Cloning and characterization of the promoters of the maxiK channel alpha and beta subunits

Large conductance, calcium-activated potassium (maxiK) channels are express ed in nerve, muscle, and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcript ional regulation of maxiK channels, we characterized the promoter regions o f the pore forming (alpha) and regulatory (beta) subunits of the human chan nel complex. Maximum promoter activity (up to 12.3-fold over control) occur red between nucleotides -567 and -220 for the a subunit (hSlo) gene. The mi nimal promoter is GC-rich with 5 Sp-1 binding sites and several TCC repeats . Other transcription factor-binding motifs, including c/EBP, NF-kB, PU.1, PEA-3, Myo-D, and E2A, were observed in the 5'-flanking sequence. Additiona lly, a CCTCCC sequence, which increases the transcriptional activity of the SM1/2 gene in smooth muscle, is located 27 bp upstream of the TATA-like se quence, a location identical to that found in the SM1/2 5'-flanking region. However, the promoter directed equivalent expression when transfected into smooth muscle and other cell types. Analysis of the hSlo beta subunit 5'-f lanking region revealed a TATA box at position -77 and maximum promoter act ivity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Bind ing sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identi fied within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity was not obs erved. The lack of tissue-specific promoter activity, particularly the find ing of promoter activity in cells from tissues in which the maxiK gene is n ot expressed, suggests a complex channel regulatory mechanism for hSlo gene s. Moreover, the lack of similarity of the promoters of the two genes sugge sts that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences. (C) 1999 Elsevier Science B.V. Al l rights reserved.