A Unitary Anesthetic Binding Site at High Resolution
2009; Elsevier BV; Volume: 284; Issue: 36 Linguagem: Inglês
10.1074/jbc.m109.017814
ISSN1083-351X
AutoresL.S. Vedula, Grace Brannigan, Nicoleta J. Economou, Xi Jin, Michael Hall, Renyu Liu, Matthew J. Rossi, William P. Dailey, Kimberly C. Grasty, Michael L. Klein, Roderic G. Eckenhoff, Patrick J. Loll,
Tópico(s)Ion channel regulation and function
ResumoPropofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABAA receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABAA receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels. Propofol is the most widely used injectable general anesthetic. Its targets include ligand-gated ion channels such as the GABAA receptor, but such receptor-channel complexes remain challenging to study at atomic resolution. Until structural biology methods advance to the point of being able to deal with systems such as the GABAA receptor, it will be necessary to use more tractable surrogates to probe the molecular details of anesthetic recognition. We have previously shown that recognition of inhalational general anesthetics by the model protein apoferritin closely mirrors recognition by more complex and clinically relevant protein targets; here we show that apoferritin also binds propofol and related GABAergic anesthetics, and that the same binding site mediates recognition of both inhalational and injectable anesthetics. Apoferritin binding affinities for a series of propofol analogs were found to be strongly correlated with the ability to potentiate GABA responses at GABAA receptors, validating this model system for injectable anesthetics. High resolution x-ray crystal structures reveal that, despite the presence of hydrogen bond donors and acceptors, anesthetic recognition is mediated largely by van der Waals forces and the hydrophobic effect. Molecular dynamics simulations indicate that the ligands undergo considerable fluctuations about their equilibrium positions. Finally, apoferritin displays both structural and dynamic responses to anesthetic binding, which may mimic changes elicited by anesthetics in physiologic targets like ion channels. Most general anesthetics alter the activity of ligand-gated ion channels, and electrophysiology, photolabeling, and transgenic animal experiments imply that this effect contributes to the mechanism of anesthesia (1.Mascia M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9305-9310Crossref PubMed Scopus (232) Google Scholar, 2.Jenkins A. Greenblatt E.P. Faulkner H.J. Bertaccini E. Light A. Lin A. Andreasen A. Viner A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; 21: RC136Crossref PubMed Google Scholar, 3.Krasowski M.D. Nishikawa K. Nikolaeva N. Lin A. Harrison N.L. Neuropharmacology. 2001; 41: 952-964Crossref PubMed Scopus (111) Google Scholar, 4.Jenkins A. Andreasen A. Trudell J.R. Harrison N.L. Neuropharmacology. 2002; 43: 669-678Crossref PubMed Scopus (57) Google Scholar, 5.Siegwart R. Jurd R. Rudolph U. J. Neurochem. 2002; 80: 140-148Crossref PubMed Scopus (119) Google Scholar, 6.Chang C.S. Olcese R. Olsen R.W. J. Biol. Chem. 2003; 278: 42821-42828Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7.Jurd R. Arras M. Lambert S. Drexler B. Siegwart R. Crestani F. Zaugg M. Vogt K.E. Ledermann B. Antkowiak B. Rudolph U. FASEB J. 2003; 17: 250-252Crossref PubMed Scopus (498) Google Scholar, 8.Schofield C.M. Harrison N.L. Brain Res. 2005; 1032: 30-35Crossref PubMed Scopus (20) Google Scholar, 9.Li G.D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (248) Google Scholar). Although the molecular mechanism for this effect is not yet clear, photolabeling studies indicate that anesthetics bind within the transmembrane regions of Cys-loop ligand-gated ion channels such as the nicotinic acetylcholine and the γ-aminobutyric acid (GABA) 2The abbreviations used are: GABAγ-aminobutyric acidHSAhuman serum albuminHSAFhorse spleen apoferritinITCisothermal calorimetryLGICligand-gated ion channelr.m.s.root mean squareRMSFroot mean squared fluctuations. 2The abbreviations used are: GABAγ-aminobutyric acidHSAhuman serum albuminHSAFhorse spleen apoferritinITCisothermal calorimetryLGICligand-gated ion channelr.m.s.root mean squareRMSFroot mean squared fluctuations. type A receptors (2.Jenkins A. Greenblatt E.P. Faulkner H.J. Bertaccini E. Light A. Lin A. Andreasen A. Viner A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; 21: RC136Crossref PubMed Google Scholar, 9.Li G.D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (248) Google Scholar, 10.Chiara D. Dangott L.J. Eckenhoff R.G. Cohen J.B. Biochemistry. 2003; 42: 13457-13467Crossref PubMed Scopus (91) Google Scholar, 11.Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1074) Google Scholar). Practical difficulties associated with overexpression, purification, and crystallization of ion channels have thus far stymied investigation of the structural and energetic bases underlying anesthetic recognition. However, general anesthetics also bind specifically to sites in soluble proteins, including firefly luciferase, human serum albumin (HSA), and horse spleen apoferritin (HSAF) (12.Franks N.P. Lieb W.R. Nature. 1984; 310: 599-601Crossref PubMed Scopus (480) Google Scholar, 13.Liu R. Meng Q. Xi J. Yang J. Ha C.E. Bhagavan N.V. Eckenhoff R.G. Biochem. J. 2004; 380: 147-152Crossref PubMed Scopus (42) Google Scholar, 14.Liu R. Loll P.J. Eckenhoff R.G. FASEB J. 2005; 19: 567-576Crossref PubMed Scopus (101) Google Scholar), and x-ray crystal structures have been determined for complexes of these proteins with several general anesthetics (14.Liu R. Loll P.J. Eckenhoff R.G. FASEB J. 2005; 19: 567-576Crossref PubMed Scopus (101) Google Scholar, 15.Franks N.P. Jenkins A. Conti E. Lieb W.R. Brick P. Biophys. J. 1998; 75: 2205-2211Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16.Bhattacharya A.A. Curry S. Franks N.P. J. Biol. Chem. 2000; 275: 38731-38738Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). In particular, HSAF is an attractive model for studying anesthetic-protein interactions because it has the highest affinity for anesthetics of any protein studied to date, has a unique anesthetic binding site, and is a multimer of 4-helix bundles, much like the putative anesthetic binding regions in ligand-gated channels. In addition, apoferritin is commercially available and crystallizes readily. Most importantly, however, the affinity of HSAF for a broad range of general anesthetics is highly correlated with anesthetic potency, confirming the utility and relevance of this model system (17.Butts C.A. Xi J. Brannigan G. Saad A.A. Venkatachalan S.P. Pearce R.A. Klein M.L. Eckenhoff R.G. Dmochowski I.J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 6501-6506Crossref PubMed Scopus (45) Google Scholar).Ferritin is a 24-mer iron-binding protein. It sequesters free iron ions, thereby helping to maintain non-toxic levels of iron in the cell and functioning as a cellular iron reservoir (18.Theil E.C. Messerschmidt A. Huber R. Poulos T. Wieghardt K. Handbook of Metalloproteins. John Wiley & Sons, Ltd., Chichester2001Google Scholar, 19.Takagi H. Shi D. Ha Y. Allewell N.M. Theil E.C. J. Biol. Chem. 1998; 273: 18685-18688Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Each subunit has a molecular mass of ∼20 kDa and adopts a 4-helix bundle fold. The 24-mer forms a hollow, roughly spherical particle with 432 symmetry. Two ferritin isoforms are found in mammals, heavy (H) and light (L), and 24-mers can contain all H chains, all L chains, or mixtures of varying stoichiometry; the biological significance of the H/L ratio is not yet clear (20.Arosio P. Levi S. Ferritins: Structural and Functional Aspects. Marcel Dekker, Inc., New York2002Crossref Google Scholar).In addition to the large central cavity, the apoferritin 24-mer contains additional, smaller cavities at the dimer interfaces; these smaller cavities are of an appropriate size to accommodate anesthetics. X-ray crystallography has confirmed that this interfacial cavity is the binding site for the inhalational anesthetics halothane and isoflurane, and isothermal titration calorimetry (ITC) measurements have shown that this interfacial site has a relatively high affinity for these anesthetics (Ka values ∼105m−1) (14.Liu R. Loll P.J. Eckenhoff R.G. FASEB J. 2005; 19: 567-576Crossref PubMed Scopus (101) Google Scholar).General anesthetics fall into at least two broad classes, inhalational and injectable. Whereas both classes of drugs can induce the amnesia, immobility, and hypnosis associated with anesthesia, molecules in the two classes differ substantially in their chemical and physical properties. Prior to this work, only one crystal structure has been available for an injectable general anesthetic complexed with a protein-propofol, bound to HSA (16.Bhattacharya A.A. Curry S. Franks N.P. J. Biol. Chem. 2000; 275: 38731-38738Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). This structure revealed that the propofol binding sites on this protein do not, by and large, overlap with the binding sites for inhalational anesthetics. This raises the question of whether the two types of drug invariably bind to separate sets of targets, or whether they could possibly transduce their effects by binding to a single protein site. To address this question we assessed whether propofol binds to the apoferritin site that had been previously identified as the binding site for inhalational anesthetics. Using x-ray crystallography, calorimetry, and molecular modeling, we show that the two types of anesthetics do indeed share a common binding site. We also investigated structure-binding relationships for a homologous series of propofol-like compounds and found that, remarkably, the energetics of binding to apoferritin precisely match the compound's abilities to potentiate GABA effects at GABAA receptors, suggesting that similar structural and physicochemical factors mediate anesthetic recognition by both apoferritin and ligand-gated ion channels. This argues for the possibility that anesthetic binding might trigger structural and dynamic alterations in GABAA receptors similar to those observed in apoferritin, and that these changes underlie anesthetic effects.DiscussionPropofol is now the most widely used of the injectable general anesthetics. It provides rapid induction of unconsciousness, swift emergence and few of the problems associated with other drugs, such as delirium and nausea (32.Kotani Y. Shimazawa M. Yoshimura S. Iwama T. Hara H. CNS Neurosci. Ther. 2008; 14: 95-106Crossref PubMed Scopus (173) Google Scholar). Its desired effects are thought to arise from potentiation of GABAergic transmission. Structure-activity relationships have been probed for various propofol analogs (33.Krasowski M.D. Jenkins A. Flood P. Kung A.Y. Hopfinger A.J. Harrison N.L. J. Pharmacol. Exp. Ther. 2001; 297: 338-351PubMed Google Scholar), but the atomic details of the structure-binding relationship, necessary to guide further drug design, have not been reported. To elucidate such details for the anesthetic-binding protein apoferritin, we examined a homologous series of compounds that all share the same phenolic skeleton, but differ in the size of the aliphatic substituents at the 2- and 6-positions.The apoferritin system provides a clear example in which polar interactions are not required for specific, high-affinity recognition of propofol and related molecules. Apart from phenol, none of the compounds studied participate in direct polar interactions with the protein, even though multiple polar residues are available within the apoferritin cavity, and despite the fact that all of the compounds are capable of participating in hydrogen bonds and polar interactions involving their pi electrons. The molecular dynamics simulations show that the ligands prefer to form hydrogen bonds to water molecules in the cavity, rather than to their protein target. It is surprising that hydrogen bonding or other polar interactions do not play key roles in propofol recognition, given that the hydroxyl group has been considered an essential component of the propofol pharmacophore (34.Sewell J.C. Sear J.W. Br. J. Anaesth. 2004; 92: 45-53Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 35.Krasowski M.D. Hong X. Hopfinger A.J. Harrison N.L. J. Med. Chem. 2002; 45: 3210-3221Crossref PubMed Scopus (86) Google Scholar). However, our results strongly suggest that the hydroxyl may serve primarily to enhance water solubility, rather than to mediate protein recognition.In the absence of strong polar interactions such as hydrogen bonds, affinity should be governed by enthalpic contributions from relatively weak van der Waals forces and entropic contributions from dehydration of the ligand and cavity upon binding. We estimated the free energy contribution from the latter effect using a value of 20 cal/mol per Å2 of hydrophobic surface area (36.Reynolds J.A. Gilbert D.B. Tanford C. Proc. Natl. Acad. Sci. U.S.A. 1974; 71: 2925-2927Crossref PubMed Google Scholar) and found excellent agreement with experimental values, indicating that the hydrophobic effect is the principal force driving anesthetic recognition by apoferritin (Fig. 5).Of the ligands studied, only the largest compound, 2, departs from this trend, with a measured binding affinity that is markedly lower than predicted by the hydrophobic effect. This discrepancy probably reflects the entropic cost of immobilizing a large ligand in the absence of strong offsetting enthalpic contributions, and is entirely consistent with the results from our molecular dynamics simulations, which suggest that “optimal” ligands still exhibit a considerable degree of mobility within the cavity.A similar enthalpy-entropy calculus governs the recognition of guest molecules by encapsulating hosts, which is also controlled by weak forces (37.Mecozzi S. Rebek Jr., J. Chem. Eur. J. 1998; 4: 1016-1022Crossref Scopus (658) Google Scholar), and suggests that optimal affinity corresponds to a packing density of guests within the host of about 0.55, similar to the packing density of most organic liquids. Looser packing fails to fully exploit favorable enthalpic contacts between host and guest, while too-tight packing restricts guest motion and exacts too great an entropic cost. Packing densities for our apoferritin-ligand complexes range from 0.35 to ∼0.52, with binding affinity peaking at a packing density of 0.48 and decreasing for higher values. Considering that apoferritin cavity volume calculations include nooks inaccessible to ligand, these values likely underestimate the true packing density and are therefore in remarkable agreement with the predictions of the host-guest model (37.Mecozzi S. Rebek Jr., J. Chem. Eur. J. 1998; 4: 1016-1022Crossref Scopus (658) Google Scholar) (Fig. 5). Unfortunately, larger ligands with higher predicted packing densities are only sparingly soluble in water, complicating experimental confirmation of this relationship.To test generality of this relationship in another anesthetic-binding protein, we examined the packing density and binding affinity of propofol for HSA (13.Liu R. Meng Q. Xi J. Yang J. Ha C.E. Bhagavan N.V. Eckenhoff R.G. Biochem. J. 2004; 380: 147-152Crossref PubMed Scopus (42) Google Scholar, 16.Bhattacharya A.A. Curry S. Franks N.P. J. Biol. Chem. 2000; 275: 38731-38738Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). Propofol binds two distinct sites in HSA; an average Ka of 15, 300 m−1 has been estimated for the two sites (13.Liu R. Meng Q. Xi J. Yang J. Ha C.E. Bhagavan N.V. Eckenhoff R.G. Biochem. J. 2004; 380: 147-152Crossref PubMed Scopus (42) Google Scholar), which is more than 10-fold weaker than the apoferritin affinity for propofol. Only one of the two HSA sites is a buried cavity and therefore permits volume determination (16.Bhattacharya A.A. Curry S. Franks N.P. J. Biol. Chem. 2000; 275: 38731-38738Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar); packing densities fall in the range 0.50–0.55. This is higher than the “optimal” value found for the apoferritin-ligand complexes, suggesting that the HSA lower affinity for propofol may stem in part from a too-small binding cavity. However, examination of other HSA-ligand complexes reveals that this cavity is capable of enlargement; the fact that it adopts the volume that it does in the HSA-propofol complex may signal that the free energy gained from a hydrogen bond formed between propofol and HSA partially compensates for the entropic cost of immobilizing the ligand.Careful analysis of both the structure and the dynamics of the apoferritin model target reveal a complex interplay between anesthetic and protein. The crystal structures clearly show that the protein cavity is able to expand, at least to a limited degree, to accommodate larger ligands (induced fit), implying that models wherein anesthetics simply bind to preformed cavities may be overly simplistic. The molecular dynamics simulations complement this structural analysis, providing an explanation for the unexpectedly large cavity volume observed for the phenol-apoferritin complex. Given that the cavity occurs at a subunit interface, it is not difficult to hypothesize that expansions of similar cavities in the transmembrane regions of multisubunit ion channels could influence the energetics of conductance or gating.The utility of complementing structural analyses with dynamics simulations is further demonstrated by the pronounced effects of anesthetic binding on residue and strand dynamics, observed even for the lowest affinity ligand. While crystallography does not identify significant global differences (other than cavity size) between unliganded and liganded apoferritin, molecular dynamics simulations reveal that the local effects of anesthetic binding translate into substantial global effects on protein dynamics, even at loci distant from the binding site (e.g. the crossover strand connecting helices 2 and 3). It is easy to envision how similar allosteric effects, triggered by anesthetic binding to LGIC targets, might modulate agonist affinity or channel function.Because apoferritin is itself unlikely to transduce anesthesia, its utility as a model system lies in its ability to predict pharmacologically relevant anesthetic effects. Structure-function data for propofol and related compounds reveal a good correlation between anesthetic potency (as measured by loss of righting reflex in Xenopus laevis tadpoles) and the ability to potentiate GABA responses at GABAA receptors (33.Krasowski M.D. Jenkins A. Flood P. Kung A.Y. Hopfinger A.J. Harrison N.L. J. Pharmacol. Exp. Ther. 2001; 297: 338-351PubMed Google Scholar), suggesting a GABAergic mechanism for propofol-like molecules. It is therefore interesting to note that in the propofol series, binding affinity for apoferritin is strongly correlated with potentiation of GABA responses (Fig. 6). Further, this correlation approximates the line of identity, implying that occupancy of an apoferritin-like site in the GABAA receptor complex is sufficient for potentiation. A significant correlation is also observed between apoferritin binding affinity and anesthetic potency in tadpoles, although not as strong as with GABA potentiation. This is not surprising, given that systems level responses such as the loss of righting reflex almost certainly reflect contributions from multiple targets.FIGURE 6Correlation between apoferritin binding affinity and the EC50 for potentiation of GABA responses at GABAA receptors (units are mol/liter). Binding affinity was measured by ITC and is expressed as the dissociation constant Kd; GABA potentiation data are taken from Ref. (33.Krasowski M.D. Jenkins A. Flood P. Kung A.Y. Hopfinger A.J. Harrison N.L. J. Pharmacol. Exp. Ther. 2001; 297: 338-351PubMed Google Scholar). The points shown correspond to compounds 1, 2, 5, 7, 8, and 9. The solid line represents a least squares fit to the data; a slope of unity (dashed line) lies within the 95% confidence interval for the slope of the regression line.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Even aside from these functional correlations, the notion that apoferritin can act as a model for the GABAA receptor is consistent with our current knowledge of LGIC architecture. Mutagenesis and photolabeling experiments (1.Mascia M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9305-9310Crossref PubMed Scopus (232) Google Scholar, 2.Jenkins A. Greenblatt E.P. Faulkner H.J. Bertaccini E. Light A. Lin A. Andreasen A. Viner A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; 21: RC136Crossref PubMed Google Scholar, 3.Krasowski M.D. Nishikawa K. Nikolaeva N. Lin A. Harrison N.L. Neuropharmacology. 2001; 41: 952-964Crossref PubMed Scopus (111) Google Scholar, 4.Jenkins A. Andreasen A. Trudell J.R. Harrison N.L. Neuropharmacology. 2002; 43: 669-678Crossref PubMed Scopus (57) Google Scholar, 5.Siegwart R. Jurd R. Rudolph U. J. Neurochem. 2002; 80: 140-148Crossref PubMed Scopus (119) Google Scholar, 6.Chang C.S. Olcese R. Olsen R.W. J. Biol. Chem. 2003; 278: 42821-42828Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7.Jurd R. Arras M. Lambert S. Drexler B. Siegwart R. Crestani F. Zaugg M. Vogt K.E. Ledermann B. Antkowiak B. Rudolph U. FASEB J. 2003; 17: 250-252Crossref PubMed Scopus (498) Google Scholar, 8.Schofield C.M. Harrison N.L. Brain Res. 2005; 1032: 30-35Crossref PubMed Scopus (20) Google Scholar, 9.Li G.D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (248) Google Scholar), coupled with homology modeling (9.Li G.D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (248) Google Scholar), have established that the anesthetic binding site of the GABAA receptor lies within its transmembrane region, at or near the interface between subunit 4-helix bundles. Similarly, the apoferritin anesthetic binding site is found in an interfacial location, sandwiched between two four-helix bundles. Obviously, the structural homology cannot extend to the atomic level, because there is no primary sequence homology between apoferritin and the GABAA receptor, nor is the 2-fold symmetry axis found in apoferritin consistent with what we know of the architecture of the transmembrane domains of ligand-gated ion channels. Nonetheless, apoferritin displays a striking level of mimicry at the secondary, tertiary, and quaternary structural levels.In conclusion, apoferritin binding affinity recapitulates anesthetic potency in a propofol-based homologous series. Recognition of these molecules, based almost entirely on van der Waals forces and the hydrophobic effect, occurs at the same binding site previously shown to bind inhalational general anesthetics such as halothane and isoflurane. When taken together with the structural mimicry described above, these results argue strongly that the apoferritin anesthetic binding site bears a high degree of physicochemical and architectural similarity to sites that exist in the GABAA receptor and other clinically relevant targets. Our results will allow the development of specific structural and dynamical hypotheses to explain anesthetic mechanisms within pharmacologically relevant ion channel targets. Most general anesthetics alter the activity of ligand-gated ion channels, and electrophysiology, photolabeling, and transgenic animal experiments imply that this effect contributes to the mechanism of anesthesia (1.Mascia M.P. Trudell J.R. Harris R.A. Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 9305-9310Crossref PubMed Scopus (232) Google Scholar, 2.Jenkins A. Greenblatt E.P. Faulkner H.J. Bertaccini E. Light A. Lin A. Andreasen A. Viner A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; 21: RC136Crossref PubMed Google Scholar, 3.Krasowski M.D. Nishikawa K. Nikolaeva N. Lin A. Harrison N.L. Neuropharmacology. 2001; 41: 952-964Crossref PubMed Scopus (111) Google Scholar, 4.Jenkins A. Andreasen A. Trudell J.R. Harrison N.L. Neuropharmacology. 2002; 43: 669-678Crossref PubMed Scopus (57) Google Scholar, 5.Siegwart R. Jurd R. Rudolph U. J. Neurochem. 2002; 80: 140-148Crossref PubMed Scopus (119) Google Scholar, 6.Chang C.S. Olcese R. Olsen R.W. J. Biol. Chem. 2003; 278: 42821-42828Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 7.Jurd R. Arras M. Lambert S. Drexler B. Siegwart R. Crestani F. Zaugg M. Vogt K.E. Ledermann B. Antkowiak B. Rudolph U. FASEB J. 2003; 17: 250-252Crossref PubMed Scopus (498) Google Scholar, 8.Schofield C.M. Harrison N.L. Brain Res. 2005; 1032: 30-35Crossref PubMed Scopus (20) Google Scholar, 9.Li G.D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (248) Google Scholar). Although the molecular mechanism for this effect is not yet clear, photolabeling studies indicate that anesthetics bind within the transmembrane regions of Cys-loop ligand-gated ion channels such as the nicotinic acetylcholine and the γ-aminobutyric acid (GABA) 2The abbreviations used are: GABAγ-aminobutyric acidHSAhuman serum albuminHSAFhorse spleen apoferritinITCisothermal calorimetryLGICligand-gated ion channelr.m.s.root mean squareRMSFroot mean squared fluctuations. 2The abbreviations used are: GABAγ-aminobutyric acidHSAhuman serum albuminHSAFhorse spleen apoferritinITCisothermal calorimetryLGICligand-gated ion channelr.m.s.root mean squareRMSFroot mean squared fluctuations. type A receptors (2.Jenkins A. Greenblatt E.P. Faulkner H.J. Bertaccini E. Light A. Lin A. Andreasen A. Viner A. Trudell J.R. Harrison N.L. J. Neurosci. 2001; 21: RC136Crossref PubMed Google Scholar, 9.Li G.D. Chiara D.C. Sawyer G.W. Husain S.S. Olsen R.W. Cohen J.B. J. Neurosci. 2006; 26: 11599-11605Crossref PubMed Scopus (248) Google Scholar, 10.Chiara D. Dangott L.J. Eckenhoff R.G. Cohen J.B. Biochemistry. 2003; 42: 13457-13467Crossref PubMed Scopus (91) Google Scholar, 11.Miyazawa A. Fujiyoshi Y. Unwin N. Nature. 2003; 423: 949-955Crossref PubMed Scopus (1074) Google Scholar). Practical difficulties associated with overexpression, purification, and crystallization of ion channels have thus far stymied investigation of the structural and energetic bases underlying anesthetic recognition. However, general anesthetics also bind specifically to sites in soluble proteins, including firefly luciferase, human serum albumin (HSA), and horse spleen apoferritin (HSAF) (12.Franks N.P. Lieb W.R. Nature. 1984; 310: 599-601Crossref PubMed Scopus (480) Google Scholar, 13.Liu R. Meng Q. Xi J. Yang J. Ha C.E. Bhagavan N.V. Eckenhoff R.G. Biochem. J. 2004; 380: 147-152Crossref PubMed Scopus (42) Google Scholar, 14.Liu R. Loll P.J. Eckenhoff R.G. FASEB J. 2005; 19: 567-576Crossref PubMed Scopus (101) Google Scholar), and x-ray crystal structures have been determined for complexes of these proteins with several general anesthetics (14.Liu R. Loll P.J. Eckenhoff R.G. FASEB J. 2005; 19: 567-576Crossref PubMed Scopus (101) Google Scholar, 15.Franks N.P. Jenkins A. Conti E. Lieb W.R. Brick P. Biophys. J. 1998; 75: 2205-2211Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar, 16.Bhattacharya A.A. Curry S. Franks N.P. J. Biol. Chem. 2000; 275: 38731-38738Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). In particular, HSAF is an attractive model for studying anesthetic-protein interactions because it has the highest affinity for anesthetics of any protein studied to date, has a unique anesthetic binding site, and is a multimer of 4-helix bundles, much like the putative anesthetic binding regions in ligand-gated channels. In addition, apoferritin is commercially available and crystallizes readily. Most importantly, however, the affinity of HSAF for a broad range of general anesthetics is highly correlated with anesthetic potency, confirming the utility and relevance of this model system (17.Butts C.A. Xi J. Brannigan G. Saad A.A. Venkatachalan S.P. Pearce R.A. Klein M.L. Eckenhoff R.G. Dmochowski I.J. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 6501-6506Crossref PubMed Scopus (45) Google Scholar). γ-aminobutyric acid human serum albumin horse spleen apoferritin isothermal calorimetry ligand-gated ion channel root mean square root mean squared fluctuations. γ-aminobutyric acid human serum albumin horse spleen apoferritin isothermal calorimetry ligand-gated ion channel r
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