Ca2+-dependent structural rearrangements within Na+-Ca2+ exchanger dimers

Abstract

Cytoplasmic Ca(2+) is known to regulate Na(+)-Ca(2+) exchanger (NCX) activity by binding to two adjacent Ca(2+)-binding domains (CBD1 and CBD2) located in the large intracellular loop between transmembrane segments 5 and 6. We investigated Ca(2+)-dependent movements as changes in FRET between exchanger proteins tagged with CFP or YFP at position 266 within the large cytoplasmic loop. Data indicate that the exchanger assembles as a dimer in the plasma membrane. Addition of Ca(2+) decreases the distance between the cytoplasmic loops of NCX pairs. The Ca(2+)-dependent movements detected between paired NCXs were abolished by mutating the Ca(2+) coordination sites in CBD1 (D421A, E451A, and D500V), whereas disruption of the primary Ca(2+) coordination site in CBD2 (E516L) had no effect. Thus, the Ca(2+)-induced conformational changes of NCX dimers arise from the movement of CBD1. FRET studies of CBD1, CBD2, and CBD1-CBD2 peptides displayed Ca(2+)-dependent movements with different apparent affinities. CBD1-CBD2 showed a Ca(2+)-dependent phenotype mirroring full-length NCX but distinct from both CBD1 and CBD2.

Fig. 1.

NCX undergoes Ca²⁺-dependent conformational changes. (A) Secondary structure of the fluorescent-tagged exchanger illustrating the relative position of the two Ca²⁺–binding domains (CBD1 and CBD2) and fluorophores (FP). Small spheres indicate bound Ca²⁺. (B) Effect of changes in intracellular Ca²⁺ on CFP (□) and YFP (■) emissions (Left) and corresponding FRET_R (Right) after coexpression of NCX^C and NCX^Y. (C) Changes in normalized FRET_R versus free [Ca²⁺] for NCX^C + NCX^Y. FRET_R values were normalized to their maximum and fitted to a single Hill function (K_h = 40 nM and Hill coefficient =1.8).

Fig. 2.

NCX exists as dimer in the plasma membrane. (A) Dependence of FRET_E on NCX^Y expression (measured as fluorescence emission intensity, a.u.) at a constant NCX^C expression level and in presence of 13 μM Ca²⁺. Points were fitted to FRET_E = (FRET_max * F_YFP)/F_YFP + K, where FRET_max is the maximal FRET_E, F_YFP is YFP emission intensity, and K is analogous to a dissociation constant (19). F test comparison between hyperbolic and linear regressions indicates that a nonlinear model best fits the points (P < 0.0001). (B) FRET_E versus protein concentration measured as the sum of YFP (before bleaching) and CFP (after YFP bleaching) fluorescence intensity. FRET_E was measured in the absence of Ca²⁺ from FluoPMS expressing a constant ratio of NCX^Y to NCX^C of 0.50 ± 0.03 (n = 20) or 4.3 ± 0.2 (n = 18) in absence of Ca²⁺ and 4.3 ± 0.1 (n = 28) in presence of 13 μM Ca²⁺. The R² values are 0.086, 0.00002, and 0.01, respectively. (C) Plot of FRET_E as function of the NCX^Y/NCX^C concentration ratio in absence or presence of 13 μM Ca²⁺. Points were fitted to a hyperbole as described in the Materials and Methods and in ref. . Each point is the average of 4–12 measurements. (D) Comparison between the experimental data shown in Fig. 2C (normalized to their maxima) and modeled FRET_E curves for dimer, trimer, and tetramer according to [(a + d)ⁿ − aⁿ − dⁿ]/[(a + d)ⁿ − aⁿ − dⁿ + ndⁿ] (24), where n is the number of molecule(s) in the oligomers, a is the number of acceptors, and d is the number of donors. The SDs of the vertical distances of the points from the lines describing the dimer, trimer, or tetramer models are 0.08, 0.18, and 0.24 (0 Ca²⁺) and 0.08, 0.17, and 0.24 (13 μM Ca²⁺).

Fig. 3.

Mutations within CBD1 abolish Ca²⁺-dependent conformational changes. (A and B) Effect of intracellular Ca²⁺ on changes in FRET_R and FRET_E between CFP- and YFP-tagged exchangers carrying the mutations D421A, E451A, and D500V (NCX^C–CBD1 + NCX^Y-CBD1). Mutations within CBD1 abolished Ca²⁺-dependent FRET changes. (C and D) FRET_R and FRET_E measurements from fluorescent-tagged exchangers with E516L in CBD2 (NCX^C–CBD2 + NCX^Y-CBD2). Note the increases in FRET_R and FRET_E upon addition of cytoplasmic Ca²⁺.

Fig. 4.

Cytoplasmic Ca²⁺ triggers movement in the isolated Ca²⁺-binding domains. (A–C) Effect of Ca²⁺ on FRET_R signals generated by ^CCBD1^Y (A), ^CCBD2^Y (B), and ^CCBD12^Y (C) in FluoPMS. Note the lower Ca²⁺ concentrations required to activate movements in CBD12. Images were acquired every 10 s. (D) Changes in normalized FRET_R value versus free [Ca²⁺]. Points, which are averaged between four and six experiments, were fitted to a Hill function. K_h values are 0.20 μM, 12 μM, and 0.010 μM for CBD1, CBD2, and CBD12. Average K_h values obtained from the single experiments are (in μM) 0.19 ± 0.01 (n = 4), 12 ± 0.01 (n = 6), and 0.014 ± 0.01 (n = 4), respectively. For CBD12, only the high Ca²⁺ affinity component was analyzed because the movements associated with higher Ca²⁺ concentrations were too small for accurate analysis. (E) Average FRET_E values in the absence and presence of 13 μM Ca²⁺ for CBD1 and CBD12 and 139 μM Ca²⁺ for CBD2. Values are 0.35 ± 0.01 and 0.23 ± 0.01 for CBD1, 0.184 ± 0.003 and 0.162 ± 0.003 for CBD2, and 0.114 ± 0.003 and 0.090 ± 0.004 for CBD12, respectively. ^CCBD1^Y-L is a lengthened construct consisting of residues 371–508, and ^CCBD12^Y-CBD1 is ^CCBD12^Y with mutations D421A, E451A, and D500V in CBD1. Values are 0.28 ± 0.01 and 0.23 ± 0.01 for ^CCBD1^Y-L and 0.14 ± 0.01 and 0.13 ± 0.01 for ^CCBD12^Y-CBD1. Number of experiments is indicated within each bar.

Fig. 5.

Removal of Ca²⁺ causes a slow conformational change in CBD12 and in the full-length NCX. (A–C) FRET_R changes upon removal of regulatory Ca²⁺ for the indicated constructs. Images were acquired every 200 or 500 ms. (D) Summary of time constants for FRET_R changes upon removal of Ca²⁺ for CBD12 and NCX. ^#Values for the individual CBDs represent an upper limit and cannot be accurately measured because of our limited frame rate (5 Hz). Faster kinetics are likely to occur as previously reported (26, 27).