Visser Frank, Valsecchi Valeria, Annunziato Lucio, Lytton Jonathan
Libin Cardiovascular Institute of Alberta and the Hotchkiss Brain Institute, Department of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada and the.
Division of Pharmacology, Department of Neuroscience, School of Medicine, Federico II University of Naples, 80131 Naples, Italy.
J Biol Chem. 2007 Feb 16;282(7):4453-4462. doi: 10.1074/jbc.M610582200. Epub 2006 Dec 15.
K(+)-dependent Na(+)/Ca(2+) exchangers (NCKX) catalyze cytosolic Ca(2+) extrusion and are particularly important for neuronal Ca(2+) signaling. Of the five mammalian isoforms, the detailed functional characteristics have only been reported for NCKX1 and -2. In the current study, the functional characteristics of recombinant NCKX3 and -4 expressed in HEK293 cells were determined and compared with those of NCKX2. Although the apparent affinities of the three isoforms for Ca(2+) and Na(+) were similar, NCKX3 and -4 displayed approximately 40-fold higher affinities for K(+) ions than NCKX2. Functional analysis of various NCKX2 mutants revealed that mutation of Thr-551 to Ala, the corresponding residue in NCKX4, resulted in an apparent K(+) affinity shift to one similar to that of NCKX4 without a parallel shift in apparent Ca(2+) affinity. In the converse situation, when Gln-476 of NCKX4 was converted to Lys, the corresponding residue in NCKX2, both the K(+) and Ca(2+) affinities were reduced. These results indicate that the apparently low K(+) affinity of NCKX2 requires a Thr residue at position 551 that may reduce the conformational flexibility and/or K(+) liganding strength of side-chain moieties on critical neighboring residues. This interaction appears to be specific to the structural context of the NCKX2 K(+) binding pocket, because it was not possible to recreate the K(+)-specific low affinity phenotype with reciprocal mutations in NCKX4. The results of this study provide important information about the structure and function of NCKX proteins and will be critical to understanding their roles in neuronal Ca(2+) signaling.
钾离子依赖型钠钙交换体（NCKX）催化胞质钙离子外流，对神经元钙信号传导尤为重要。在五种哺乳动物亚型中，仅报道了NCKX1和NCKX2的详细功能特性。在本研究中，测定了在HEK293细胞中表达的重组NCKX3和NCKX4的功能特性，并与NCKX2进行了比较。尽管这三种亚型对钙离子和钠离子的表观亲和力相似，但NCKX3和NCKX4对钾离子的亲和力比NCKX2高约40倍。对各种NCKX2突变体的功能分析表明，将苏氨酸-551突变为丙氨酸（NCKX4中的对应残基）会导致表观钾离子亲和力转变为与NCKX4相似，而表观钙离子亲和力没有平行转变。在相反的情况下，当NCKX4的谷氨酰胺-476转变为NCKX2中的对应残基赖氨酸时，钾离子和钙离子的亲和力均降低。这些结果表明，NCKX2明显较低的钾离子亲和力需要551位的苏氨酸残基，该残基可能会降低关键相邻残基上侧链部分的构象灵活性和/或钾离子配位强度。这种相互作用似乎特定于NCKX2钾离子结合口袋的结构背景，因为在NCKX4中进行相互突变无法重现钾离子特异性低亲和力表型。本研究结果提供了有关NCKX蛋白结构和功能的重要信息，对于理解它们在神经元钙信号传导中的作用至关重要。
