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Membrane Targeting of L-type Calcium Channels

ROLE OF PALMITOYLATION IN THE SUBCELLULAR LOCALIZATION OF THE β2a SUBUNIT*
  • Andy J. Chien
    Footnotes
    Affiliations
    Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
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  • Tianyan Gao
    Affiliations
    Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
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  • Edward Perez-Reyes
    Affiliations
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  • M. Marlene Hosey
    Correspondence
    To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, 303 E. Chicago Ave., S215, Chicago, IL 60611. Tel.: 312-503-3692; Fax: 312-503-5349;
    Affiliations
    Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Medical School, Chicago, Illinois 60611
    Search for articles by this author
  • Author Footnotes
    * This work was supported by National Institutes of Health Grants HL23306 (to M. M. H.) and HL46702 (to E. P. R.).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.
    ‡ Recipient of National Institute of Mental Health National Research Service Award Predoctoral Fellowship MH10770.
Open AccessPublished:September 04, 1998DOI:https://doi.org/10.1074/jbc.273.36.23590
      In this study, we report that palmitoylation was a critical determinant of the subcellular localization of the rat β2a subunit of voltage-dependent calcium channels. Immunohistochemical staining of transfected cells revealed that a palmitoylation-deficient β2a subunit exhibited a diffuse intracellular staining pattern, in contrast to the plasma membrane distribution seen with the wild-type β2asubunit. Unexpectedly, mutations in regions distal to the palmitoylation sites at Cys3 and Cys4 affected palmitoylation of the β2a protein. Mutations in ansrc homology 3 motif of the β2a subunit affected both palmitoylation and subcellular localization of the β2a protein. A mutation in the β interaction domain, which disrupted interactions between the expressed α1 and β subunits, also resulted in a decreased palmitoylation and diffuse intracellular localization of the β2a protein. Studies of chimeric proteins revealed that the 16-amino acid N terminus of the β2a subunit was sufficient to confer palmitoylation to the nonpalmitoylated β1b and β3 isoforms. However, palmitoylation of chimeric β subunits was by itself insufficient to restore the plasma membrane localization observed with the wild-type β2a protein. Treatment of transfected cells with brefeldin A increased the amount of palmitic acid incorporated in the β2a protein, suggesting that palmitoylation of β2a occurs during or shortly after protein synthesis. Two other β2 variants, the rabbit β2a and β2b, which lack the palmitoylation sties at Cys3 and Cys4, exhibited a diffuse intracellular staining pattern and were not palmitoylated.
      PCR
      polymerase chain reaction
      SH3
      src homology 3
      BID
      β interaction domain.
      Voltage-dependent calcium channels are heteromultimeric proteins composed of a pore-forming α1subunit, which determines many of the biophysical and pharmacological properties of the channel, and at least two other modulatory subunits, termed α2δ and β (
      • De Waard M.
      • Gurnett C.A.
      • Campbell K.P.
      ,
      • Hosey M.M.
      • Chien A.J.
      • Puri T.S.
      ). Although these channels have been extensively studied electrophysiologically, less is known about the biochemical properties of these proteins due to their rarity in native tissues. The α2δ and β subunits contain no homology to any known proteins and are involved in the modulation of channel properties. To date, four separate β isoforms have been identified, each of which contains a central conserved core flanked by unique N- and C-terminal regions specific to each isoform (
      • De Waard M.
      • Gurnett C.A.
      • Campbell K.P.
      ). Although the β subunits are highly hydrophilic proteins with no predicted membrane-spanning domains, we recently demonstrated that the cardiac β2a isoform was localized to the plasma membrane even in the absence of an α1 subunit (
      • Chien A.J.
      • Zhao X.
      • Shirokov R.E.
      • Puri T.S.
      • Chang C.F.
      • Sun D.
      • Rios E.
      • Hosey M.M.
      ).
      Co-expression of an accessory β subunit with an α1subunit in heterologous mammalian cell systems results in an increase in the number of drug/toxin binding sites (
      • Chien A.J.
      • Zhao X.
      • Shirokov R.E.
      • Puri T.S.
      • Chang C.F.
      • Sun D.
      • Rios E.
      • Hosey M.M.
      ,
      • Mitterdorfer J.
      • Froschmayr M.
      • Grabner M.
      • Striessnig J.
      • Glossmann H.
      ,
      • Nishimura S.
      • Takeshima H.
      • Hofmann F.
      • Flockerzi V.
      • Imoto K.
      ,
      • Perez-Reyes E.
      • Castellano A.
      • Kim H.S.
      • Bertrand P.
      • Baggstrom E.
      • Lacerda A.E.
      • Wei X.Y.
      • Birnbaumer L.
      ,
      • Williams M.E.
      • Feldman D.H.
      • McCue A.F.
      • Brenner R.
      • Velicelebi G.
      • Ellis S.B.
      • Harpold M.M.
      ), an increase in peak current amplitude (
      • Chien A.J.
      • Zhao X.
      • Shirokov R.E.
      • Puri T.S.
      • Chang C.F.
      • Sun D.
      • Rios E.
      • Hosey M.M.
      ,
      • Chien A.J.
      • Carr K.M.
      • Shirokov R.E.
      • Rios E.
      • Hosey M.M.
      ,
      • Perez-Garcia M.T.
      • Kamp T.J.
      • Marban E.
      ,
      • Kamp T.J.
      • Perez-Garcia M.T.
      • Marban E.
      ,
      • Josephson I.R.
      • Varadi G.
      ), and an increase in the number of channels at the cell surface (
      • Chien A.J.
      • Zhao X.
      • Shirokov R.E.
      • Puri T.S.
      • Chang C.F.
      • Sun D.
      • Rios E.
      • Hosey M.M.
      ,
      • Chien A.J.
      • Carr K.M.
      • Shirokov R.E.
      • Rios E.
      • Hosey M.M.
      ,
      • Kamp T.J.
      • Perez-Garcia M.T.
      • Marban E.
      ,
      • Josephson I.R.
      • Varadi G.
      ). The increase in channels at the plasma membrane has been demonstrated both biochemically (
      • Chien A.J.
      • Zhao X.
      • Shirokov R.E.
      • Puri T.S.
      • Chang C.F.
      • Sun D.
      • Rios E.
      • Hosey M.M.
      ) and electrophysiologically (
      • Chien A.J.
      • Carr K.M.
      • Shirokov R.E.
      • Rios E.
      • Hosey M.M.
      ,
      • Kamp T.J.
      • Perez-Garcia M.T.
      • Marban E.
      ,
      • Josephson I.R.
      • Varadi G.
      ), and probably accounts for the increased drug/toxin binding sites and the increase in peak current amplitude observed upon β subunit co-expression. In addition, it has been demonstrated recently that calcium currents, charge movements, and the number of dihydropyridine receptors were largely reduced in skeletal muscle myotubes of β1 subunit null mice (
      • Beurg M.
      • Sukhareva M.
      • Strube C.
      • Powers P.A.
      • Gregg R.G.
      • Coronado R.
      ) but could be restored by transfection of the β1 subunit, suggesting that the β subunit played important roles in maintaining the expression of the α1 subunits. The major identified α1-β interaction site involved regions conserved among all known α1 and β subunit isoforms (
      • Pragnell M.
      • De Waard M.
      • Mori Y.
      • Tanabe T.
      • Snutch T.P.
      • Campbell K.P.
      ,
      • De Waard M.
      • Pragnell M.
      • Campbell K.P.
      ), and further characterization of this domain revealed that interactions between different β subunits and a specific α1 subunit were fairly similar in affinity (
      • De Waard M.
      • Witcher D.R.
      • Pragnell M.
      • Liu H.
      • Campbell K.P.
      ). Likewise, it has been shown that all four known β subunit isoforms were capable of modulating currents from channels containing the cardiac α1C subunit (
      • Perez-Reyes E.
      • Castellano A.
      • Kim H.S.
      • Bertrand P.
      • Baggstrom E.
      • Lacerda A.E.
      • Wei X.Y.
      • Birnbaumer L.
      ,
      • De Waard M.
      • Witcher D.R.
      • Pragnell M.
      • Liu H.
      • Campbell K.P.
      ,
      • Castellano A.
      • Wei X.
      • Birnbaumer L.
      • Perez-Reyes E.
      ,
      • Castellano A.
      • Wei X.
      • Birnbaumer L.
      • Perez-Reyes E.
      ,
      • Castellano A.
      • Perez-Reyes E.
      ,
      • Singer D.
      • Biel M.
      • Lotan I.
      • Flockerzi V.
      • Hofmann F.
      • Dascal N.
      ).
      Recently, we identified sites of palmitoylation in the rat β2a subunit of voltage-dependent calcium channels (
      • Chien A.J.
      • Carr K.M.
      • Shirokov R.E.
      • Rios E.
      • Hosey M.M.
      ). Palmitoylation is a post-translational modification involving the reversible addition of a 16-carbon palmitic acid group to the cysteine residues of proteins through a labile thioester linkage (
      • Mumby S.M.
      ). The mechanisms involved in the addition and removal of palmitic acid are still unclear, although palmitoyl transferases that exhibit catalytic selectivity for palmitic acid have been recently purified (
      • Berthiaume L.
      • Resh M.D.
      ,
      • Camp L.A.
      • Verkruyse L.A.
      • Afendis S.J.
      • Slaughter C.A.
      • Hofmann S.L.
      ). Palmitoylation can be dynamically regulated due to the labile nature of the thioester bond, and studies have demonstrated the receptor-regulated depalmitoylation of certain proteins (
      • Wedegaertner P.B.
      • Bourne H.R.
      ,
      • Robinson L.J.
      • Busconi L.
      • Michel T.
      ). Three other known β subunit isoforms (β1, β3, and β4) were found not to be palmitoylated (
      • Chien A.J.
      • Carr K.M.
      • Shirokov R.E.
      • Rios E.
      • Hosey M.M.
      ), making palmitoylation the first identified biochemical modification unique to a specific β subunit isoform. Consequently, palmitoylation could provide a mechanism for the selective regulation of voltage-dependent calcium channels containing a β2a subunit.
      Palmitoylation of β2a required both the Cys3 and Cys4 residues in the N terminus, since site-directed substitution of either of these residues resulted in the loss of palmitate incorporation (
      • Chien A.J.
      • Carr K.M.
      • Shirokov R.E.
      • Rios E.
      • Hosey M.M.
      ). The mutation of these residues in the palmitoylation-deficient β2a(C3S/C4S) protein resulted in dramatic changes in whole-cell ionic conductance without affecting the number of functional channels at the cell surface (
      • Chien A.J.
      • Carr K.M.
      • Shirokov R.E.
      • Rios E.
      • Hosey M.M.
      ). The studies presented herein describe the effects of palmitoylation on the subcellular localization of the β2a protein and further define the determinants of palmitoylation in the β2a sequence.

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