The smooth muscle cell-restricted KCNMB1 ion channel subunit is a direct transcriptional target of serum response factor and myocardin.
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 alpha and beta 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.
Long
Xiaochun
X
Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.
Tharp
Darla L
DL
Georger
Mary A
MA
Slivano
Orazio J
OJ
Lee
Monica Y
MY
Wamhoff
Brian R
BR
Bowles
Douglas K
DK
Miano
Joseph M
JM
D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D005260:Female; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D017403:In Situ Hybridization; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026362:Serum Response Factor / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D000099251:Myocardin
D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D009687:Nuclear Proteins; D026362:Serum Response Factor; D015534:Trans-Activators; D000099251:Myocardin; C531905:KCNMB1 protein, human; C543091:Kcnmb1 protein, mouse