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Calmodulin Regulates Assembly and Trafficking of SK4/IK1 Ca2+-activated K+ Channels*

  • William J. Joiner
    Footnotes
    Affiliations
    From the Departments of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine New Haven, Connecticut 06520
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  • Rajesh Khanna
    Footnotes
    Affiliations
    Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, and Department of Physiology, University of Toronto, Toronto, Ontario M5T 2S8, Canada
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  • Lyanne C. Schlichter
    Correspondence
    To whom correspondence should be addressed: MC9-415, Toronto Western Hospital, 399 Bathurst St., Toronto, Ontario M5T 2S8, Canada. Tel.:416-603-5800 ext. 2052; Fax: 416- 603-5745;
    Affiliations
    Division of Cellular and Molecular Biology, Toronto Western Research Institute, University Health Network, and Department of Physiology, University of Toronto, Toronto, Ontario M5T 2S8, Canada
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  • Leonard K. Kaczmarek
    Affiliations
    From the Departments of Pharmacology and Cellular and Molecular Physiology, Yale University School of Medicine New Haven, Connecticut 06520
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  • Author Footnotes
    * This work was funded in part by Canadian Institutes for Health Research (CIHR) Grant MT13657, a Natural Sciences and Engineering Research Council Grant (to L. C. S.), and National Institutes of Health Grant DC01919 (to L. K. K.). Part of this work was published previously as abstracts (42, 43). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    § Both authors contributed equally to this work.
    ‖ Supported by an Ontario Graduate Scholarship and a University of Toronto Margaret J. Santalo Scholarship. Current address: Dept. of Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1751.
Open AccessPublished:October 12, 2001DOI:https://doi.org/10.1074/jbc.M104965200
      Calmodulin (CaM) regulates gating of several types of ion channels but has not been implicated in channel assembly or trafficking. For the SK4/IK1 K+ channel, CaM bound to the proximal C terminus (“Ct1 ” domain) acts as the Ca2+ sensor. We now show that CaM interacting with the C terminus of SK4 also controls channel assembly and surface expression. In transfected cells, removing free CaM by overexpressing the CaM-binding domain, Ct1, redistributed full-length SK4 protein from the plasma membrane to the cytoplasm and decreased whole-cell currents. Making more CaM protein available by overexpressing the CaM gene abrogated the dominant-negative effect ofCt1 and restored both surface expression of SK4 protein and whole-cell currents. The distal C-terminal domain (“Ct2”) also plays a role in assembly, but is not CaM-dependent. Co-immunoprecipitation experiments demonstrated that multimerization of SK4 subunits was enhanced by CaM and inhibited by removal of CaM, indicating that CaM regulates trafficking of SK4 by affecting the assembly of channels. Our results support a model in which CaM-dependent association of SK4 monomers at their Ct1 domains regulates channel assembly and surface expression. This appears to represent a novel mechanism for controlling ion channels, and consequently, the cellular functions that depend on them.
      SK channel
      small-conductance KCa channel
      KCa channel
      Ca2+-activated K+ channel
      Ct1
      proximal C terminus
      CaM
      calmodulin
      CHO
      Chinese hamster ovary
      HA
      hemagglutinin
      NM
      N terminus + 6 transmembrane domains
      Ct2
      distal C terminus
      PCR
      polymerase chain reaction
      PBS
      phosphate-buffered saline
      Kv channel
      voltage-gated K+channel
      GFP
      green fluorescent protein
      In neurons, small-conductance Ca2+-activated K+ (SK)1 channels participate in the slow after-hyperpolarization that regulates action-potential frequency, spike frequency adaptation, and long term potentiation (
      • Goh J.W.
      • Pennefather P.S.
      ,
      • Pedarzani P.
      • Mosbacher J.
      • Rivard A.
      • Cingolani L.A.
      • Oliver D.
      • Stocker M.
      • Adelman J.P.
      • Fakler B.
      ,
      • Sah P.
      ,
      • Sah P.
      • Davies P.
      ). In non-excitable tissues, a related isoform, called IK (due to its intermediate conductance) contributes to volume regulation and the secondary immune response in lymphocytes (
      • Schlichter L.C.
      • Sakellaropoulos G.
      ,
      • Vandorpe D.H.
      • Shmukler B.E.
      • Jiang L.
      • Lim B.
      • Maylie J.
      • Adelman J.P.
      • de Franceschi L.
      • Cappellini M.D.
      • Brugnara C.
      • Alper S.L.
      ,
      • Khanna R.
      • Chang M.C.
      • Joiner W.J.
      • Kaczmarek L.K.
      • Schlichter L.C.
      ,
      • Ghanshani S.
      • Wulff H.
      • Miller M.J.
      • Rohm H.
      • Neben A.
      • Gutman G.A.
      • Cahalan M.D.
      • Chandy K.G.
      ,
      • Fanger C.M.
      • Rauer H.
      • Neben A.L.
      • Miller M.J.
      • Wulff H.
      • Rosa J.C.
      • Ganellin C.R.
      • Chandy K.G.
      • Cahalan M.D.
      ), salt and water transport in colonic and airway epithelia (
      • Devor D.C.
      • Singh A.K.
      • Lambert L.C.
      • DeLuca A.
      • Frizzell R.A.
      • Bridges R.J.
      ,
      • McCann J.D.
      • Matsuda J.
      • Garcia M.
      • Kaczorowski G.
      • Welsh M.J.
      ), activation of brain microglia (
      • Khanna R.
      • Roy L.
      • Zhu X.
      • Schlichter L.C.
      ), and pathophysiological conditions, such as Diamond-Blackfan anemia and sickle cell anemia in erythrocytes (
      • Brugnara C.
      • De Franceschi L.
      • Armsby C.C.
      • Saadane N.
      • Trudel M.
      • Beuzard Y.
      • Rittenhouse A.
      • Rifai N.
      • Platt O.
      • Alper S.L.
      ,
      • Ghanshani S.
      • Coleman M.
      • Gustavsson P.
      • Wu A.C.
      • Gargus J.J.
      • Gutman G.A.
      • Dahl N.
      • Mohrenweiser H.
      • Chandy K.G.
      ).
      The existence of a Ca2+-activated K+ (KCa) conductance was first demonstrated in erythrocytes by Gardos (
      • Gardos G.
      ). The gene underlying this conductance was cloned by us and others. It encodes a KCa channel that has been variously called SK4, IK1, KCa4, and KCNN4 (
      • Ghanshani S.
      • Coleman M.
      • Gustavsson P.
      • Wu A.C.
      • Gargus J.J.
      • Gutman G.A.
      • Dahl N.
      • Mohrenweiser H.
      • Chandy K.G.
      ,
      • Joiner W.J.
      • Wang L.Y.
      • Tang M.D.
      • Kaczmarek L.K.
      ,
      • Ishii T.M.
      • Silvia C.
      • Hirschberg B.
      • Bond C.T.
      • Adelman J.P.
      • Maylie J.
      ,
      • Logsdon N.J.
      • Kang J.
      • Togo J.A.
      • Christian E.P.
      • Aiyar J.
      ). This channel, like other members of the SK family, is tethered tightly at its proximal C terminus (Ct1domain) to calmodulin (CaM), which opens the channel in the presence of elevated intracellular Ca2+ (
      • Khanna R.
      • Chang M.C.
      • Joiner W.J.
      • Kaczmarek L.K.
      • Schlichter L.C.
      ,
      • Xia X.M.
      • Fakler B.
      • Rivard A.
      • Wayman G.
      • Johnson-Pais T.
      • Keen J.E.
      • Ishii T.
      • Hirschberg B.
      • Bond C.T.
      • Lutsenko S.
      • Maylie J.
      • Adelman J.P.
      ,
      • Fanger C.M.
      • Ghanshani S.
      • Logsdon N.J.
      • Rauer H.
      • Kalman K.
      • Zhou J.
      • Beckingham K.
      • Chandy K.G.
      • Cahalan M.D.
      • Aiyar J.
      ). The demonstration that SK channels and CaM can be activated by divalent metal ions in the same order of preference (
      • Cao Y.J.
      • Houamed K.M.
      ) has been interpreted to mean that CaM is both necessary and sufficient to account for Ca2+-dependent gating of SK channels. Recently, an x-ray crystallographic study suggested a structural model for gating of SK channels that involved coordinated, Ca2+-dependent interactions between CaM andCt1 domains (
      • Schumacher M.A.
      • Rivard A.F.
      • Bachinger H.P.
      • Adelman J.P.
      ).
      Beyond the role of Ct1 in CaM binding and channel gating, very little is known about domains involved in SK function, such as channel assembly. SK channels lack the conserved N-terminal tetramerization domains of voltage-gated K+ channels (
      • Shen N.V.
      • Pfaffinger P.J.
      ,
      • Xu J.
      • Yu W.
      • Jan Y.N.
      • Jan L.Y.
      • Li M.
      ,
      • Tu L.
      • Santarelli V.
      • Sheng Z.
      • Skach W.
      • Pain D.
      • Deutsch C.
      ) and the C-terminal multimerization domains of human ether-a-go-go-related gene (
      • Kupershmidt S.
      • Snyders D.J.
      • Raes A.
      • Roden D.M.
      ) and big Ca-activated K+ channels (
      • Jiang Y.
      • Pico A.
      • Cadene M.
      • Chait B.T.
      • MacKinnon R.
      ). Here we provide the first evidence that multimerization of SK channel subunits is regulated by the same molecule responsible for channel gating: CaM. We find that CaM regulates the surface expression of SK4/IK1 protein by regulating the multimerization of channels through intersubunit interactions between apposed Ct1 domains.

      ACKNOWLEDGEMENTS

      We thank Xiaoping Zhu (in the Schlichter laboratory) for expert technical assistance. Dr. Diane M. Papazian (Dept. of Physiology, UCLA) provided helpful comments on the manuscript.

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