The mitochondrial Na+/Ca2+ exchanger upregulates glucose dependent Ca2+ signalling linked to insulin secretion.

Mitochondria mediate dual metabolic and Ca(2+) shuttling activities. While the former is required for Ca(2+) signalling linked to insulin secretion, the role of the latter in β cell function has not been well understood, primarily because the molecular identity of the mitochondrial Ca(2+) transporters were elusive and the selectivity of their inhibitors was questionable. This study focuses on NCLX, the recently discovered mitochondrial Na(+)/Ca(2+) exchanger that is linked to Ca(2+) signalling in MIN6 and primary β cells. Suppression either of NCLX expression, using a siRNA construct (siNCLX) or of its activity, by a dominant negative construct (dnNCLX), enhanced mitochondrial Ca(2+) influx and blocked efflux induced by glucose or by cell depolarization. In addition, NCLX regulated basal, but not glucose-dependent changes, in metabolic rate, mitochondrial membrane potential and mitochondrial resting Ca(2+). Importantly, NCLX controlled the rate and amplitude of cytosolic Ca(2+) changes induced by depolarization or high glucose, indicating that NCLX is a critical and rate limiting component in the cross talk between mitochondrial and plasma membrane Ca(2+) signalling. Finally, knockdown of NCLX expression was followed by a delay in glucose-dependent insulin secretion. These findings suggest that the mitochondrial Na(+)/Ca(2+) exchanger, NCLX, shapes glucose-dependent mitochondrial and cytosolic Ca(2+) signals thereby regulating the temporal pattern of insulin secretion in β cells

A Common Ca2+-Driven Interdomain Module Governs Eukaryotic NCX Regulation

Na+/Ca2+ exchanger (NCX) proteins mediate Ca2+-fluxes across the cell membrane to maintain Ca2+ homeostasis in many cell types. Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ 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 Ca2+. 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 Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-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 Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants

Structure-Based Function and Regulation of NCX Variants: Updates and Challenges

The plasma-membrane homeostasis Na+/Ca2+ exchangers (NCXs) mediate Ca2+ extrusion/entry to dynamically shape Ca2+ signaling/in biological systems ranging from bacteria to humans. The NCX gene orthologs, isoforms, and their splice variants are expressed in a tissue-specific manner and exhibit nearly 104-fold differences in the transport rates and regulatory specificities to match the cell-specific requirements. Selective pharmacological targeting of NCX variants could benefit many clinical applications, although this intervention remains challenging, mainly because a full-size structure of eukaryotic NCX is unavailable. The crystal structure of the archaeal NCX_Mj, in conjunction with biophysical, computational, and functional analyses, provided a breakthrough in resolving the ion transport mechanisms. However, NCX_Mj (whose size is nearly three times smaller than that of mammalian NCXs) cannot serve as a structure-dynamic model for imitating high transport rates and regulatory modules possessed by eukaryotic NCXs. The crystal structures of isolated regulatory domains (obtained from eukaryotic NCXs) and their biophysical analyses by SAXS, NMR, FRET, and HDX-MS approaches revealed structure-based variances of regulatory modules. Despite these achievements, it remains unclear how multi-domain interactions can decode and integrate diverse allosteric signals, thereby yielding distinct regulatory outcomes in a given ortholog/isoform/splice variant. This article summarizes the relevant issues from the perspective of future developments

Structure-Functional Basis of Ion Transport in Sodium–Calcium Exchanger (NCX) Proteins

The membrane-bound sodium–calcium exchanger (NCX) proteins shape Ca2+ homeostasis in many cell types, thus participating in a wide range of physiological and pathological processes. Determination of the crystal structure of an archaeal NCX (NCX_Mj) paved the way for a thorough and systematic investigation of ion transport mechanisms in NCX proteins. Here, we review the data gathered from the X-ray crystallography, molecular dynamics simulations, hydrogen–deuterium exchange mass-spectrometry (HDX-MS), and ion-flux analyses of mutants. Strikingly, the apo NCX_Mj protein exhibits characteristic patterns in the local backbone dynamics at particular helix segments, thereby possessing characteristic HDX profiles, suggesting structure-dynamic preorganization (geometric arrangements of catalytic residues before the transition state) of conserved α1 and α2 repeats at ion-coordinating residues involved in transport activities. Moreover, dynamic preorganization of local structural entities in the apo protein predefines the status of ion-occlusion and transition states, even though Na+ or Ca2+ binding modifies the preceding backbone dynamics nearby functionally important residues. Future challenges include resolving the structural-dynamic determinants governing the ion selectivity, functional asymmetry and ion-induced alternating access. Taking into account the structural similarities of NCX_Mj with the other proteins belonging to the Ca2+/cation exchanger superfamily, the recent findings can significantly improve our understanding of ion transport mechanisms in NCX and similar proteins