The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin

Xiaochun Long, Darla L. Tharp, Mary A. Georger, Orazio J. Slivano, Monica Y. Lee, Brian R. Wamhoff, Douglas K. Bowles, Joseph M. Miano

Research output: Contribution to journalArticle

Abstract

Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise α and β subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype.

Original languageEnglish (US)
Pages (from-to)33671-33682
Number of pages12
JournalJournal of Biological Chemistry
Volume284
Issue number48
DOIs
StatePublished - Nov 27 2009
Externally publishedYes

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Serum Response Factor
Large-Conductance Calcium-Activated Potassium Channels
Ion Channels
Smooth Muscle Myocytes
Muscle
Cells
Genes
Transcriptional Activation
Assays
Potassium
Excitation Contraction Coupling
Modulation
Chromatin Immunoprecipitation
Luciferases
Introns
Muscle Cells
Transcription
Rodentia
Swine
Down-Regulation

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin. / Long, Xiaochun; Tharp, Darla L.; Georger, Mary A.; Slivano, Orazio J.; Lee, Monica Y.; Wamhoff, Brian R.; Bowles, Douglas K.; Miano, Joseph M.

In: Journal of Biological Chemistry, Vol. 284, No. 48, 27.11.2009, p. 33671-33682.

Research output: Contribution to journalArticle

Long, X, Tharp, DL, Georger, MA, Slivano, OJ, Lee, MY, Wamhoff, BR, Bowles, DK & Miano, JM 2009, 'The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin', Journal of Biological Chemistry, vol. 284, no. 48, pp. 33671-33682. https://doi.org/10.1074/jbc.M109.050419
Long, Xiaochun ; Tharp, Darla L. ; Georger, Mary A. ; Slivano, Orazio J. ; Lee, Monica Y. ; Wamhoff, Brian R. ; Bowles, Douglas K. ; Miano, Joseph M. / The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 48. pp. 33671-33682.
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T1 - The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin

AU - Long, Xiaochun

AU - Tharp, Darla L.

AU - Georger, Mary A.

AU - Slivano, Orazio J.

AU - Lee, Monica Y.

AU - Wamhoff, Brian R.

AU - Bowles, Douglas K.

AU - Miano, Joseph M.

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N2 - Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise α and β subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype.

AB - Large conductance calcium-activated potassium (MaxiK) channels play a pivotal role in maintaining normal arterial tone by regulating the excitation-contraction coupling process. MaxiK channels comprise α and β subunits encoded by Kcnma and the cell-restricted Kcnmb genes, respectively. Although the functionality of MaxiK channel subunits has been well studied, the molecular regulation of their transcription and modulation in smooth muscle cells (SMCs) is incomplete. Using several model systems, we demonstrate down-regulation of Kcnmb1 mRNA upon SMC phenotypic modulation in vitro and in vivo. As part of a broad effort to define all functional CArG elements in the genome (i.e. the CArGome), we discovered two conserved CArG boxes located in the proximal promoter and first intron of the human KCNMB1 gene. Gel shift and chromatin immunoprecipitation assays confirmed serum response factor (SRF) binding to both CArG elements. A luciferase assay showed myocardin (MYOCD)-mediated transactivation of the KCNMB1 promoter in a CArG element-dependent manner. In vivo analysis of the human KCNMB1 promoter disclosed activity in embryonic heart and aortic SMCs; mutation of both conserved CArG elements completely abolished in vivo promoter activity. Forced expression of MYOCD increased Kcnmb1 expression in a variety of rodent and human non-SMC lines with no effect on expression of the Kcnma1 subunit. Conversely, knockdown of Srf resulted in decreases of endogenous Kcnmb1. Functional studies demonstrated MYOCD-induced, iberiotoxin-sensitive potassium currents in porcine coronary SMCs. These results reveal the first ion channel subunit as a direct target of SRF-MYOCD transactivation, providing further insight into the role of MYOCD as a master regulator of the SMC contractile phenotype.

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