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A common Ca2+-driven interdomain module governs eukaryotic NCX regulation.

ABSTRACT: Na(+)/Ca(2+) exchanger (NCX) proteins mediate Ca(2+)-fluxes across the cell membrane to maintain Ca(2+) homeostasis in many cell types. Eukaryotic NCX contains Ca(2+)-binding regulatory domains, CBD1 and CBD2. Ca(2+) binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca(2+). To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca(2+) binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca(2+)-driven interdomain switch controls slow dissociation of "occluded" Ca(2+) from the primary sensor and thus dictates Ca(2+) sensing dynamics. In the Ca(2+)-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca(2+)-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.

SUBMITTER: Giladi M

PROVIDER: S-EPMC3386913 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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A common Ca2+-driven interdomain module governs eukaryotic NCX regulation.

Giladi Moshe M Sasson Yehezkel Y Fang Xianyang X Hiller Reuben R Buki Tal T Wang Yun-Xing YX Hirsch Joel A JA Khananshvili Daniel D

PloS one 20120629 6

Na(+)/Ca(2+) exchanger (NCX) proteins mediate Ca(2+)-fluxes across the cell membrane to maintain Ca(2+) homeostasis in many cell types. Eukaryotic NCX contains Ca(2+)-binding regulatory domains, CBD1 and CBD2. Ca(2+) binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca(2+). To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammal ...[more]

PMID: 22768191

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Project description:We have cloned the squid neuronal Na+-Ca2+ exchanger, NCX-SQ1, expressed it in Xenopus oocytes, and characterized its regulatory and ion transport properties in giant excised membrane patches. The squid exchanger shows 58% identity with the canine Na+-Ca2+ exchanger (NCX1.1). Regions determined to be of functional importance in NCX1 are well conserved. Unique among exchanger sequences to date, NCX-SQ1 has a potential protein kinase C phosphorylation site (threonine 184) between transmembrane segments 3 and 4 and a tyrosine kinase site in the Ca2+ binding region (tyrosine 462). There is a deletion of 47 amino acids in the large intracellular loop of NCX-SQ1 in comparison with NCX1. Similar to NCX1, expression of NCX-SQ1 in Xenopus oocytes induced cytoplasmic Na+-dependent 45Ca2+ uptake; the uptake was inhibited by injection of Ca2+ chelators. In giant excised membrane patches, the NCX-SQ1 outward exchange current showed Na+-dependent inactivation, secondary activation by cytoplasmic Ca2+, and activation by chymotrypsin. The NCX-SQ1 exchange current was strongly stimulated by both ATP and the ATP-thioester, ATP gamma S, in the presence of F- (0.2 mM) and vanadate (50 microM), and both effects reversed on application of a phosphatidylinositol-4',5'-bisphosphate antibody. NCX1 current was stimulated by ATP, but not by ATP gamma S. Like NCX1 current, NCX-SQ1 current was strongly stimulated by phosphatidylinositol-4',5'-bisphosphate liposomes. In contrast to results in squid axon, NCX-SQ1 was not stimulated by phosphoarginine (5-10 mM). After chymotrypsin treatment, both the outward and inward NCX-SQ1 exchange currents were more strongly voltage dependent than NCX1 currents. Ion concentration jump experiments were performed to estimate the relative electrogenicity of Na+ and Ca2+ transport reactions. Outward current transients associated with Na+ extrusion were much smaller for NCX-SQ1 than NCX1, and inward current transients associated with Ca2+ extrusion were much larger. For NCX-SQ1, charge movements of Ca2+ transport could be defined in voltage jump experiments with a low cytoplasmic Ca2+ (2 microM) in the presence of high extracellular Ca2+ (4 mM). The rates of charge movements showed "U"-shaped dependence on voltage, and the slopes of both charge-voltage and rate-voltage relations (1,600 s-1 at 0 mV) indicated an apparent valency of -0.6 charges for the underlying reaction. Evidently, more negative charge moves into the membrane field in NCX-SQ1 than in NCX1 when ions are occluded into binding sites.

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A common Ca2+-driven interdomain module governs eukaryotic NCX regulation.

Publications

A common Ca2+-driven interdomain module governs eukaryotic NCX regulation.

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