG, but with ∼1000-fold lower sensitivity (IC50 = 201 μm) (Fig. 3B). To test whether OAG-mediated inhibition observed with C. elegans KQT channels is conserved with vertebrate KCNQ channels, we examined the OAG sensitivity of currents carried by all the vertebrate KCNQ channels (KCNQ1–5) expressed as homomeric channels in Xenopus oocytes. Of these vertebrate channels, KCNQ5 currents were uniquely sensitive to inhibition by OAG at submicromolar concentrations (IC50 = 0.1 μm), similar to C. elegans KQT-1 (Fig. 3C). Other KCNQ currents examined were far less sensitive to OAG inhibition, similar to C. elegans KQT-3, requiring ∼1000-fold greater OAG concentrations for inhibition (KCNQ1, IC50 = ∼717 μm; KCNQ3, IC50 =∼1.1 mm; (Fig. 3C) (KCNQ2 and KCNQ4 were similarly uninhibited by 125 μm OAG (data not shown)). Because KCNQ5 forms heteromeric channels with KCNQ3 in heterologous expression systems (16Lerche C. Scherer C.R. Seebohm G. Derst C. Wei A.D. Busch A.E. Steinmeyer K. J. Biol. Chem. 2000; 275: 22395-22400Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 17Schroeder B.C. Hechenberger M. Weinreich F. Kubisch C. Jentsch T.J. J. Biol. Chem. 2000; 275: 24089-24095Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar), and most likely in vivo (31Hadley J.K. Passmore G.M. Tatulian L. Al-Qatari M. Ye F. Wickenden A.D. Brown D.A. J. Neurosci. 2003; 23: 5012-5019Crossref PubMed Google Scholar), we examined the OAG sensitivity of currents produced by co-expression of KCNQ5 and KCNQ3 cRNAs. In agreement with these previous reports, we observed that KCNQ3/5 coinjected oocytes produced currents with amplitudes 5–8 times greater than that predicted by the linear sum of individually injected KCNQ3 and KCNQ5 oocytes, consistent with efficient heteromeric channel formation. Moreover, heteromeric KCNQ3/5 currents, like homomeric KCNQ5 currents, were effectively suppressed by OAG at submicromolar concentrations (IC50 = 0.6 μm) (Fig. 3D). Vertebrate KCNQ5 and C. elegans KQT-1 subunits thus define a conserved molecular subclass of M-channels with high sensitivity to OAG. Furthermore, KCNQ5 confers high OAG sensitivity to heteromeric channels formed with KCNQ3 subunits. Suppression by OAG Is Mediated Through the COOH-terminal Tail of OAG-sensitive KQT/KCNQ Subunits—To investigate the structural regions necessary for OAG-mediated inhibition of channels formed by KCNQ and KQT subunits, we generated a series of chimeric constructs between subunits with high and low sensitivity. Other functional classes of potassium and non-selective cation channels gated by cytosolic ligands (Slo, SK, and CNG) are encoded by subunits that appear to be modularly composed of a core voltage-sensing and pore-forming domain (S1–S6), linked to unique carboxyl-terminal tail structures that modify gating (20Wei A. Solaro C. Lingle C. Salkoff L. Neuron. 1994; 13: 671-681Abstract Full Text PDF PubMed Scopus (227) Google Scholar, 32Jan L.Y. Jan Y.N. Nature. 1994; 371: 119-122Crossref PubMed Scopus (252) Google Scholar, 33Jiang Y. Lee A. Chen J. Cadene M. Chait B.T. MacKinnon R. Nature. 2002; 417: 515-522Crossref PubMed Scopus (1196) Google Scholar). By analogy, we reasoned that OAG may exert its effects through carboxyl-terminal domains conserved in OAG-sensitive KCNQ and KQT subunits. We first examined chimeric channels formed by exchanging carboxyl-terminal tail domains between C. elegans KQT-1 and KQT-3 subunits. Attaching the tail from the OAG-sensitive KQT-1 subunit to the core of KQT-3 created a functional chimeric subunit (K3CR/K1TL) with high OAG sensitivity, essentially identical to wild-type KQT-1 channels (Fig. 3E). To address if this structural requirement for inhibition by OAG may employ an evolutionarily conserved mechanism, an analogous chimeric subunit was generated attaching the tail from the vertebrate OAG-sensitive subunit, KCNQ5, to the core of C. elegans KQT-3 (K3CR/Q5TL). As with K3CR/K1TL, the interspecies K3CR/Q5TL chimeric subunit also produced channels with high OAG sensitivity, and an OAG dose-response profile essentially identical to wild-type KCNQ5 (Fig. 3F). High OAG sensitivity could thus be conferred to C. elegans KQT-3 by substituting the tail from the OAG-sensitive channel subunits from either vertebrate or C. elegans species. Conversely, low OAG sensitivity was also conferred by attaching the tail from vertebrate KCNQ1 to the core of C. elegans KQT-1 (K1CR/Q1TL) (Fig. 3G), although the analogous intraspecies chimera K1CR/K3TL failed to express (see construct 1.1, Fig. 4). Results from additional interspecies core and tail chimeric combinations were consistent with a determinative role of the tail in OAG sensitivity. Thus, chimeric subunits with the core of C. elegans KQT-1 and tail of vertebrate KCNQ5 (K1CR/Q5TL) exhibited high OAG sensitivity (IC50 = 0.032 μm), whereas chimera with the core of C. elegans KQT-3 and tail of vertebrate KCNQ1 (K3CR/Q1TL) exhibited low OAG sensitivity (IC50 = 200 μm) (data not shown). Taken together, these results are consistent with OAG sensitivity being determined by the carboxyl-terminal tails of KCNQ/KQT subunits. Although we determined that the full-length carboxyl-terminal tails fully confer either high or low OAG sensitivities, attempts to localize OAG sensitivities to a smaller domain within the tails were unsuccessful (Fig. 4). No single tail subregion was able to confer complete OAG sensitivity. Our results suggest that the structural requirements for OAG sensitivity are possibly complex and not mediated through a single subregion of the carboxyl-terminal tail, despite the apparent functional modularity of the entire tail domain. Suppression Is Not Mediated by Direct Binding of OAG to the Channel Protein—Because DAG directly interacts to alter the gating of cyclic-GMP gated cation channels (34Gordon S.E. Downing-Park J. Tam B. Zimmerman A.L. Biophys. J. 1995; 69: 409-417Abstract Full Text PDF PubMed Scopus (45) Google Scholar, 35Crary J.I. Dean D.M. Nguitragool W. Kurshan P.T. Zimmerman A.L. J. Gen. Physiol. 2000; 116: 755-768Crossref PubMed Scopus (26) Google Scholar, 36Crary J.I. Dean D.M. Maroof F. Zimmerman A.L. J. Gen. Physiol. 2000; 116: 769-780Crossref PubMed Scopus (8) Google Scholar) and a subset of TRPC cation channels (37Hardie R.C. Annu. Rev. Physiol. 2003; 65: 7" @default.
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• W2000483745 title "KCNQ-like Potassium Channels in Caenorhabditis elegans" @default.
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