Dhulipala P D, Kotlikoff M I
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6046, USA.
Biochim Biophys Acta. 1999 Feb 16;1444(2):254-62. doi: 10.1016/s0167-4781(98)00276-0.
Large conductance, calcium-activated potassium (maxiK) channels are expressed in nerve, muscle, and other cell types and are important determinants of smooth muscle tone. To determine the mechanisms involved in the transcriptional regulation of maxiK channels, we characterized the promoter regions of the pore forming (alpha) and regulatory (beta) subunits of the human channel complex. Maximum promoter activity (up to 12.3-fold over control) occurred between nucleotides -567 and -220 for the alpha subunit (hSlo) gene. The minimal 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. Additionally, 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 sequence, 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'-flanking region revealed a TATA box at position -77 and maximum promoter activity (up to 11.0-fold) in a 200 bp region upstream from the cap site. Binding sites for GATA-1, Myo-D, c-myb, Ets-1/Elk-1, Ap-1, and Ik-2 were identified within this sequence. Two CCTCCC elements are present in the hSlo beta subunit promoter, but tissue-specific transcriptional activity was not observed. The lack of tissue-specific promoter activity, particularly the finding of promoter activity in cells from tissues in which the maxiK gene is not expressed, suggests a complex channel regulatory mechanism for hSlo genes. Moreover, the lack of similarity of the promoters of the two genes suggests that regulation of coordinate expression of the subunits does not occur through equivalent cis-acting sequences.
大电导钙激活钾通道（maxiK通道）在神经、肌肉和其他细胞类型中表达，是平滑肌张力的重要决定因素。为了确定参与maxiK通道转录调控的机制，我们对人通道复合物的孔形成（α）亚基和调节（β）亚基的启动子区域进行了表征。α亚基（hSlo）基因在核苷酸-567至-220之间出现最大启动子活性（比对照高12.3倍）。最小启动子富含GC，有5个Sp-1结合位点和几个TCC重复序列。在5'侧翼序列中观察到其他转录因子结合基序，包括c/EBP、NF-κB、PU.1、PEA-3、Myo-D和E2A。此外，一个CCTCCC序列位于类TATA序列上游27 bp处，该序列可增加平滑肌中SM1/2基因的转录活性，其位置与在SM1/2 5'侧翼区域中发现的位置相同。然而，当转染到平滑肌和其他细胞类型中时，该启动子指导等量表达。对hSloβ亚基5'侧翼区域的分析显示，在-77位有一个TATA盒，并且在帽位点上游200 bp区域有最大启动子活性（高达11.0倍）。在该序列中鉴定出GATA-1、Myo-D、c-myb、Ets-1/Elk-1、Ap-1和Ik-2的结合位点。hSloβ亚基启动子中有两个CCTCCC元件，但未观察到组织特异性转录活性。缺乏组织特异性启动子活性，特别是在maxiK基因未表达的组织来源的细胞中发现启动子活性，提示hSlo基因存在复杂的通道调节机制。此外，两个基因启动子缺乏相似性表明亚基协同表达的调控不是通过等效的顺式作用序列发生的。
