Electrogenic Partial Reactions of the SR-Ca-ATPase Investigated by a Fluorescence Method

Abstract

Abstract.A fluorescence method was adapted to investigate active ion transport in membrane preparations of the SR-Ca-ATPase. The styryl dye RH421 previously used to investigate the Na,K-ATPase was replaced by an analogue, 2BITC, to obtain optimized fluorescence changes upon substrate-induced partial reactions. Assuming changes of the local electric field to be the source of fluorescence changes that are produced by uptake/ release or by movement of ions inside the protein, 2BITC allowed the determination of electrogenic partial reactions in the pump cycle. It was found that Ca 2+ binding on the cytoplasmic and on the lumenal side of the pump is electrogenic while phosphorylation and conformational transition showed only minor electrogenicity. Ca 2+ equilibrium titration experiments at pH 7.2 in the two major conformations of the protein indicated cooperative binding of two Ca 2+ ions in state E 1 with an apparent half-saturation concentration, K M of 600 nM. In state P-E 2 two K M values, 5 M and 2.2 mM, were determined and are in fair agreement with published data. From Ca 2+ titrations in buffers with various pH and from pH titrations in P-E 2 , it could be demonstrated that H + binding is electrogenic and that Ca 2+ and H + compete for the same binding site(s). Tharpsigargin-induced inhibition of the Ca-ATPase led to a state with a specific fluorescence level comparable to that of state E 1 with unoccupied ion sites, independent of the buffer composition.

Detection of Charge Movements in Ion Pumps by a Family of Styryl Dyes

“…The cuvette holder was thermostated at 20°C and equipped with a magnetic stirrer. Experiments to study ion-pump speci®c responses with the styryl dyes were performed as so-called standard experiments, which were described previously in the case of Na, K-ATPase (Schneeberger & Apell, 1999) and of SR CaATPase (Butscher et al, 1999). In a ®rst set of experiments, the excitation wavelength dependence of the¯uorescence signals was recorded in steady-state conditions of the Na, K-ATPase and of the Ca-ATPase, which could be maintained by de®ned substrate compositions of the ion pumps.…”

“…2A. A correspondent standard experiment was also introduced for the SR Ca-ATPase (Butscher et al, 1999): in bu er initially containing 25 mM tricine, 50 mM KCl, 1 mM MgCl 2 , and approximately 500 nM Ca 2+ (according to Fura-2 measurements), pH 7.2, 200 nM¯uorescent dye and protein (19 lg/ml). The initial state of the ion pump in this bu er condition was not well de®ned due to the unde®ned Ca 2+ concentration, therefore, we named it Ca x E 1 with approximately x £ 1.…”

“…Each``half cycle'' consists of an ordered sequence of experimentally identi®ed steps: ion binding, ion occlusion (together with enzyme phosphorylation or dephosphorylation), transition between both principal conformations, E 1 ®E 2 or vice versa, ion deocclusion and release to the aqueous phase on the other side of the membrane. In principle, the ions could be moved through the interior of the protein dielectric to a certain extent at each of these steps; however, recent investigations revealed that a charge movement occurred mainly during the ion binding and release steps (Apell, Schneeberger & Sokolov, 1998;Butscher, Roudna & Apell, 1999;Apell & Karlish, 2001). Those reaction steps are termed`e lctrogenic'' (LaÈ uger & Apell, 1989;Apell, 1989).…”

“…In the case of the CaATPase of the sarcoplasmatic reticulum (SR) the situation is di erent: direct electric measurements are not possible since the membrane of the SR is extremely leaky for monovalent cations, a property that short-circuits both sides of the membrane. Therefore, investigations to identify and analyze the electrogenic reaction steps with native membranes containing the SR Ca-ATPase so far could be performed successfully only with the use of styryl dyes (Butscher et al, 1999;Peinelt & Apell, 2002). Originally, styryl dyes were used to monitor fast changes of transmembrane electric potentials (Loew et al, 1979;Loew & Simpson, 1981;Loew, 1982;Grinvald et al, 1983Grinvald et al, , 1987Ehrenberg, Meiri & Loew, 2000).…”

“…The chromophore itself as well as the hydrophobic tail and the spacer between the chromophore and polar``head'' of the dye are known to a ect the sensitivity of the¯uorescence response on charge-translocating reaction steps of the ion pumps (Fedosova et al, 1995, Butscher et al, 1999. This observation led to the conception of this paper to study systematically the e ect of charge movements produced by the action of the Na,K-ATPase and the Ca-ATPase on the¯uorescence responses of a family of styryl dyes, named nXITC, with various lengths of the hydrophobic tail and of the spacer between polar head and chromophore (Fig.…”