Long-Range Allosteric Regulation of Pumps and Transporters: What Can We Learn from Mammalian NCX Antiporters?

The Ca2+-dependent allosteric regulation of ion-channels, pumps, and transporters is still a subject of multidisciplinary research due to its fundamental significance. The mammalian Na+/Ca2+ exchangers (NCX1–3) and their splice-variants are expressed in a tissue-specific manner to extrude Ca2+ in diverse cell types. Since NCX proteins are involved in regulating numerous physiological and pathophysiological events, their selective pharmacological targeting is a long-wanted objective, although this intervention remains challenging due to our poor understanding of the underlying mechanisms. Eukaryotic NCXs are strongly regulated by cytosolic [Ca2+] oscillations, where Ca2+ interacts with the regulatory domains, CBD1 and CBD2. Recent evidence suggests that the CBD1–CBD2 interface controls Ca2+driven tethering of CBDs, which is associated with Ca2+ occlusion (and slow dissociation) at the primary Ca2+ sensor (Ca3–Ca4 sites), thereby driving the dynamic coupling of CBDs. This mechanism seems to be common for all isoform/splice variants. The primary allosteric Ca2+ sensor on CBD1 is highly conserved among all NCX variants, whereas the “tissue-specific” splicing segment located on CBD2 modifies not only the affinity and kinetic properties of Ca3–Ca4 sites but also the essence of the primary signal, resulting either the activation, inhibition, or no response to regulatory Ca2+ in a given variant. By using hydrogen-deuterium exchange mass-spectrometry (HDX-MS), small-angle X-ray scattering (SAXS), equilibrium 45Ca2+ binding, and stopped-flow techniques, we found that Ca2+ binding to CBD1 rigidifies the backbone flexibility of CBD2 (but not for CBD1), whereas CBD2 stabilizes the apo-CBD1 structure. The extent and strength of Ca2+-dependent rigidification of CBD2 is splice-variant dependent, where the backbone rigidification spans from Ca3–Ca4 sites of CBD1 up to the tip of CBD2 (>50 Å), or alternatively, it stops at the CBD2 helix in the splice variant exhibiting inhibitory response to Ca2+. These findings provide a structure-dynamic basis by which alternative splicing diversifies responses to Ca2+ and controls the propagation of allosteric signals over long distances.