CHARACTERIZATION OF TISSUE-EXPRESSED ALPHA-SUBUNITS OF THE HIGH-CONDUCTANCE CA2+-ACTIVATED K+ CHANNEL
CHARACTERIZATION OF TISSUE-EXPRESSED ALPHA-SUBUNITS OF THE HIGH-CONDUCTANCE CA2+-ACTIVATED K+ CHANNEL
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DOI：
10.1074/jbc.270.38.22434
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发表时间：
1995-09-22
影响因子：
4.8
通讯作者：
GARCIA, ML
中科院分区：
文献类型：
--
作者：
KNAUS, HG;EBERHART, A;GARCIA, ML
来自气管平滑肌的纯化的高电导钙激活钾（maxi-K）通道已被证明由一个60-70-kDa的α亚基和一个31-kDa的β亚基组成，α亚基由α基因编码。虽然β亚基的大小是编码这种蛋白质的基因产物的预期大小，但α亚基的大小小于从β编码区预测的大小。为了确定这种差异的基础，已经提出了针对β的序列导向抗体。这些抗体特异性地沉淀出mglutamine的体外翻译产物，其产生预期分子量（135 kDa）的α亚基。使用平滑肌肌膜、骨骼肌T-小管以及来自GH（3）细胞的膜的免疫染色实验揭示存在表观分子量为125 kDa的Lu亚基，这些膜和纯化制剂中表达的α亚基大小的差异是由于高度可重复的蛋白水解衰变造成的，这种衰变主要发生在maxi-g通道纯化的后期阶段。在研究的纯化maxi-K通道制剂中，全长α亚基，使用适当的抗体可以检测到90 kDa的中间大小产物和65-kDa多肽，以及其它较小的片段。蛋白水解仅发生在β-内酰胺酶的长C-末端尾内的两个不同位置。此外，提供了在膜结合以及纯化的maxi-It通道中不同剪接变体的组织表达的证据。
Purified high conductance calcium-activated potassium (maxi-K) channels from tracheal smooth muscle have been shown to consist of a 60-70-kDa alpha subunit, encoded by the slo gene, and a 31-kDa beta subunit. Although the size of the beta subunit is that expected for the product of the gene encoding this protein, the size of the alpha subunit is smaller than that predicted from the slo coding region, To determine the basis for this discrepancy, sequence-directed antibodies have been raised against slo. These antibodies specifically precipitate the in vitro translation product of mslo, which yields an alpha subunit of the expected molecular mass (135 kDa), Immunostaining experiments employing smooth muscle sarcolemma, skeletal muscle T-tubules, as well as membranes derived from GH(3) cells reveal the presence of an Lu subunit with an apparent molecular mass of 125 kDa, The difference in size of the alpha subunit as expressed in these membranes and the purified preparations is due to a highly reproducible proteolytic decay that occurs mostly at an advanced stage of the maxi-g channel purification, In the purified maxi-K channel preparations investigated, the full-length alpha subunit, an intermediate size product of 90 kDa, and the 65-kDa polypeptide, as well as other smaller fragments can be detected using appropriate antibodies, Proteolysis occurs exclusively at two distinct positions within the long C-terminal tail of slo, In addition, evidence for the tissue expression of distinct splice variants in membrane-bound as well as purified maxi-It channels is presented.