Dynamic features of allosteric Ca²⁺ sensor in tissue-specific NCX variants

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• 作者：Moshe Giladi^a ; Hilla Bohbot^a ; Tal Buki^a ; Dan H. Schulze^b ; Reuben Hiller^a ; Daniel Khananshvili^a ; ^{dhanan@post.tau.ac.il}
• 关键词：NCX ; Ca2+ occlusion ; Allosteric sensor ; Regulation ; Splice variants
• 刊名：Cell Calcium
• 出版年：2012
• 期刊代码：7_01434160
• 类别：bio
• 出版时间：June, 2012
• 卷：51
• 期：6
• 页码：478-485
• 文件大小：1016 K

摘要

The Na⁺-Ca²⁺ exchanger (NCX) mediated Ca²⁺ fluxes are essential for handling Ca²⁺ homeostasis in many cell-types. Eukaryotic NCX variants contain regulatory CBD1 and CBD2 domains, whereas in distinct variants the Ca²⁺ binding to Ca3-Ca4 sites of CBD1 results either in sustained activation, inhibition or no effect. CBD2 contains an alternatively spliced segment, which is expressed in a tissue-specific manner although its impact on allosteric regulation remains unclear. Recent studies revealed that the Ca²⁺ binding to Ca3-Ca4 sites results in interdomain tethering of CBDs, which rigidifies CBDs movements with accompanied slow dissociation of 鈥渙ccluded鈥?Ca²⁺. Here we investigate the effects of CBD2 variants on Ca²⁺ occlusion in the two-domain construct (CBD12). Mutational studies revealed that both sites (Ca3 and Ca4) contribute to Ca²⁺ occlusion, whereas after dissociation of the first Ca²⁺ ion the second Ca²⁺ ion becomes occluded. This mechanism is common for the brain, kidney and cardiac splice variants of CBD12, although the occluded Ca²⁺ exhibits 20-50-fold difference in off-rates among the tested variants. Therefore, the spliced exons on CBD2 affect the rate-limiting step of the occluded Ca²⁺ dissociation at the primary regulatory sensor to shape dynamic features of allosteric regulation in NCX variants.