Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095-1751, USA.
Proc Natl Acad Sci U S A. 2011 Jan 25;108(4):1699-704. doi: 10.1073/pnas.1016114108. Epub 2011 Jan 5.
Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.
细胞质中的 Ca(2+) 被认为通过结合位于跨膜片段 5 和 6 之间的大细胞内环中的两个相邻的 Ca(2+) 结合域 (CBD1 和 CBD2) 来调节 Na(+)-Ca(2+) 交换器 (NCX) 的活性。我们研究了 Ca(2+) 依赖性运动，即通过在大细胞质环内位置 266 处标记的交换蛋白之间的 FRET 变化来检测。数据表明，交换器在质膜中组装成二聚体。Ca(2+) 的加入会减小 NCX 对之间细胞质环的距离。在 CBD1 中的 Ca(2+) 协调位点突变 (D421A、E451A 和 D500V) 后，检测到的配对 NCX 之间的 Ca(2+) 依赖性运动被消除，而 CBD2 中的主要 Ca(2+) 协调位点突变 (E516L) 没有影响。因此，NCX 二聚体的 Ca(2+) 诱导构象变化来自 CBD1 的运动。CBD1、CBD2 和 CBD1-CBD2 肽的 FRET 研究显示出具有不同表观亲和力的 Ca(2+) 依赖性运动。CBD1-CBD2 显示出与全长 NCX 相似的 Ca(2+) 依赖性表型，但与 CBD1 和 CBD2 不同。
