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 Ca(2+)-binding sites contribute to NCX regulation. Two of them are located on CBD1 (K(d) = approximately 0.2 microM), and one is on CBD2 (K(d) = approximately 5 microM). 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 Ca(2+) dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca(2+) ions (k(r) = 280 s(-1), k(f) = 7 s(-1), and k(s) = 0.4 s(-1)), whereas the dissociation of two Ca(2+) ions from CBD1 (k(f) = 16 s(-1)) and one Ca(2+) ion from CBD2 (k(r) = 125 s(-1)) is monophasic. Insertion of seven alanines into the linker (CBD12-7Ala) abolishes slow dissociation of Ca(2+), whereas the kinetic and equilibrium properties of three Ca(2+) sites of CBD12-7Ala and CBD1 + CBD2 are similar. Therefore, the linker-dependent interactions in CBD12 decelerate the Ca(2+) on/off kinetics at a specific CBD1 site by 50-80-fold, thereby representing Ca(2+) "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.