Promoter cloning and characterization of the rabbit BK channel beta1 subunit gene.
The beta1 subunit of the voltage-dependent and Ca(2+)-activated large-conductance K(+) channel (BK) in mammalian smooth muscle cells (SMCs) plays an important role in regulating smooth muscle tone and is closely linked with a series of smooth muscle tone associated diseases. However, knowledge of the transcriptional regulation of the BK beta1 is still largely unclear. For the first time, we cloned and characterized the full-length genomic sequence of the rabbit BK beta1 containing a 5'-flanking region of 2021 bp. The full-reading frame of the BK beta1 spans ~7.7 kb and is organized into 4 exons and 3 introns. All of the exon/intron junction sequences contain the GT/AG consensus junction sequence. The transcription initiation site (+1G) is located at 447 bp upstream of the translation initiation codon. Bioinformatics analysis indicated that, without any canonical TATA-box, the 5'-flanking region possesses a high GC content and contains a number of putative transcription factor binding sites. 5'-deletion analysis demonstrated that the region of -93/+30 potentially functions as a core promoter region. A gel mobility shift assay and chromatin immunoprecipitation assay revealed that Sp1 specifically interacts with a putative Sp1-binding site (-91/-85) in vitro and in vivo. Mutation of this site significantly diminished the promoter activities. Over-expression of Sp1 in smooth muscle cells of rabbit sphincter of Oddi enhanced the promoter activities of the BK beta1 in a dose-dependent manner. Thus, we suggest that the Sp1-binding site (-91/-85) is essential to the basal transcription of the rabbit BK beta1. Our studies provide a basic knowledge of the transcription regulation of the rabbit BK beta1.
Zhang
Xiao-Yong
XY
Department of Radiology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China.
Wang
Sha
S
Yan
Zhen
Z
Zhang
Ying-Qi
YQ
Wan
Yi
Y
Zhang
Bin
B
Wang
Li-Feng
LF
Chai
Yu-Bo
YB
Wei
Jing-Guo
JG
D024506:5' Flanking Region; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D018387:Codon, Initiator; D019295:Computational Biology / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D024202:Electrophoretic Mobility Shift Assay; D005091:Exons; D007438:Introns; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics* / Q000378:metabolism*; D049410:Luciferases, Renilla / Q000032:analysis / Q000378:metabolism; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000166:cytology / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021122:Protein Subunits / Q000235:genetics* / Q000378:metabolism; D012313:RNA / Q000302:isolation & purification; D011817:Rabbits / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism; D009803:Sphincter of Oddi / Q000166:cytology; D024363:Transcription Initiation Site; D014158:Transcription, Genetic; D014162:Transfection
D018387:Codon, Initiator; D051036:Large-Conductance Calcium-Activated Potassium Channels; D021122:Protein Subunits; D016329:Sp1 Transcription Factor; D012313:RNA; D004247:DNA; D049410:Luciferases, Renilla