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How Do Volatile Anesthetics Inhibit Ca2+-ATPases?(*)

  • Maria M. Lopez
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  • Danuta Kosk-Kosicka
    Correspondence
    To whom the correspondence should be addressed: Dept. of Anesthesiology/CCM, The Johns Hopkins University, 600 N. Wolfe St., Blalock 1404, Baltimore, MD 21287
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  • Author Footnotes
    * This work was supported by Grant GM 447130 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Volatile anesthetics at concentrations that are used in clinical practice to induce anesthesia selectively inhibit activity of the plasma membrane Ca2+-transport ATPase (Kosk-Kosicka, D., and Roszczynska, G.(1993) Anesthesiology 79, 774-780). We have investigated the mechanism of the inhibitory action of several anesthetics on the purified erythrocyte Ca2+-ATPase by employing fluorescence spectroscopy measurements that report changes in the environment of intrinsic tryptophans and of an extrinsic probe attached in the active site of the enzyme. We have shown that the observed inhibition of the Ca2+-dependent activation of the enzyme correlates well with the elimination of the Ca2+-induced conformational change that is important for the proper function of the enzyme. Analysis of the anesthetics effects on the total tryptophan fluorescence indicates a significant effect on enzyme conformation. Similar changes have been observed in the sarcoplasmic reticulum Ca2+-ATPase. We propose that volatile anesthetics inhibit Ca2+-ATPase by interacting with nonpolar sites in protein interior, in analogy to the binding demonstrated for myoglobin, hemoglobin, and adenylate kinase (Schoenborn, B. P., and Featherstone, R. M.(1967) Adv. Pharmacol. 5, 1-17; Tilton, R. F., Kuntz, I. D., and Petsko, G. A.(1984) Biochemistry 23, 2849-2857). Such binding is expected to modify conformational substate(s) of the enzyme and perturb its function. We view this process as an example of a general phenomena of interaction of small molecules with internal sites in proteins.

      INTRODUCTION

      The molecular site and mechanism of action of inhaled volatile anesthetics is not known despite intensive studies of over 30 years. Various hypothesis have been developed ranging from lipids to proteins as molecular targets and unspecific to specific sites of action (for review, see Miller(1985), Koblin(1990), and Franks and Lieb(1994)). In recent years the focus has shifted to membrane proteins, including ion channels and receptors for which different anesthetic effects have been reported, however, neither a specific nor an unifying mechanism of action has been described. We have previously identified another intrinsic membrane protein, the plasma membrane Ca2+ pump, as a potential target for anesthetic action (Kosk-Kosicka and Roszczynska, 1993; Kosk-Kosicka, 1994). Its Ca2+-ATPase activity and Ca2+ transport are inhibited by a variety of inhaled anesthetics, both clinically used and experimental ones (KoskKosicka and Roszczynska, 1993; Franks et al., 1995).(
      Kosk-Kosicka, D., Fomitcheva, I., and Lopez, M. M.,(1995) Biochemistry, in press.
      ) We have described the effects of four halogenated volatile anesthetics that are used in clinical anesthesia on the normal process of enzyme activation by either calmodulin binding or dimerization (Kosk-Kosicka and Roszczynska, 1993). All four drugs inhibited these two activation pathways in a dose-dependent manner and with a similar I50. The inhibition was observed at their clinical concentrations suggesting that Ca2+-ATPase was not only a good model target of an intrinsic membrane protein but could also be a pharmacological in vivo target for this group of general anesthetics. In subsequent studies we have demonstrated that the inhibition was selective as judged by the following criteria. First, two other groups of general anesthetics, barbiturates and alkanols, did not inhibit the enzyme at their anesthetic concentrations (Kosk-Kosicka et al., 1992).1 Second, several other ATPases including Mg2+-ATPase and Na+,K+-ATPase showed significantly lower sensitivity to the volatile anesthetics than did the Ca2+-ATPase.(
      I. Fomitcheva and D. Kosk-Kosicka, submitted for publication.
      ) Third, the above phenomena were observed in three distinct plasma membranes, ranging from erythrocytes to neuronal and endothelial cell types (Kosk-Kosicka et al., 1995b). As the site and mechanism of anesthetic action have not been explained and only a few proteins among the several reported to be targeted by anesthetics in vitro are affected at clinically relevant concentrations the observed inhibition of the Ca2+-ATPase activity deserves a close attention.
      In the present study we have investigated the mechanism of enzyme inhibition by volatile anesthetics by two experimental approaches: Ca2+-ATPase activity assay and fluorescence spectroscopy measurements. We have used dimeric Ca2+-ATPase whose activation is independent of a modulatory protein calmodulin since we have previously established that both normal modes of enzyme activation (by calmodulin binding to enzyme monomers and by the self-association of monomers to dimers) are equally sensitive to this group of general anesthetics (Kosk-Kosicka and Roszczynska, 1993; Kosk-Kosicka and Bzdega, 1988; Kosk-Kosicka et al., 1990). We have assessed the effects of the anesthetics on the Ca2+-dependent conformational changes of the enzyme by monitoring the Ca2+-dependent changes in fluorescence intensity of two probes: 1) intrinsic tryptophan(s) that reflect a conformational change which the Ca2+-ATPase undergoes upon binding the substrate Ca2+ in the initial step of enzymatic cycle, and 2) an external probe, fluorescein 5′-isothiocyanate (FITC)(
      The abbreviations used are: FITC
      fluorescein 5′-isothiocyanate
      C12E8
      octaethylene glycol mono-n-dodecyl ether
      MAC
      minimum alveolar concentration
      SR
      sarcoplasmic reticulum.
      ) attached to lysine 601 in the active site that normally binds ATP (Dupont, 1976; Inesi et al., 1980; Kosk-Kosicka and Inesi, 1985; Kosk-Kosicka et al., 1989). We demonstrate that the anesthetics inhibit both measures in a dose-dependent manner and there is a good correlation between the attenuation of the Ca2+-dependent conformational changes and the inhibition of the Ca2+-ATPase activity. In addition, the observed changes in the total tryptophan fluorescence also suggest that the anesthetics affect enzyme conformation. We analyze these findings with respect to the demonstrated binding of small ligands such as xenon (a very potent experimental inhaled anesthetic) in interior spaces of metmyoglobin which affects the internal motions and substates of the protein (Schoenborn and Featherstone, 1967; Tilton et al., 1984). As proteins undergo motions small molecules enter and interact with nonpolar sites in the protein interior (Englander et al., 1972; Lakowicz and Weber, 1973; Eftink and Ghiron, 1976; Cooper, 1976; Cohen et al., 1977; Lim and Sauer, 1991; Eriksson et al., 1992; Lim et al., 1994). We postulate that interaction of anesthetic molecules with nonpolar sites in the Ca2+-ATPase molecule modifies conformational substate(s) of the protein which results in impairment of its enzymatic function. We consider binding of small molecules in nonpolar internal protein spaces a general phenomena whose occurrence, however, requires compatibility between the nonpolar sites available in the protein and the invading molecule. In the case of gaseous anesthetic such an interaction may or may not have functional consequences at clinical anesthetic concentrations depending on the structure (flexibility) and function of a given protein, as it does for the Ca2+-ATPases but apparently not for myoglobin or hemoglobin. The lack of a significant functional effect of the demonstrated anesthetic binding in myoglobin or hemoglobin and their relationship to anesthesia apparently eliminated it from consideration as a mechanism of anesthetic action. In contrast, both function and conformation of the Ca2+-ATPase are significantly disturbed by the anesthetics and their action on this intrinsic membrane protein which controls intracellular Ca2+ homeostasis could certainly contribute to the pharmacological anesthetic effects. Our model needs to be treated as hypothetical until future advances in NMR or x-ray crystallography allow for its verification.

      MATERIALS AND METHODS

      Egg yolk phosphatidylcholine (P5763) and CNBr-activated Sepharose 4B were purchased from Sigma; octaethylene glycol mono-n-dodecylether (C12E8) was obtained from Nikko (Tokyo, Japan). Coupling of bovine calmodulin to Sepharose was performed in accordance with Pharmacia Biotech Inc. instructions as described earlier (Kosk-Kosicka and Bzdega, 1988). Enflurane and isoflurane were obtained from Anaquest (Liberty Corner, NJ); methoxyflurane was from Abbott Hospital Products; and thymol-free halothane from Halocarbon Laboratories (River Edge, NJ). Halothane is a two-carbon alkane halogenated derivative, the other three anesthetics belong to halogenated methyl ethyl ether derivatives as shown inFig. 1.
      Figure thumbnail gr1
      Figure 1:Chemical structures of volatile anesthetics used in the study.
      The methods used for preparation of erythrocyte ghost membranes and sarcoplasmic reticulum (SR), purification of the Ca2+-ATPase from erythrocyte membranes, determination of protein, and Ca2+ concentration were as described previously (Kosk-Kosicka et al., 1983, 1986; Kosk-Kosicka and Bzdega, 1988). Free Ca2+ concentrations were calculated (Fabiato and Fabiato, 1979) from total calcium and EGTA concentrations, based on the constants given by Schwartzenbach et al.(1957). Total calcium was measured by atomic absorption. Sarcoplasmic reticulum was prepared from rabbit skeletal muscle in the laboratory of Dr. Inesi (Eletr and Inesi, 1972).

       The Ca-ATPase Activity

      This was determined by colorimetric measurement of inorganic phosphate production, generally as described previously (Kosk-Kosicka and Bzdega, 1988; Kosk-Kosicka et al., 1983). The assay was performed in sealed (to maintain constant concentration of the anesthetic) 0.65-ml polypropylene tubes in a total reaction volume of 0.33 ml. After addition of all reagents and protein, volatile anesthetic was delivered to the tube in an air-tight Hamilton syringe and the reaction was started with 3 mM ATP. After vortexing, samples were placed in a water bath at either 37 or 25°C for 30 min. The reaction was terminated with ammonium molybdate/metavanadate at individual times. The aliquots of volatile anesthetics delivered to the assay were taken from solutions of saturated volatile anesthetics in reaction mixture, which were prepared daily from the stock of pure volatile anesthetic under nitrogen gas at room temperature. Activity of the purified dimeric Ca-transport ATPase was assayed at 70 nM enzyme concentration in a reaction mixture containing 50 mM Tris maleate, pH 7.4, 120 mM KCl, 8 mM MgCl2, 3 mM ATP, 1 mM EGTA, and CaCl2 in concentrations yielding the required free Ca2+. The concentration of C12E8 was kept constant at 150 μM. For activity assay of the Ca2+-transport ATPase in membranes, C12E8 was omitted. The activity was calculated as a difference between the activity determined in the above reaction mixture and the reaction mixture in which Ca2+ was omitted. 65 μg of ghost membranes and 1.2 μg of sarcoplasmic reticulum were used per tube. In addition, the reaction mixture for sarcoplasmic reticulum contained the divalent cation ionophore A23187 (10 μM).

       Fluorescence Measurements

      Fluorescence intensity was measured at equilibrium using a FluoroMax spectrofluorometer with DM3000F software. Tryptophan fluorescence was excited at 290 nm and the emission was recorded at 330 nm. Excitation and emission wavelengths used for FITC Ca2+-ATPase were 484 and 515 nm. The Ca2+-ATPase in erythrocyte ghosts was labeled with FITC as described previously (Kosk-Kosicka and Bzdega, 1990). The labeled enzyme was then purified by our standard procedure. The stoichiometry of labeling was 1 mol of FITC/1 mol of enzyme. Fluorescence measurements were done in the medium containing 100 mM Tris-HCl, pH 7.4, 150 μM C12E8, 130 mM KCl, 8 mM MgCl2, and 1 mM EGTA at 25°C in the total volume of 1.1 ml in a sealed cuvette. Protein concentration was 10 μg/ml for the purified plasma membrane Ca2+-ATPase (70 nM) and SR. For measurements of the calcium-dependent change in fluorescence CaCl2 was added after the anesthetic and the calcium-induced decrease of FITC fluorescence was related to the total initial fluorescence at each anesthetic concentration (Fi). Because total tryptophan fluorescence decreased with time we have differentiated between Fio (at time 0) and Fi (measured right before the addition of CaCl2). Free Ca2+ was 17.5 μM. The solution was gently stirred during the experiment which lasted 4-5 min. Stirring did not cause any perturbation of the fluorescence signal. Corrections for the dilution effect were made when necessary.

       Decays of Fluorescence Intensity

      The frequency domain data were analyzed by the method of nonlinear least squares (Gratton et al., 1984; Lakowicz et al., 1984). The measured values were compared with values predicted from a model. For a multiexponential decay, I(t) is given by,
      I(t)=αie-t/τi
      (Eq. 1)


      where αi is the pre-exponential factor and τi is the lifetime. The fractional intensity of each component in the decay is given by:
      fi=αiτijαjτj
      (Eq. 2)


      the best fit between the data and the calculated value is indicated by a minimum value for the goodness-of-fit parameter χ2R:
      χ2R=1Vw(ϕw-ϕcwδϕ)2+1Vw(mw-mcwδm)2
      (Eq. 3)


      where ν is the number of degrees of freedom and δφ and φm are uncertainties in the phase and modulation values, respectively.

       Determination of Concentrations of Anesthetics

      In parallel to the activity or fluorescence assay, samples were incubated under identical conditions and used for determination of the effective anesthetic concentrations during the assay. Halothane, isoflurane, and enflurane were extracted with heptane (methoxyflurane with octane), measured by gas chromatography, and the concentrations were calculated as described earlier (Kosk-Kosicka and Roszczynska, 1993). 1 MAC equals minimum alveolar concentration of an agent that produces immobility in 50% of those subjects exposed to a noxious stimulus as defined by Eger (1978).
      Data are expressed as the mean ± S.E. of three to six independent experiments performed in duplicates. The data points were fitted by the nonlinear regression method.

      RESULTS

      We have demonstrated a strong correlation between the general anesthetic potency (as expressed in MAC values) of several halogenated volatile anesthetics and their ability to inhibit the Ca2+-ATPase activity (Kosk-Kosicka and Roszczynska, 1993). The anesthetic concentrations in our activity assays were expressed in volume % at 37°C for easy comparison with their anesthetic potency. At present we have recalculated the values into millimolar concentrations (in addition, original data for methoxyflurane have been included) and compared the concentrations of the anesthetics in the reaction mixture that half-maximally inhibit the purified erythrocyte Ca2+-ATPase with their concentrations in blood to which the enzyme may be exposed when 1 MAC of a given anesthetic is administered in clinical anesthesia.Fig. 2 shows that the concentrations of all four agents that the enzyme may encounter during anesthesia inhibit enzyme function.
      Figure thumbnail gr2
      Figure 2:Correlation between anesthetic and inhibitory potencies of four inhalation anesthetics: isoflurane (•), methoxyflurane ( ), enflurane (■), and halothane (*) at 37°C. The anesthetic potencies, expressed in millimolar concentrations, in blood were calculated from published values of MAC taking into consideration the appropriate blood/gas partition coefficients (Eger, 1974; Koblin, 1990; Kosk-Kosicka and Roszczynska, 1993). Inhibitory potencies are expressed in millimolar concentrations (in the reaction mixture) that half-maximally inhibit Ca2+-ATPase activity of the purified dimeric enzyme. Both sets of values were obtained at 37°C, i.e. human body temperature.
      The anesthetics decrease both Vmax and Ca2+ affinity of the Ca2+-ATPase activity (Fig. 3). As shown inFig. 3 isoflurane at a concentration that half-maximally inhibits the enzyme causes a small yet statistically significant shift in the apparent K12 for calcium from pCa 7.05 ± 0.05 to 6.9 ± 0.04. Similar results were obtained with other anesthetics (data not shown).
      Figure thumbnail gr3
      Figure 3:Calcium dependence of Ca2+-ATPase activity in the presence (•) and absence of isoflurane (○). The Ca2+-ATPase activity was assayed as described under “Materials and Methods.” The reaction mixture contained 50 mM Tris maleate, pH 7.4, 120 mM KCl, 8 mM MgCl2, 150 μM C12E8, 1 mM EGTA, and 3 mM ATP. Various amounts of CaCl2 were added to obtain the calcium concentrations specified in the horizontal axis. Enzyme concentration was 70 nM. The assays were performed at 37°C. Inset shows normalized data of one typical experiment, where 100% is the maximal activity either with or without isoflurane. Six independent experiments were performed in duplicates. The mean K12 for the enzyme in the presence of isoflurane was significantly different from the control (p < 0.04).
      The effects of volatile anesthetics on the enzyme were subsequently examined at 25°C by two experimental approaches: Ca2+-ATPase activity assay and fluorescence spectroscopy measurements.Fig. 4 shows that at this temperature the activity is also inhibited in a dose-dependent manner, similar to the patterns observed at 37°C except that higher concentrations are required for a comparable extent of inhibition. This temperature dependence is in agreement with nonpolar interactions between each anesthetic and the enzyme (Collins and Washabaugh, 1985; Makhatadze and Privalov, 1995).
      Figure thumbnail gr4
      Figure 4:Inhibition of the Ca2+-ATPase activity by isoflurane (•), enflurane (■), methoxyflurane ( ), and halothane (*). The Ca2+-ATPase activity was assayed as described under “Materials and Methods.” The reaction mixture contained 50 mM Tris maleate, pH 7.4, 120 mM KCl, 8 mM MgCl2, 150 μM C12E8, 1 mM EGTA, 17.5 μM free Ca2+, and 3 mM ATP. Ca2+-ATPase concentration was 70 nM. The assays were performed at 25°C. The specific activity (100% activity) was 220 ± 9 μmol of Pi/mg of protein/h.
      The effect of anesthetics on the functionally important enzyme conformation has been assessed by measuring the calcium-dependent increase in intrinsic tryptophan fluorescence induced by substrate Ca2+ binding and in addition, by the decrease of the fluorescence of an external probe, FITC, bound in the active site of the enzyme. As shown inFig. 5 the Ca2+-induced increase in tryptophan fluorescence is attenuated by all three anesthetics in a similar dose-dependent manner. Halothane effect could not be determined accurately due to the high extent of quenching of tryptophan fluorescence by this compound. The half-maximal reduction is observed at ~0.16-0.26 mM concentrations of the agents in the order of efficiency shown inFig. 5; the difference between their F50 values may be not statistically significant. The Ca2+-induced fluorescence increase is readily reversed by addition of EGTA both in the presence and absence of anesthetics. Similar dose dependence is observed in the attenuation of the Ca2+-dependent decrease in FITC fluorescence, even though the ΔF/ΔFTotal does not proceed to 0 (Fig. 6). Thus, there is a good correlation between the inhibitory action of the anesthetics on the Ca2+-ATPase activity (I50 = 0.20-0.27 mM) and the Ca2+-induced conformational change assessed by either tryptophan or FITC fluorescence measurements (F50 = 0.16-0.23 mM).
      Figure thumbnail gr5
      Figure 5:Concentration dependence of the suppressing effect of volatile anesthetics on the Ca2+-dependent increase in the intrinsic tryptophan fluorescence. The fluorescence measurements were performed at 25°C as described under “Materials and Methods.” Enzyme concentration was 70 nM. Fluorescence increase recorded at 330 nm was induced upon addition of 1.2 μM free Ca2+. The Ca2+-dependent change in tryptophan fluorescence in the control in the absence of anesthetics (1.2-2% of the total tryptophan fluorescence) is shown as 100%. The ΔF/ΔFTotal at each anesthetic concentration is expressed in percent of the Ca2+-dependent fluorescence increase in the presence of anesthetic as compared to the control experiment. The increase was reversible upon addition of EGTA both in the absence and presence of anesthetics. Symbols used for anesthetics are as described in the legend to.
      Figure thumbnail gr6
      Figure 6:Concentration dependence of the suppressing effect of volatile anesthetics on the Ca2+-dependent decrease of FITC fluorescence. Labeling of the Ca2+-ATPase with FITC and fluorescence spectroscopy measurements were performed as described under “Materials and Methods.” Fluorescence decrease was induced upon addition of 1.2 μM free Ca2+ to the reaction mixture containing 70 nM FITC-labeled enzyme. The Ca2+ dependent change in FITC fluorescence of the control in the absence of anesthetics (1-2% of the total fluorescence) is shown as 100%. The ΔF/ΔFTotal at each anesthetic concentration is expressed in percent of the Ca2+-dependent fluorescence decrease in the presence of anesthetic as compared to the control experiment. Symbols used for anesthetics are as described in the legend to.
      We have also analyzed the anesthetics effects on the total tryptophan fluorescence intensity of Ca2+-ATPase. All four studied compounds decrease the fluorescence in a dose-dependent linear fashion (Fig. 7). In order to distinguish which type of quenching process is taking place, lifetime fluorescence intensity measurements were performed in the presence and absence of isoflurane. As shown inTable 1 intensity decays are very heterogenous and three exponentials are needed to fit the data. This is expected for a protein with several tryptophans and was observed previously for the SR Ca2+-ATPase (Lakowicz et al., 1986; Gryczynski et al., 1989; Wang et al., 1992). In the presence of isoflurane the lifetimes decrease (compare exponential fits χ2R and <τ> inTable 1). These changes in lifetimes are characteristic of collisional quenching (Pesce et al., 1971; Lakowicz, 1991). On the other hand the apparent quenching constants, Kapp, calculated from the decrease in fluorescence intensity (inFig. 7 andTable 2) are larger than those possible if collisional quenching were entirely responsible for the observed fluorescence decrease (for example, for other multitryptophan proteins β-trypsin and pepsin the Kapp are 2.4 M-1 and 9.5 M-1), indicating contributions from other processes such as binding of the quencher and conformational changes (Sellers and Ghiron, 1973; Eftink and Ghiron, 1976; Lakowicz, 1991). Accordingly, steady-state fluorescence intensity spectra recorded for the enzyme in the absence and presence of increasing concentrations of isoflurane show a progressive decrease of intensity and a red shift of up to 6 nm at 0.52 mM isoflurane concentration (Fig. 8), suggesting an increased polarity of the tryptophan environment that could result from a conformational change caused by the interacting anesthetic. It has been shown that conformational changes caused by partial denaturation of protein result in fluorescence intensity and lifetime decreases and a red shift of the spectrum (Gryczynski et al., 1988). To assess the contribution of a direct quenching of tryptophans by the anesthetics we have tested the effect of anesthetics on the fluorescence of the heat-denaturated enzyme and on free tryptophan in the same reaction mixture, including detergent. The free tryptophan fluorescence was not affected while there was a decrease in the fluorescence of the denatured enzyme. In addition, no fluorescence resonance energy transfer was observed between tryptophan and anesthetics (no overlap of absorption and emission spectra). All these findings point to a substantial contribution of conformational change of the enzyme in the anesthetic effect on the total tryptophan fluorescence.
      Figure thumbnail gr7
      Figure 7:Concentration dependence of the total intrinsic tryptophan fluorescence intensity of the Ca2+-ATPase in the presence of halothane (*), methoxyflurane (♦), isoflurane (•), and enflurane (■). Fo is the total fluorescence intensity recorded at 330 nm in the absence of a volatile anesthetic; Fio is the total fluorescence intensity at each concentration of the anesthetic (both at time zero). The experiment was performed at 25°C in 100 mM Tris-HCl, pH 7.4, 8 mM MgCl2, 150 μM C12E8, 130 mM KCl, and 1 mM EGTA.
      Figure thumbnail gr8
      Figure 8:Steady-state fluorescence emission spectra of erythrocyte Ca2+-ATPase in the absence (upper) and presence (lower) of 0.52 mM isoflurane. Excitation wavelength was 290 nm. Spectra were recorded at 25°C in 100 mM Tris-HCl, pH 7.4, 8 mM MgCl2, 150 μM C12E8, 130 mM KCl, and 1 mM EGTA.
      To examine the contribution of lipids in the decrease of total tryptophan fluorescence due to anesthetics we investigated their action on the sarcoplasmic reticulum Ca2+-ATPase. This enzyme could be studied in the native membrane because it constitutes ≈80% of the total membrane protein as compared to ≈0.01% of the membrane protein represented by the Ca2+-ATPase in the plasma membrane. Tryptophan fluorescence of the SR enzyme is reduced by the four anesthetics with the same order of efficiency as observed for the purified plasma membrane Ca2+-ATPase (Table 2). Also similar to the plasma membrane enzyme, the apparent quenching constants are too large for an exclusively diffusion controlled process. They are approximately two times lower than for the purified plasma membrane enzyme. The difference could arise from either an interference of the surrounding lipids or differences in the enzyme protein molecules such as existence of fewer sites for interaction with the anesthetics in the SR enzyme or their lower affinity than in the plasma membrane enzyme. The fact that about 2-fold lower anesthetic concentrations are required for inhibition of the purified plasma membrane Ca2+-ATPase activity as compared to the enzyme in the erythrocyte ghost membrane (Fig. 9: I50 = 0.26 and 0.50 mM halothane) suggests that the lower apparent quenching constants for the enzyme in SR may be caused by a decrease of tryptophans accessibility to the anesthetics by the lipid bilayer. A similar difference in halothane sensitivity of the Ca2+-ATPase in ghost membrane versus purified was observed previously at 37°C (Kosk-Kosicka and Roszczynska, 1993). As shown inFig. 9, the Ca2+-ATPase in SR requires a 40-50% higher halothane concentration for half-maximal inhibition than the enzyme in erythrocyte membrane. This result and the fact that at concentrations up to 0.2 mM halothane the enzyme in SR is activated, in contrast to the plasma membrane Ca2+-ATPase (Fig. 9), indicate that in addition to the lipid effect, the nonpolar sites in the SR enzyme may have a lower affinity for the anesthetics than comparable sites in the plasma membrane enzyme.
      Figure thumbnail gr9
      Figure 9:Comparison of the effect of halothane on the Ca2+-ATPase activity in the purified Ca2+-ATPase (*), Ca2+-ATPase in erythrocyte ghost membranes (○), and Ca2+-ATPase in sarcoplasmic reticulum (□). The activity assay was performed at 25°C as described under “Materials and Methods.” The reaction mixture for sarcoplasmic reticulum contained the divalent cation ionophore A23187 (10 μM) and the reaction time was 15 min as compared to 30 min for the plasma membrane Ca2+-ATPase. For both membranes the Ca2+-ATPase activity was calculated as the difference between the activity detected in the presence of both Mg2+ and Ca2+ ions and absence of Ca2+ ions. The specific Ca2+-ATPase activities (in μmol of Pi/mg of protein/h) were 100 in SR and 0.35 in erythrocyte ghosts.

      DISCUSSION

      We have shown that volatile anesthetics affect both the conformational state and activity of the Ca2+-ATPase. There is a strong correlation between the attenuation of the Ca2+-induced functionally important conformational change of the protein and impairment of its enzymatic function by anesthetics. Also our data on anesthetic effects on the total tryptophan fluorescence, including the decrease of fluorescence intensity, the red shift, and the decrease in lifetimes, indicate that anesthetics produce conformational changes in the enzyme molecule. To explain these effects we suggest that the anesthetics enter the protein and interact with nonpolar amino acids in its interior. To accommodate the intruding anesthetic the protein molecule undergoes structural rearrangement that results in the loss of its Ca2+-ATPase activity. In proposing this mechanism of anesthetic action on the Ca2+-ATPase we are considering multiple x-ray diffraction and NMR data that have demonstrated binding of gaseous anesthetics and other molecules, including urea and steroids, in hydrophobic “cavities” in the interior of several proteins (Schoenborn, 1965; Schoenborn et al., 1965; Schoenborn and Featherstone, 1967; Nunes and Schoenborn, 1973; Brown et al., 1976; Sachsenheimer et al., 1977; Hibbard and Tulinsky, 1978; Tilton and Kuntz, 1982; Tilton et al., 1984; Otting et al., 1991; Arevalo et al., 1994; Jain et al., 1994; Williams et al., 1994; Hubbard et al., 1994).

       Inhibition of the Ca2+-ATPase as a Consequence of Anesthetic Binding in Protein Interior

      Extensive studies on myoglobin and hemoglobin using NMR spectroscopy measurements of protein in solution and x-ray diffraction studies of crystals saturated with gaseous anesthetics have demonstrated that xenon, cyclopropane, dichloromethane, and halothane bind at discrete sites in the protein interior through van der Waals contacts with nonpolar amino acids and histidines (Schoenborn, 1969, 1976; Settle, 1973; Tilton et al., 1984, 1986; Nunes and Schoenborn, 1973). Generally, the binding appears stronger for smaller and more polarizable molecules (Nunes and Schoenborn, 1973). The findings suggest selectivity in the interactions between anesthetics and proteins and a requirement for compatibility between the nonpolar sites available in the protein (proper steric arrangement of amino acid side chains) and the intruding molecule (size, shape, and polarizability).
      The Ca2+-ATPase seems to have a fitting nonpolar site(s) available to all five volatile anesthetics studied in our laboratory (present paper and in Kosk-Kosicka and Roszczynska, 1993) and to xenon (Franks et al., 1995) as judged by their inhibitory effects. Temperature effects (on the Ca2+-ATPase activity) support a nonpolar nature of interactions between the anesthetics and the amino acids in the Ca2+-ATPase. It would be worthwhile to investigate effects of bigger or/and less polarizable anesthetic molecules on the enzyme to further test our hypothesis.
      If volatile anesthetics invade the interior of the Ca2+-ATPase molecule their binding is expected to affect internal motions and conformational substates of the enzyme (Schoenborn, 1965; Brown et al., 1976; Hibbard and Tulinsky, 1978; Tilton et al., 1984; Lim et al., 1994). The observed effects on total tryptophan fluorescence might then reflect the invasion that results in subtle conformational changes including tryptophan environment. The lack of the Ca2+-sensitive increase in tryptophan fluorescence in the presence of anesthetics would reflect the increased stability of the substate characteristic of a nonactive enzyme: enzyme with bound anesthetic cannot attain the conformation necessary for its normal activation. As a result no Ca2+-ATPase activity can be detected. Recent studies, including investigation of the effects of amino acid mutations in the hydrophobic core of λ-repressor on internal packing interactions, have demonstrated that the protein accommodates the potentially disruptive residues with shifts in its α-helical arrangement and becomes more stable since its packing is improved. However, the rearrangements cause repositioning of functional residues, which results in reduced function of the protein (Lim and Sauer, 1991; Lim et al., 1994). By analogy, a distortion of amino acid side chain(s) (resulting from anesthetic binding to the Ca2+-ATPase) that normally coordinate to Ca2+ may cause a change in Ca2+ binding and this slightly changed substate may not reveal the expected tryptophan fluorescence increase. The small shift observed in Ca2+ affinity at 37°C favors this interpretation as opposed to a total loss of Ca2+ binding in the presence of an anesthetic. Alternatively, insertion of the anesthetic and the resulting rearrangement may affect not Ca2+ binding but the coupling between the Ca2+ binding and induction of a proper conformation that in the active enzyme is detected as the Ca2+-dependent change in tryptophan fluorescence. Anesthetics effects on the conformational change (normally reflected in Ca2+-dependent changes in fluorescence of either tryptophan or FITC) correlate well with their effects on the Ca2+-ATPase activity in support of the interpretation that anesthetics prevent the enzyme from assuming a functionally proper active conformation. This suggestion is consistent with the observation that two activation pathways of the enzyme, by dimerization of enzyme monomers and by calmodulin binding to enzyme monomers, are inhibited by anesthetics to a comparable extent implying a “basic” defect in the activation mechanism. In contrast, the two pathways are affected distinctly different by two other groups of general anesthetics, barbiturates and alkanols (Kosk-Kosicka et al., 1995a).1 These compounds with the structural properties of detergents are postulated to inhibit Ca2+-ATPase monomers by binding to an exposed nonpolar patch on the enzyme surface to which calmodulin binds to activate monomers and which is not easily accessible in enzyme dimers.

       Selectivity of Anesthetic Action on the Ca2+-ATPase

      The reviewed effects on the plasma membrane Ca2+-ATPase occur at clinical anesthetic concentrations. The Ca2+-ATPase in sarcoplasmic reticulum is also sensitive to the anesthetics in this concentration range. Its activity is inhibited half-maximally by halothane at an approximately 50% higher concentration than the Ca2+-ATPase in the plasma membrane (Fig. 9; see also Kosk-Kosicka and Roszczynska(1993)). Also for this Ca2+-ATPase the large quenching constants support conformational changes in the presence of anesthetics (Table 2). We expect then that the inhibition results from the anesthetic binding in nonpolar sites which correspond to those in the plasma membrane Ca2+-ATPase (although in SR they may have lower affinity for the anesthetics) and the final consequences of restriction of protein motions are similar in the two Ca2+-transporting enzymes. We have, however, demonstrated a significant difference between the response of the two enzymes to lower anesthetic concentrations. The initial activation phase observed only for the SR Ca2+-ATPase (Fig. 9) which correlates with its effect of the Ca2+-induced tryptophan fluorescence(
      M. M. Lopez and D. Kosk-Kosicka, unpublished data.
      ) may be due to changes in its oligomeric size (from bigger to smaller oligomers, as suggested by Karon et al.(1994) based on the time-resolved fluorescence anisotropy data).
      In contrast to Ca2+-ATPase, two other plasma membrane ATPases, the Na+,K+-ATPase and Mg2+-ATPase, require at least 3-5 times higher concentrations of volatile anesthetics.2 Several other membrane proteins that have been reported as potential targets for anesthetics, including voltage-gated sodium, potassium, and calcium channels, are half-maximally inhibited only at halothane concentrations ranging from 4 to 30 times higher than its clinical potency, as summarized in a recent review of the last 10 years of search for the target of anesthetic action (Franks and Lieb, 1994). From among many proteins shown to be affected by the anesthetics only ligand-gated ion channels, including GABA receptors and possibly nicotinic acetylcholine receptor, are also targeted at clinical anesthetic concentrations. Studies performed in many laboratories imply GABAA receptors channel complex as a major target for the general anesthetics (for review, see Franks and Lieb(1994)). The pharmacological target is expected to be affected by anesthetics at their clinical concentrations (≈1 MAC) because the dose-response curves for the induction of anesthesia with volatile anesthetics are very steep (concentrations 20% above 1 MAC anesthetize almost all subjects) and because concentrations 2-4 times higher have damaging side effects on the organism. Our findings indicate that the anesthetics action on the Ca2+-ATPase is selective at their pharmacological concentrations. It remains to be tested whether anesthetic action on the Ca2+-ATPases and the receptors at the molecular level is exerted by their binding in protein interior. Our hypothesis cannot be verified at present without x-ray structure data.
      The finding that volatile anesthetics inhibit the Ca2+ATPases in vitro at their clinical concentrations while several other membrane proteins are affected by the anesthetics only at concentrations significantly exceeding those used in the clinical situation opens a possibility that the enzyme is perturbed during anesthesia and this anesthetic action has important physiological consequences. The consequences would be multiple as so many cellular events and cascades depend on precisely controlled intracellular Ca2+ concentrations, ranging from regulation of cell shape in erythrocytes, to neuronal transmission to depression of contractile force in heart.
      In conclusion, we propose that the observed correlation between the attenuation of the functionally important conformational change of the enzyme and inhibition of its Ca2+-ATPase activity could be explained by permeation of the protein molecule by volatile anesthetic and stabilization of its nonactive conformational substate. We view this process as an example of a general phenomena of discriminating binding of small ligands in protein interior.

      Acknowledgments

      We thank Dr. Ignacy Gryczynski for valuable suggestions. Lifetime experiments were performed at the Center of Fluorescence Spectroscopy, University of Maryland at Baltimore.