Protein-protein interaction between cPLA2 and splice variants of α-subunit of BK channels
Protein-protein interaction between cPLA2 and splice variants of α-subunit of BK channels
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DOI：
10.1152/ajpcell.00221.2009
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发表时间：
2010-02-01
影响因子：
5.5
通讯作者：
Denson, Donald D.
中科院分区：
文献类型：
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作者：
Li, Juan;Al-Khalili, Otor;Denson, Donald D.
Li J，Al-Khalili O，Ramosevac S，Eaton DC，Denson DD. cPLA（2）与BK通道α亚基剪接变体之间的蛋白质-蛋白质相互作用。美国生理学杂志细胞生理学298：C251-C262，2010年。首次发表于2009年11月25日; doi：10.1152/ajpcell.00221.2009。改变大电导Ca 2+激活钾（BK）通道的剪接变体组成可以改变其活性和对Ca 2+和其他活性调节剂的表观敏感性。我们推测，GH 3和GH4细胞对花生四烯酸的反应性差异是由于两种剪接变体的差异，一种存在于GH 3细胞中，另一种存在于GH4细胞中。两种剪接变体的序列在几个方面彼此不同，但最大的差异是BK α亚基的COOH末端是否存在27个氨基酸。含有27个氨基酸的变体的开放概率被花生四烯酸显著增加，而缺少27个氨基酸的变体对花生四烯酸不敏感。此外，BK通道对花生四烯酸的敏感性取决于胞浆磷脂酶A（2）（cPLA 2）。在这里，我们使用哺乳动物Matchmaker双杂交测定和具有[rSlo（27）]和不具有[rSlo（0）] 27-氨基酸基序的两种BK α-亚基构建体来确定cPLA（2）是否与一种构建体[ rSlo（27）]缔合而不与另一种缔合。我们假设cPLA（2）的差异性结合可能解释了两种构建体以及GH 3和GH4细胞对花生四烯酸的不同反应性。我们发现cPLA（2）与rSlo（27）的COOH末端强相关，而与rSlo（0）的相关性非常弱。我们还发现花生四烯酸对rSlo（0）的亲和力低于对rSlo（27）的亲和力。我们的结论是，GH4细胞中BK通道对花生四烯酸缺乏反应，部分原因是cPLA（2）与rSlo（0）α亚基的COOH末端结合不良，这与花生四烯酸不敏感的GH4细胞中发现的剪接变体非常相似。
Li J, Al-Khalili O, Ramosevac S, Eaton DC, Denson DD. Protein-protein interaction between cPLA(2) and splice variants of alpha-subunit of BK channels. Am J Physiol Cell Physiol 298: C251-C262, 2010. First published November 25, 2009; doi: 10.1152/ajpcell.00221.2009.-Altering the splice variant composition of large-conductance Ca2+-activated potassium (BK) channels can alter their activity and apparent sensitivity to Ca2+ and other regulators of activity. We hypothesized that differences in the responsiveness to arachidonic acid of GH3 and GH4 cells was due to a difference in two splice variants, one present in GH3 cells and the other in GH4 cells. The sequences of the two splice variants differ from one another in several ways, but the largest difference is the presence or absence of 27 amino acids in the COOH terminus of the BK alpha-subunit. Open probability of the variant containing the 27 amino acids is significantly increased by arachidonic acid, while the variant lacking the 27 amino acids is insensitive to arachidonic acid. In addition, sensitivity of BK channels to arachidonic acid depends on cytosolic phospholipase A(2) (cPLA2). Here we used the Mammalian Matchmaker two-hybrid assay and two BK alpha-subunit constructs with [rSlo(27)] and without [rSlo(0)] the 27-amino acid motif to determine whether cPLA(2) associates with one construct [ rSlo(27)] and not the other. We hypothesized that differential association of cPLA(2) might explain the differing responsiveness of the two constructs and GH3 and GH4 cells to arachidonic acid. We found that cPLA(2) is strongly associated with the COOH terminus of rSlo(27) and only very weakly associated with rSlo(0). We also found that arachidonic acid has a lower affinity for rSlo(0) than for rSlo(27). We conclude that the lack of response of BK channels in GH4 cells to arachidonic acid can be explained, in part, by the poor binding of cPLA(2) to the COOH terminus of the rSlo(0) alpha-subunit, which is very similar to the splice variant found in the arachidonic acid-insensitive GH4 cells.