Calmodulin binds and modulates K+-dependent Na+/Ca2+-exchanger isoform 4, NCKX4

The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ 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 Ca2+-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 Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ 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. The family of K+-dependent Na+/Ca2+-exchangers, NCKX, are important mediators of cellular Ca2+ 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 Ca2+-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 Ca2+ binding-deficient calmodulin mutant, suppressed NCKX4 activation in a time-dependent manner. We propose that Ca2+ 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. Cellular Ca2+ signaling events underlie a broad range of physiological processes in all cells, particularly excitable cells such as neurons (1Brini M. Cali T. Ottolini D. Carafoli E. Neuronal calcium signaling: function and dysfunction.Cell Mol. Life Sci. 2014; 71: 2787-2814Crossref PubMed Scopus (249) Google Scholar). In all these environments, Ca2+ influx must be balanced by efflux, both to terminate signals and to provide longer-term homeostasis. The family of K+-dependent Na+/Ca2+-exchangers, NCKX, has emerged as a particularly important pathway for modulating Ca2+ efflux in a variety of tissues, particularly ones associated with sensory transduction (2Hassan M.T. Lytton J. Potassium-dependent sodium-calcium exchanger (NCKX) isoforms and neuronal function.Cell Calcium. 2020; 86: 102135Crossref PubMed Scopus (5) Google Scholar). There are five known members of the NCKX family, NCKX1-5, each being a product of a different SLC24A gene. NCKX proteins couple both Na+ and K+ gradients to the movement of Ca2+ with a stoichiometry of 4 Na+ ions in exchange for 1 K+ ion and 1 Ca2+ ion and are thus thermodynamically poised to extrude Ca2+ over a broad range of physiological ionic conditions (3Lytton J. Li X.F. Dong H. Kraev A. K(+)-dependent Na(+)/Ca(2+) exchangers in the brain.Ann. N. Y. Acad. Sci. 2002; 976: 382-393Crossref PubMed Scopus (54) Google Scholar). The NCKX proteins share that comprises that of a of five a cytosolic loop of and a of five Lytton J. of the brain K(+)-dependent Na(+)/Ca(2+) exchanger N. Y. Acad. Sci. 2002; 976: PubMed Scopus Google Scholar). family the of the particularly the the found in the the and the protein of K(+)-dependent Na(+)/Ca(2+) exchangers Calcium. 2020; 86: PubMed Scopus (5) Google Scholar). NCKX proteins function in a variety of cells and tissues, a range of physiological was the family to be and for role in Ca2+ visual adaptation in in Calcium. PubMed Scopus Google Scholar). was identified as a to Ca2+ in central system neurons and important for X.F. M. Lytton J. of K+-dependent in and PubMed Scopus Google M. D. Li Lytton J. of a sodium-calcium exchanger from Scopus Google Scholar). In and NCKX4 on and to be in the brain as as in A. Li X.F. Dong H. M. Lytton J. of a of the sodium-calcium exchanger family, PubMed Scopus Google X.F. Kraev Lytton J. of a of the sodium-calcium exchanger family, 2002; PubMed Scopus Google Scholar). NCKX4 with was to be critical for termination and adaptation in T. A. H. J. The exchanger NCKX4 for PubMed Scopus Google Scholar). NCKX4 was demonstrated to have in sensory termination and adaptation in olfactory neurons M. J. H. The Na(+)/Ca(2+) exchanger NCKX4 termination and adaptation of the olfactory Scopus Google in melanocortin-dependent satiety signaling in the of the X.F. Lytton J. role for the K+-dependent in 2014; PubMed Scopus Google and in dental of the exchanger, in PubMed Scopus Google H. Y. H. of in a exchanger, as a of J. PubMed Scopus Google Scholar). identified as the the was to have a important role in in a exchanger, in and PubMed Scopus Google Scholar). in associated with the Cellular of the exchanger and the role of the loop in in PubMed Scopus Google Scholar). in to and for for J. of calcium in the of and PubMed Scopus Google Scholar). and NCKX4 be the in A. Li X.F. Dong H. M. Lytton J. of a of the sodium-calcium exchanger family, PubMed Scopus Google X.F. Kraev Lytton J. of a of the sodium-calcium exchanger family, 2002; PubMed Scopus Google cellular location has not been The role of NCKX isoforms in adaptation and termination of sensory signals, as as in that temporal of might be important for to physiological (2Hassan M.T. Lytton J. Potassium-dependent sodium-calcium exchanger (NCKX) isoforms and neuronal function.Cell Calcium. 2020; 86: 102135Crossref PubMed Scopus (5) Google Scholar). in to with the a protein to different M. of and exchangers in PubMed Scopus Google of or exchanger with as with PubMed Scopus Google Scholar). have been for in of or exchanger with as with PubMed Scopus Google Scholar). have that neuronal be by protein kinase C Lytton J. kinase of of brain in HEK293 PubMed Scopus Google Scholar). both and NCKX4 be by purinergic be by as a signaling with from Lytton J. stimulation of K+-dependent exchanger 4 activation by and PubMed Scopus Google Scholar). purinergic stimulation has been to activation of both and kinase Lytton J. stimulation of K+-dependent exchanger 4 activation by and PubMed Scopus Google activation for purinergic stimulation of Lytton J. kinase of of brain in HEK293 PubMed Scopus Google Scholar). 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