Barry E L, Gesek F A, Froehner S C, Friedman P A
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, NH 03755-3835, USA.
Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10914-8. doi: 10.1073/pnas.92.24.10914.
Osteoblasts express calcium channels that are thought to be involved in the transduction of extracellular signals regulating bone metabolism. The molecular identity of the pore-forming subunit (alpha 1) of L-type calcium channel(s) was determined in rat osteosarcoma UMR-106 cells, which express an osteoblast phenotype. A homology-based reverse transcriptase-polymerase chain reaction cloning strategy was employed that used primers spanning the fourth domain. Three types of cDNAs were isolated, corresponding to the alpha 1S (skeletal), alpha 1C (cardiac), and alpha 1D (neuroendocrine) isoforms. In the transmembrane segment IVS3 and the extracellular loop formed by the IVS3-S4 linker, a single pattern of mRNA splicing was found that occurs in all three types of calcium channel transcripts. Northern blot analysis revealed an 8.6-kb mRNA that hybridized to the alpha 1C probe and 4.8- and 11.7-kb mRNAs that hybridized to the alpha 1S and alpha 1D probes. Antisense oligonucleotides directed to the calcium channel alpha 1D transcript, but not those directed to alpha 1S or alpha 1C transcripts, inhibited the rise of intracellular calcium induced by parathyroid hormone. However, alpha 1D antisense oligonucleotides had no effect on the accumulation of cAMP induced by parathyroid hormone. When L-type calcium channels were activated with Bay K 8644, antisense oligonucleotides to each of the three isoforms partially inhibited the rise of intracellular calcium. The present results provide evidence for the expression of three distinct calcium channel alpha 1-subunit isoforms in an osteoblast-like cell line. We conclude that the alpha 1D isoform is selectively activated by parathyroid hormone.
成骨细胞表达钙通道，这些钙通道被认为参与调节骨代谢的细胞外信号转导。在表达成骨细胞表型的大鼠骨肉瘤UMR-106细胞中，确定了L型钙通道孔形成亚基（α1）的分子身份。采用了基于同源性的逆转录酶-聚合酶链反应克隆策略，该策略使用跨越第四结构域的引物。分离出三种类型的cDNA，分别对应于α1S（骨骼肌型）、α1C（心脏型）和α1D（神经内分泌型）同工型。在跨膜片段IVS3和由IVS3-S4连接子形成的细胞外环中，发现了一种单一的mRNA剪接模式，这种模式出现在所有三种类型的钙通道转录本中。Northern印迹分析显示，一个8.6 kb的mRNA与α1C探针杂交，4.8 kb和11.7 kb的mRNA与α1S和α1D探针杂交。针对钙通道α1D转录本的反义寡核苷酸，而不是针对α1S或α1C转录本的反义寡核苷酸，抑制了甲状旁腺激素诱导的细胞内钙升高。然而，α1D反义寡核苷酸对甲状旁腺激素诱导的cAMP积累没有影响。当用Bay K 8644激活L型钙通道时，针对三种同工型的反义寡核苷酸部分抑制了细胞内钙的升高。目前的结果为在成骨细胞样细胞系中表达三种不同的钙通道α1亚基同工型提供了证据。我们得出结论，α1D同工型被甲状旁腺激素选择性激活。
