Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism.
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.
Hilge
Mark
M
Protein Biophysics, Institute for Molecules and Materials, Radboud University Nijmegen, 6525 GA Nijmegen, The Netherlands. markhilge@mac.com
Aelen
Jan
J
Foarce
Alice
A
Perrakis
Anastassis
A
Vuister
Geerten W
GW
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004285:Dogs; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism; D019831:Sodium-Calcium Exchanger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055672:Static Electricity; D013816:Thermodynamics
D020033:Protein Isoforms; C491739:Slc8a2 protein, mouse; C491101:Slc8a3 protein, mouse; D019831:Sodium-Calcium Exchanger; C110374:sodium-calcium exchanger 1; D012964:Sodium; D002118:Calcium