Kinetic and equilibrium properties of regulatory Ca²⁺-binding domains in sodium-calcium exchangers 2 and 3

In mammals, three sodium-calcium exchanger (NCX) protein isoforms (NCX1, NCX2, and NCX3) mediate Ca²⁺ fluxes across the membrane to maintain cellular Ca²⁺ homeostasis. NCX isoforms and their splice variants are expressed in a tissue-specific manner to meet physiological demands. NCX1 is ubiquitously expressed, NCX2 is expressed in the brain and spinal cord, and NCX3 is expressed in the brain and skeletal muscle. Eukaryotic NCXs contain two cytosolic regulatory Ca²⁺-binding domains, CBD1 and CBD2, which form a two-domain tandem (CBD12) through a short linker. Ca²⁺ binding to the CBDs underlies allosteric regulation of NCX. Previous structural and functional studies in NCX1 have shown that the CBDs synergistically interact, where their interactions are modulated in a splice variant-specific manner by splicing segment at CBD2. Here, we analyze the equilibrium and kinetic properties of Ca²⁺ binding to purified preparations of CBD1, CBD2, and CBD12 from NCX2 and from NCX3 splice variants. We show that CBD1 interacts with CBD2 in the context of the CBD12 tandem in all NCX isoforms, where these interactions specifically modulate Ca²⁺ sensing at the primary sensor of CBD1 to meet the physiological requirements. For example, the rate-limiting slow dissociation of "occluded" Ca²⁺ from the primary allosteric sensor of variants expressed in skeletal muscle is ~10-fold slower than that of variants expressed in the brain. Notably, these kinetic differences between NCX variants occur while maintaining a similar Ca²⁺ affinity of the primary sensor, since the resting [Ca²⁺]_i levels are similar among different cell types.