Essential role of the CBD1-CBD2 linker in slow dissociation of Ca 2+ from the regulatory two-domain tandem of NCX1

Abstract

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 (Kd=∼0.2 μM), and one is on CBD2 (K d=∼5 μM). 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 comparableKd values but differ dramatically in their Ca2+ dissociation kinetics. CBD12 exhibits multiphasic kinetics for the dissociation of three Ca2+ ions (kr + 280 s-1, kf + 7 s-1, and ks = 0.4 s-1), whereas the dissociation of two Ca2+ ions from CBD1 (kf=16 s -1) and one Ca2+ ion from CBD2 (kr = 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. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.