Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism

Abstract

Regulation of ion-transport in the Na(+)/Ca(2+) exchanger (NCX) occurs via its cytoplasmic Ca(2+)-binding domains, CBD1 and CBD2. Here, we present a mechanism for NCX activation and inactivation based on data obtained using NMR, isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). We initially determined the structure of the Ca(2+)-free form of CBD2-AD and the structure of CBD2-BD that represent the two major splice variant classes in NCX1. Although the apo-form of CBD2-AD displays partially disordered Ca(2+)-binding sites, those of CBD2-BD are entirely unstructured even in an excess of Ca(2+). Striking differences in the electrostatic potential between the Ca(2+)-bound and -free forms strongly suggest that Ca(2+)-binding sites in CBD1 and CBD2 form electrostatic switches analogous to C(2)-domains. SAXS analysis of a construct containing CBD1 and CBD2 reveals a conformational change mediated by Ca(2+)-binding to CBD1. We propose that the electrostatic switch in CBD1 and the associated conformational change are necessary for exchanger activation. The response of the CBD1 switch to intracellular Ca(2+) is influenced by the closely located cassette exons. We further propose that Ca(2+)-binding to CBD2 induces a second electrostatic switch, required to alleviate Na(+)-dependent inactivation of Na(+)/Ca(2+) exchange. In contrast to CBD1, the electrostatic switch in CBD2 is isoform- and splice variant-specific and allows for tailored exchange activities.

Fig. 1.

Model of intact NCX and CBD2 NMR structures, representing the two major NCX1 splice variant classes. (A) Hypothetical NCX model consisting of four domains: Transmembrane domain (gray; residues 1–216 and 706–903), catenin-like domain (blue; residues 217–370 and 651–705), CBD1 (red; residues 371–500) and CBD2 (green; residues 501–650) with the numbering based on the canine NCX1 AD-splice variant (NCX1.4). At high intracellular Ca²⁺ concentrations four Ca²⁺ ions are bound to CBD1 whereas two Ca²⁺ ions are coordinated by CBD2-AD. The CBD2 ribbon diagram depicts the location of the regions encoding the mutually exclusive exon A or B in blue and the cassette exons in yellow. (B) Ensemble of NMR structures displaying the Ca²⁺-binding sites of CBD2-AD in the absence of Ca²⁺. Residues 578–581 and 648–657 are disordered and colored yellow. In the blow-up of the central chain, the crucial Lys-585 maintains a salt-bridge with Asp-552 and thereby stabilizes the CBD2-AD Ca²⁺-binding sites. (C) Ensemble of CBD2-BD NMR structures with an increased number of disordered residues (yellow) despite the presence of 10 mM CaCl₂.

Fig. 2.

Thermodynamical analyses of CBD1 and CBD2 Ca²⁺-binding sites using ITC. (A) ITC curves of NCX1, NCX2, and NCX3 CBD1, obtained at pH 7 in 30 mM NaCl and 130 mM KCl, confirm binding of four Ca²⁺ ions with likely comparable affinities. (B) Isoform- and splice variant-specific key-residues 552, 578, and 585 that form the CBD2 Ca²⁺-binding sites between the CD- and EF-loops. (C) Comparison of the NCX1 CBD2-AD binding isotherm (5) with those of NCX2 CBD2, NCX3 CBD2-AC, and NCX3 CBD2-B suggests binding of one, two, and three Ca²⁺ ions, respectively. (D) Comparison of NCX1 CBD12-AD and CBD12-ACDEF Ca²⁺-binding isotherms.

Fig. 3.

Electrostatic potentials of the CBD1 and CBD2 Ca²⁺-binding sites in the presence and absence of Ca²⁺ shown in topview. (A) Ca²⁺-bound state of NCX1 CBD1 (PDB code: 2DPK) (6). (B) Ca²⁺-free state of NCX1 CBD1, calculated from the Ca²⁺-bound form with its four Ca²⁺ ions removed. (C) NCX1 CBD2-AD coordinating two Ca²⁺ ions (PDB code: 2QVM) (7). (D) Ca²⁺-free state of NCX1 CBD2-AD as determined by NMR (this work, PDB code: 2KLS) and (E) NCX1 CBD2-BD (this work, PDB code: 2KLT) incapable of Ca²⁺ binding despite the presence of 10 mM CaCl₂. The red and blue meshes represent isocontours of the electrostatic potential at −5 and +5 kT/e that were calculated with the program apbs (29) in the presence of 0.15 M KCl and visualized using the python molecular viewer PMV (30).

Fig. 4.

SAXS shape reconstructions. Analyses of brain CBD12-AD (A) and heart CBD12-ACDEF (B) constructs in the presence and absence of Ca²⁺ reveal a conformational change induced by Ca²⁺ binding to CBD1. Pink beads reflect constructs in the absence of Ca²⁺ whereas yellow beads represent the Ca²⁺ bound forms. All beads and rigid-body models are superimposed on the CBD1 beads of the CBD12-AD construct.

Fig. 5.

Hypothetical dual electrostatic switch mechanism in NCX regulation. (A) Inactive, Ca²⁺-free NCX in extended conformation. (B) Submicromolar Ca²⁺ concentrations induce a conformational change via the electrostatic switch in CBD1 that results in a compaction of the Ca²⁺-binding domains and probably reduces tension on the linker regions to the CLD. (C) Binding of Ca²⁺ to CBD2 allows for sustained Na⁺/Ca²⁺ exchange and removes counteracting Na⁺-dependent inactivation.