Charge movement and transcription regulation of L-type calcium channel alpha(1S) in skeletal muscle cells.
Several factors, such as Ca(2+), trophic factors and ageing, regulate dihydropyridine-sensitive receptor (DHPR) alpha(1) subunit expression. However, basic mechanisms of DHPR alpha(1S) expression are unknown. To better understand the regulatory elements that control transcription, the 1.2 kb 5'-flanking region fragment immediately upstream of the mouse L-type Ca(2+) channel or DHPR alpha(1S) gene was isolated and sequenced. Luciferase reporter constructs driven by different promoter regions of mouse DHPR alpha(1S) gene were used for transient transfection assays in muscle C2C12 cells. In these preparations we found that three regions corresponding to CREB, GATA-2 and SOX-5 consensus sequence within the 5'-flanking region of the DHPR alpha(1S) gene are important for DHPR alpha(1S) gene transcription. Antisense oligonucleotides against CREB, GATA-2 and SOX-5 significantly reduced charge movement in C2C12 cells. Charge movement was recorded in the whole-cell configuration of the patch clamp technique. Results from cells transfected with antisense (AS) and sense (S) oligonucleotides and nontransfected cells were compared. Charge movement experiments were fitted to a Boltzmann equation. Maximum charge movement (Q(max)) (nC microF(-1), mean +/- S.E.M.) for S- and AS-CREB was 70.3 +/- 2.9 and 52.8 +/- 3.3, respectively (P < 0.05). The same parameter for S- and AS-GATA-2 was 71.3 +/- 3.9 and 48.2 +/- 2.3, respectively (P < 0.05) and for S- and AS-SOX-5 was 70.4 +/- 4.2 and 45.1 +/- 3.2, respectively (P < 0.05). Values recorded in cells transfected with sense S-CREB, S-GATA-2 and S-SOX-5 oligonucleotides were not significantly different from those recorded in nontransfected cells. This study demonstrates that the transcription factors CREB, GATA-2 and SOX-5 play a significant role in the expression of the skeletal muscle DHPR or L-type Ca(2+) channel alpha(1S).
Zheng
Zhenlin
Z
Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA.
Wang
Zhong-Min
ZM
Delbono
Osvaldo
O
D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D017362:Cyclic AMP Response Element-Binding Protein / Q000502:physiology; D004268:DNA-Binding Proteins / Q000502:physiology; D004594:Electrophysiology; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation / Q000502:physiology; D008156:Luciferases / Q000235:genetics; D051379:Mice; D018482:Muscle, Skeletal / Q000166:cytology / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000502:physiology; D023282:Nuclease Protection Assays; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055755:SOXD Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology; D014162:Transfection
D020746:Calcium Channels, L-Type; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; C484171:Gata4 protein, mouse; D009687:Nuclear Proteins; D016376:Oligonucleotides, Antisense; D055755:SOXD Transcription Factors; C089186:Sox5 protein, mouse; D014157:Transcription Factors; D008156:Luciferases