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.
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.
Second, several other ATPases including Mg2+
-ATPase and Na+
-ATPase showed significantly lower sensitivity to the volatile anesthetics than did the Ca2+
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
octaethylene glycol mono-n-dodecyl ether
minimum alveolar concentration
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.