Dissociation of Ca²⁺from Sarcoplasmic Reticulum Ca²⁺-ATPase and Changes in Fluorescence of Optically Selected Trp Residues. Effects of KCl and NaCl and Implications for Substeps in Ca²⁺Dissociation

Abstract

Sequential dissociation of the two Ca2+ ions bound to non-phosphorylated sarcoplasmic reticulum Ca2+-ATPase was triggered by addition, in a stopped-flow experiment, of quin2, which acted both as a high-affinity chelator and as a Ca2+-sensitive fluorescent probe. The kinetics of Ca2+ dissociation were deduced from the observed changes in quin2 fluorescence in the visible region (with lambdaex = 313 nm), while fluorescence detection in the UV region (with lambdaex = 290 nm) made it possible to monitor the tryptophan fluorescence changes accompanying this dissociation under the same ionic conditions. In the absence of KCl or NaCl, at pH 6 or 7, the observed changes in quin2 fluorescence were monoexponential, with rate constants very close to those of the changes in ATPase tryptophan fluorescence, which also appeared monophasic. In the presence of 100 mM KCl, quin2 fluorescence changes, although still monoexponential, were faster than in the absence of the monovalent ions but distinctly slower than the changes in tryptophan fluorescence, which were accelerated to a larger extent. In addition, the apparent kinetics of the Trp fluorescence changes depended on the excitation wavelength. Using an excitation wavelength of 296 nm, the Trp fluorescence drop was still faster than with an excitation wavelength of 290 nm, and in the presence of NaCl it even displayed a clear undershoot. We conclude that in the presence of KCl or NaCl and with an excitation wavelength of 290 nm, the rapid drop in tryptophan fluorescence mainly monitors the dissociation of the first of the two Ca2+ ions to be released from Ca2+-ATPase, while excitation at 296 nm optically selects a subpopulation of Trp residues whose fluorescence level is lower in the ATPase species with one Ca2+ ion bound than in the Ca2+-deprived ATPase species. The latter conditions result in an initial drop in Trp fluorescence whose apparent rate constant (in single-exponential analysis) is faster than the true rate of dissociation of the first Ca2+ ion and in a subsequent slower rise related to dissociation of the second Ca2+ ion. The difference between results obtained in the absence and in the presence of K+ or Na+ is due to an antagonizing effect of these cations on proton-induced conformational rearrangement of Ca2+-free ATPase, a conformational rearrangement which changes the ATPase Trp fluorescence level and significantly affects the cooperativity of Ca2+ binding at equilibrium.

Roles of Tyr122-hydrophobic Cluster and K+ Binding in Ca2+-releasing Process of ADP-insensitive Phosphoenzyme of Sarcoplasmic Reticulum Ca2+-ATPase

“…Possible Structural Role of K ϩ for Reducing Ca 2ϩ Affinity and Lumenal Gating-K ϩ is known to markedly accelerate the E2P hydrolysis (35,36) and also to modulate the E2 to E1Ca 2 transition in the non-phosphorylated Ca 2ϩ -ATPase (46,47). In the present study, we further found that the K ϩ binding is important for reducing the affinity for Ca 2ϩ and lumenal gating thus for Ca 2ϩ release from E2PCa 2 .…”

Importance of Transmembrane Segment M3 of the Sarcoplasmic Reticulum Ca2+-ATPase for Control of the Gateway to the Ca2+ Sites

“…18). Assuming that the two calcium ions dissociate through a common gateway, k Ϫ2 ϭ 0.5 ⅐ k Ϫ3 (23,24). The value k Ϫ1 ϭ 50 s Ϫ1 was assigned to reproduce as accurately as possible the apparently monoexponential time dependence of phosphorylation starting from the Ca 2ϩ -deprived enzyme (Fig.…”

“…9. In these experiments, we found it advantageous to change the pH to 6.0 to reduce the rate of Ca 2ϩ dissociation in the mutants as well as the wild type (23,24), thereby allowing the full time course to be monitored. A double mixing procedure was used in which EGTA is added to enzyme preincubated with Ca 2ϩ , followed by the addition of [␥-32 P]ATP at the indicated time interval, t, and acid quenching 34 ms later.…”

A Remarkably Stable Phosphorylated Form of Ca2+-ATPase Prepared from Ca2+-loaded and Fluorescein Isothiocyanate-labeled Sarcoplasmic Reticulum Vesicles

“…The nonionic detergents C 12 E 8 and DM were obtained from Nikko and Calbiochem (or Anatrace), respectively. Free Ca 2ϩ concentrations were computed as described previously (8,11).…”