Department of Biochemistry & Molecular Biology, Libin Cardiovascular Institute and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
Department of Biochemistry & Molecular Biology, Libin Cardiovascular Institute and Hotchkiss Brain Institute, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada.
J Biol Chem. 2021 Jan-Jun;296:100092. doi: 10.1074/jbc.RA120.015037. Epub 2020 Nov 23.
The family of K-dependent Na/Ca-exchangers, NCKX, are important mediators of cellular Ca efflux, particularly in neurons associated with sensory transduction. The NCKX family comprises five proteins, NCKX1-5, each being the product of a different SLC24 gene. NCKX4 (SLC24A4) has been found to have a critical role in termination and adaptation of visual and olfactory signals, melanocortin-dependent satiety signaling, and the maturation of dental enamel. To explore mechanisms that might influence the temporal control of NCKX4 activity, a yeast two-hybrid system was used to search for protein interaction partners. We identified calmodulin as a partner for NCKX4 and confirmed the interaction using glutathione-S-transferase fusion pull-down. Calmodulin binding to NCKX4 was demonstrated in extracts from mouse brain and in transfected HEK293 cells. Calmodulin bound in a Ca-dependent manner to a motif present in the central cytosolic loop of NCKX4 and was abolished by the double-mutant I328D/F334D. When cotransfected in HEK293 cells, calmodulin bound to NCKX4 under basal conditions and induced a ∼2.5-fold increase in NCKX4 abundance, but did not influence either cellular location or basal activity. When purinergic stimulation of NCKX4 was examined in these cells, coexpression of wild-type calmodulin, but not a Ca binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca binding to calmodulin prepositioned on NCKX4 induces a slow conformational rearrangement that interferes with purinergic stimulation of the exchanger, possibly by obscuring T331, a previously identified potential protein kinase C site.
K 依赖性 Na/Ca 交换器家族（NCKX）是细胞内 Ca 外流的重要调节剂，特别是在与感觉转导相关的神经元中。NCKX 家族由五个蛋白组成，即 NCKX1-5，它们分别是不同 SLC24 基因的产物。已经发现 NCKX4（SLC24A4）在视觉和嗅觉信号的终止和适应、黑皮质素依赖性饱腹感信号传递以及牙釉质的成熟中起着关键作用。为了探索可能影响 NCKX4 活性时间控制的机制，我们使用酵母双杂交系统来搜索蛋白相互作用伙伴。我们发现钙调蛋白是 NCKX4 的一个伴侣，并通过谷胱甘肽 S-转移酶融合下拉实验证实了这种相互作用。钙调蛋白与来自小鼠大脑的提取物和转染的 HEK293 细胞中的 NCKX4 结合。钙调蛋白以 Ca 依赖性方式与 NCKX4 中心胞质环中的一个基序结合，并被双突变 I328D/F334D 所消除。当在 HEK293 细胞中共转染时，钙调蛋白在基础条件下与 NCKX4 结合，并使 NCKX4 的丰度增加约 2.5 倍，但不影响细胞内位置或基础活性。当在这些细胞中检查嘌呤能刺激 NCKX4 时，与野生型钙调蛋白共表达，但不是 Ca 结合缺陷型钙调蛋白突变体，以时间依赖性方式抑制 NCKX4 的激活。我们提出，钙调蛋白与 NCKX4 上预先结合的 Ca 结合诱导缓慢的构象重排，可能通过掩盖先前鉴定的潜在蛋白激酶 C 位点 T331，干扰嘌呤能对交换器的刺激。
