Effects of volatile anesthetic on the Ca²⁺‐ATPase activation by dimerization

Abstract

The phenomenological distance-dependent quenching (DDQ) model was employed to investigate the character of the interaction between volatile anesthetics (VAs) and the plasma membrane Ca 21 -ATPase (PMCA). The simultaneous analysis of the frequency-domain and steady-state data of tryptophan (Trp) fluorescence quenching by a VA points to a specific character of the apparent quenching effect of the VA, possibly arising from a significant contribution of static quenching. The apparent contributions of both static and dynamic quenching may be due to VA binding in the PMCA, which results in the modification of the conformational substates of the enzyme. To characterize further the molecular consequences of VA binding, we investigated its effects on the process of PMCA activation by self-association. VA shifted the equilibrium from enzyme dimers to monomers, as monitored by the loss of fluorescence energy transfer. The shift was apparently due to the VA-induced decrease in the affinity of PMCA molecules for self-association. Addition of a large molecular mass dextran to increase the proximity between enzyme monomers induced re-association of the VA-impaired PMCA, while the Ca 21 -ATPase activity was not recovered. The results are congruent with a dual VA effect on PMCA, a shift in the monomer/dimer equilibrium, and an inactivation of both monomers and dimers. -induced conformational change that PMCA undergoes in the initial step of its enzymatic cycle and the decrease in the intrinsic Trp fluorescence [4,5]. We have proposed that, upon binding to PMCA, the VAs disturb the sequence of conformational changes the enzyme normally undergoes in the process of catalytic activation, thus resulting in the inhibition of Ca 21 -ATPase activity [4,6]. Recently, we have shown that quenching of the total PMCA-intrinsic Trp fluorescence by halothane is dose-dependent, is saturable, and can be fitted to a binding curve [7].In this study we used two approaches to investigate the VA±PMCA interactions and their effects on enzyme activation. First, we employed the exponential distance-dependent quenching (DDQ) model (with parameters bimolecular rate constant of quenching k a , diffusion D and distances a and r e ) to analyze the Trp fluorescence quenching of PMCA by VA, which results in the loss of Ca 21 -ATPase activity. The DDQ model was developed to explain experimental frequency-domain and steady-state intensity decay data for which not only collisional but also through-space quenching was expected [8]. In using the DDQ model for our system we expanded our earlier spectroscopic studies [4] in which several characteristics of steady-state and preliminary frequency-domain data suggested that the PMCA±VA interaction is not just the result of mere collisional contacts. The frequency-domain data have been analyzed in more detail (see Materials and methods). Steadystate and frequency-domain Trp fluorescence intensity decays of the dimeric enzyme were measured under comparable experimental conditions at up to 5 mm isoflurane concentrations. S...