Essential Role of the CBD1-CBD2 Linker in Slow Dissociation of Ca2+ 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 Ca , 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.The Na ϩ /Ca 2ϩ exchanger proteins (NCX1-3) and their splice variants are expressed in a tissue-specific manner (1-3) and mediate Ca 2ϩ entry/extrusion depending on various factors (4, 5). NCX is "autoregulated" by cytosolic Ca 2ϩ at sites that do not directly participate in the ion translocation (6, 7). For example, the cardiac/neuronal NCX1 variants respond to 0.3-10 M cytosolic Ca 2ϩ , whereas the kidney variant does not (8, 9). Allosteric regulation of NCX involves the interaction of cytosolic Ca 2ϩ with the regulatory loop-f of NCX (10 -14). Although the high affinity regulatory site of NCX1 was discovered 16 years ago (15), only recent studies identified two Ca 2ϩ regulatory domains, CBD1 and CBD2, which form a tandem (CBD12) through a short linker (amino acids 501-504) (16). High resolution x-ray and NMR revealed C 2 -type protein folding in both CBDs (16 -20) that display four Ca 2ϩ sites (Ca1-Ca4) on CBD1 (17,18,20) and two Ca 2ϩ sites (CaI and CaII) on CBD2 (16, 19). The NCX splice variants arise from a combination of 6 small exons (A, B, C, D, E, F), while the splice variant region is exclusively restricted to the CBD2 domain (2-4, 16, 19, 24). Excitable tissues, such as those of the brain (AD splice variant) and heart (ACDEF splice variant) contain exon A, whereas kidney, stomach, and skeletal muscle tissues comprise NCX with exon B (2-5).Equilibrium binding studies have shown that isolated CBD1 protein contains two sites with high affinity for Ca ]-dependent regulation by cytosolic Ca 2ϩ in cellular systems, whereas the primary site of CBD2 (CaI) may contribute to the Ca 2ϩ -dependent relief of Na ϩ -dependent inactivation of intact NCX (23-25). Importantly, the segment that undergoes alternative splicing is located solely within the CBD2 domain, meaning that specific domain-domain interactions may shape the regulatory properties of primary Ca 2ϩ sites at CBD1 (16 -20, 24). Fluorescence resonance energy transfer studies demonstrated that CBD domains of NCX can undergo significant conformational changes in the cell within the excitation-contraction coupling (22,27), although dynamic aspects of relevant conformational transitions, as well as the contribution of each specific Ca 2ϩ site to regulatory even...

Proton-sensing Ca2+ Binding Domains Regulate the Cardiac Na+/Ca2+ Exchanger

“…Earlier stopped-flow experiments have shown that Ca 2ϩ dissociation from the two high affinity binding sites of CBD12 occurs in two kinetic phases, fast (k f ϭ 4 -7 s Ϫ1 ) and slow (k s ϭ 0.3-0.5 s Ϫ1 ) (17). Because the slow phase of Ca 2ϩ dissociation from CBD12 largely represents the synergistic interactions between the two CBD domains (16,17), we investigated how protons affect Ca 2ϩ off-rates of CBD12.…”

“…Stopped-flow Experiments-Quin-2 was used in the stoppedflow experiments to monitor Ca 2ϩ off rates (16,17). In the stopped-flow machine SFM-3 (BioLogic), 150 l (syringe A) of proteins in buffer (100 mM KCl and 10 mM Bistris propane) were mixed with 150 l of buffer plus 200 -600 M Quin-2 (syringe B).…”

“…Proteins-The DNA constructs of CBD1, CBD2, and CBD12 (AD-splice variant) were cloned into a pET23b vector and expressed in Escherichia coli Rosetta2 (DE3) competent cells (Novagen) as described (16,17). Overexpressed proteins were purified on nickel beads (Ͻ95% purity, judged by SDS-PAGE).…”

“…All Ca 2ϩ binding assays were done at 22-23°C. The 45 Ca 2ϩ titration curves were fitted to a Hill or Adair equation (16,17).…”

“…[Ca 2ϩ ] i , pH i , and I NCX were measured in single ventricular myocytes (13)(14)(15). 45 Ca 2ϩ binding to purified preparations of CBD1 and CBD2 was assayed by ultrafiltration and Ca 2ϩ off rates were measured by stopped-flow assay (16,17). We have identified and characterized the Ca 2ϩ sensitivity of NCX at CBD1 and CBD2 and discovered a process that is profoundly modulated by pH.…”