Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ from the Regulatory Two-domain Tandem of NCX1

In NCX proteins CBD1 and CBD2 domains are connected through a short linker (3 or 4 amino acids) forming a regulatory tandem (CBD12). Only three of the six CBD12 Ca2+-binding sites contribute to NCX regulation. Two of them are located on CBD1 (K-d = similar to 0.2 mu M), and one is on CBD2 (K-d = similar to 5 mu M). Here we analyze how the intrinsic properties of individual regulatory sites are affected by linker-dependent interactions in CBD12 (AD splice variant). The three sites of CBD12 and CBD1 + CBD2 have comparable K-d values but differ dramatically in their Ca2+ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca2+ ions (k(r) = 280 s(-1), k(f) = 7 s(-1), and k(s) = 0.4 s(-1)), whereas the dissociation of two Ca2+ ions from CBD1(k(f) = 16 s(-1)) and one Ca2+ ion from CBD2 (k(r) = 125 s(-1)) is monophasic. Insertion of seven alanines into the linker (CBD12-7Ala) abolishes slow dissociation of Ca2+, whereas the kinetic and equilibrium properties of three Ca2+ sites of CBD12-7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca2+ on/off kinetics at a specific CBD1 site by 50-80-fold, thereby representing Ca2+ occlusion at CBD12. Notably, the kinetic and equilibrium properties of the remaining two sites of CBD12 are linker-independent, so their intrinsic properties are preserved in CBD12. In conclusion, the dynamic properties of three sites are specifically modified, conserved, diversified, and integrated by the linker in CBD12, thereby generating a wide range dynamic sensor.