![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 61, Issue 5, 945-952, May 2002
Departments of Anesthesia (Y.I., R.P., R.G.E.), Physiology (R.G.E.), and Biochemistry and Biophysics (P.A.L.), University of Pennsylvania Medical Center, Philadelphia, Pennsylvania
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The molecular pharmacology of inhalational anesthetics remains poorly understood. Despite accumulating evidence suggesting that neuronal membrane proteins are potential targets of inhaled anesthetics, most currently favored membrane protein targets lack any direct evidence for anesthetic binding. We report herein the location of the binding site for the inhaled anesthetic halothane at the amino acid residue level of resolution in the ligand binding cavity in a prototypical G protein-coupled receptor, bovine rhodopsin. Tryptophan fluorescence quenching and direct photoaffinity labeling with [14C]halothane suggested an interhelical location of halothane with a stoichiometry of 1 (halothane/rhodopsin molar ratio). Radiosequence analysis of [14C]halothane-labeled rhodopsin revealed that halothane contacts an amino acid residue (Trp265) lining the ligand binding cavity in the transmembrane core of the receptor. The predicted functional consequence, competition between halothane and the ligand retinal, was shown here by spectroscopy and is known to exist in vivo. These data suggest that competition with endogenous ligands may be a general mechanism of the action of halothane at this large family of signaling proteins.
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
The
mechanisms of general anesthetic action at the molecular level remain
poorly understood, despite their use in millions of patients each year.
Understanding the molecular mechanisms by which inhaled anesthetics
produce behavioral effects, such as loss of consciousness and
analgesia, is thus an important goal with therapeutic implications.
Accumulating evidence suggests that these drugs act at multiple
neuronal membrane proteins that function as ion channels and
neurotransmitter receptors (Franks and Lieb, 1994
). However,
classification as an anesthetic target requires evidence of direct
binding, and most currently favored targets lack any direct evidence
for anesthetic binding. One of the major difficulties in demonstrating
direct binding is the weak binding energetics of the inhaled anesthetic
(Eckenhoff and Johansson, 1997). Weak binding, although consistent with
the relatively featureless molecules and the high aqueous
EC50 for general anesthesia in mammals (0.2-1.0
mM) (Franks and Lieb, 1994), essentially precludes conventional
radioligand binding studies. Furthermore, there have been few good
model systems for studying the actions of inhaled anesthetics in
biological membranes (Forman et al., 1997). In addition to plausible
roles in central nervous system signaling, it is important for
potential model proteins to be available in sufficient abundance and
purity to permit direct binding and high-resolution structural studies.
A large superfamily of G protein-coupled receptors (GPCRs) modulates
most signaling in central and peripheral nervous systems. In
particular, the rhodopsin family of GPCRs includes many
neurotransmitter receptors, such as muscarinic acetylcholine,
noradrenaline, dopamine, adenosine, and opioid receptors (Baldwin et
al., 1997). These receptors have highly conserved regions in the
transmembrane portion (Baldwin et al., 1997), and the ligand-receptor
interactions in the core formed by the seven 
-helices are thought to
be similar in the GPCRs of this family (Strader et al., 1994; Ji et
al., 1998). Functionally, cholinergic neurotransmission is known to influence awareness, sleep, and learning and memory (Durieux, 1996).
The 2-adrenergic receptor seems to play a role
in antinociceptive responses as well as in the state of arousal (Bol et
al., 1999). In fact, agonists and/or antagonists that work through
these GPCRs have been reported to significantly alter anesthetic
requirements in humans and animals (Segal et al., 1988; Seitz et al.,
1990; Glass et al., 1997; Ishizawa et al., 2000a). Although this
might include unrelated, parallel effects on the central nervous
system, recent studies show that inhaled anesthetics can interfere with GPCR signaling in vitro (Durieux, 1995; Honemann et al., 1998; Schotten
et al., 1998), suggesting direct anesthetic effects.
Halothane, a clinically used volatile anesthetic, has two features that
allow monitoring of binding. First, the photolabile carbon-bromine bond
allows photolabeling (Eckenhoff and Johansson, 1997); second, the
bromine atom can quench intrinsic protein fluorescence if it is near
the fluorophore (Johansson et al., 1995). Both features allow
determination of location of the anesthetic within the protein matrix.
Because the abundance of rhodopsin in native retinal membrane preparation facilitates direct binding approaches, we used bovine rhodopsin as a structural homolog for other neuronal GPCRs to characterize the binding domain for this inhaled anesthetic. We reported previously that halothane binds to rhodopsin but not to its
associated G protein (Ishizawa et al., 2000b). In this study,
using a higher resolution approach, we provide evidence for halothane
binding to the endogenous ligand binding site in rhodopsin.
| |
Materials and Methods |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Rod Disk Membranes Preparation.
Fresh bovine retinas were
dissected in room light. Rod disk membranes (RDM) were prepared by
sucrose flotation in isotonic buffer (20 mM MOPS, 100 mM KCl, 6 mM
MgCl2, pH 7.0). Peripheral proteins were stripped
by washing in hypotonic buffer (10 mM MOPS, 2 mM
MgCl2, 100 µM GTP, pH 7.0) (Panico et al.,
1990). Estimated molar ratio of the protein in the RDM was 24:1
(rhodopsin/transducin) based on the relative mass in SDS-PAGE using
reflective density (GS-710; Bio-Rad Laboratories, Hercules, CA). Molar
ratio of tryptophan residues was then 12:1 (rhodopsin/transducin).
Tryptophan residues in cyclic GMP phosphodiesterase and arrestin were
estimated to be lower than 3% and 0.5% of the total residues in RDM,
respectively. The RDM were regenerated using 3-fold molar excess of
9-cis-retinal (Sigma Chemical Co., St. Louis, MO) for
12 h on ice in the dark followed by a further 1 h incubation
at room temperature, as reported previously, to provide almost 100% of
chromophore regeneration (Gibson et al., 1998).
Steady-State Fluorescence and Absorption Spectra. All fluorescence measurements were performed with a spectrofluorophotometer (RF-5301PC; Shimadzu Scientific Instruments, Inc. Columbia, MD) using a 10-mm-pathlength quartz cell at 25°C. Bleached and regenerated RDM samples were equilibrated in sodium phosphate buffer (130 mM NaCl and 20 mM Na2HPO4, pH 7.0) at a rhodopsin concentration of 0.5 µM with increasing concentrations of halothane (0-15 mM) in a gas-tight 4.0-ml cell. RDM samples were excited at 295 nm to probe tryptophan fluorescence.
For the fluorescence time-based measurements, the RDM samples were equilibrated in sodium phosphate buffer at a rhodopsin concentration of 0.5 µM with increasing concentrations of halothane (0-6.0 mM). Then the RDM were excited with 295 nm light and 3-fold molar excess of 9-cis-retinal (1.5 µl) was added to the sample cell (4.0 ml) with continuous stirring. Data were recorded at 330 nm at 30-s intervals for 60 min, with the excitation shutter closed between acquisitions to minimize exposure to the excitation beam. Halothane and 9-cis-retinal, the two principle ligands in this study, do not absorb appreciably at the excitation (295 nm) or emission (~330 nm) wavelength for tryptophan, so inner filter corrections were not performed. All UV/visible spectra were measured with a spectrophotometer (Cary300Bio; Varian Instruments, Walnut Creek, CA) using a 10-mm-pathlength 1.8-ml quartz cell at 25°C. RDM samples were equilibrated in sodium phosphate buffer at a rhodopsin concentration of 5.0 µM with increasing concentrations of halothane (0-4.0 mM). The time course of the increase in absorbance at 487 nm after addition of 9-cis-retinal (3-fold molar excess) was measured for 180 min.Photoaffinity Labeling of [14C]Halothane. Bleached and regenerated RDM samples were incubated with [14C]halothane (2-bromo-2-chloro-1,1,1-[1-14C]trifluoroethane; specific activity, 51 mCi/mmol; PerkinElmer Life Sciences, Boston, MA) and with increasing concentrations of unlabeled halothane in isotonic MOPS buffer in 2-ml quartz cuvettes at 25°C. The samples were exposed to 254-nm light at a distance of ~5 mm for 30 s with continuous stirring. The final concentrations in the photolabeling solution were 1.5 µM rhodopsin, 9.7 µM [14C]halothane, and 0 to 8.5 mM unlabeled halothane. SDS-PAGE of the labeled membranes was performed in modified Laemmli gels, and the gels were stained with Coomassie Brilliant Blue. The relative protein mass in the rhodopsin bands on SDS-PAGE was determined by reflective density. Then rhodopsin bands in the dried gels were excised and dissolved by incubating with 30% hydrogen peroxide at 60°C for 5 h. Stoichiometry of label incorporation into opsin or isorhodopsin was determined in the dissolved gel slices by scintillation counting, and disintegrations per minute were normalized to relative mass of rhodopsin.
For proteolytic digestion and radiosequence analysis, the bleached and regenerated RDM samples were incubated with [14C]halothane at 0.75 mM in isotonic MOPS buffer. The samples were exposed to 254-nm light for 40 s. The labeled samples were washed with the buffer, and the pellets were then used for proteolytic digestion.Proteolysis and Radiosequence Analysis. The pellets of the photolabeled RDM were resuspended in 15 mM Tris, 0.1% SDS, pH 8.1. For digestion with Staphylococcus aureus glutamyl endopeptidase (V8 protease; ICN Biomedicals Inc., Aurora, OH), V8 protease was added to the final concentration of 1:1 (w/w) protease/rhodopsin into the sample solution and incubated at 37°C for 3 h. For endoproteinase Lys-C (EndoLysC) digestion (Roche Applied Science, Indianapolis, IN), EndoLysC was added at ~2 mU:1 µg (protease: rhodopsin), and incubated at 37°C for 6 to 8 hours. The suspension of proteolytic fragments from enzymatic digestion was diluted in sample loading buffer. SDS/PAGE was performed in the modified Laemmli gels, and the gel was subsequently electroblotted to a polyvinylidene difluoride membrane (Problott Membranes; Applied Biosystems, Foster City, CA).
Automated N-terminal sequence analysis was performed on an Applied Biosystems model 473A protein sequencer (Foster City, CA) with an in-line 120A PTH analyzer. Blotted samples were directly loaded onto the chamber, and sequencing was performed using gas-phase trifluoroacetic acid to minimize possible hydrolysis. After conversion of the released amino acids to PTH-amino acids, the suspension was divided into two parts. One portion, approximately 30%, went to the PTH analyzer, whereas the remaining 70% was collected for scintillation counting. Yield of PTH amino acids was calculated from peak height compared with standards using the model 610A Data Analysis Program. Cysteine was not included in the standards. The analysis was done at least twice for each fragment.| |
Results |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Halothane Binding to Opsin and Regenerated Isorhodopsin.
Halothane binding to rhodopsin in RDM was initially studied using
intrinsic protein fluorescence. Halothane decreased tryptophan fluorescence of the bleached RDM by 80% with a
Kd value of 2.3 mM (95% CI,
1.8~2.8) (Fig. 1A). Tryptophan
fluorescence of the RDM was also decreased by nearly 75% upon
regeneration of chromophore (isorhodopsin) with
9-cis-retinal. In the regenerated RDM, halothane further
decreased tryptophan fluorescence with a
Kd value of 4.1 mM (95% CI,
1.0~6.4), but only by 11% (Fig. 1A).
N-Acetyl-tryptophan-amide (NATA) was used as a control for
random collisional quenching of soluble tryptophan. Halothane decreased
tryptophan fluorescence for NATA in a linear fashion, with a maximum
quenching of 19 ± 2% at 15 mM. Stern-Volmer plots for these
halothane effects are shown in Fig. 1B. The tryptophan emission
wavelength maximum of bleached RDM was 329.6 ± 0.2 nm, and
regeneration with 9-cis-retinal shifted it to 330.8 ± 0.2 nm. Halothane red-shifted the emission maximum in both bleached and
regenerated RDM (Fig. 1C). Because fluorescence quenching by halothane
is considered a short-range phenomenon (Basu et al., 1993), these
changes indicate that halothane binds near most of the five tryptophan
residues present in this molecule and that those residues are located
in hydrophobic environments.
|
). These samples regained 105 ± 4% and 90 ± 10% of its tryptophan fluorescence for 2 mM and 12 mM halothane, respectively, compared with a control sample treated in the same manner
(mean ± S.E.M., n = 3 for each). The tryptophan
emission maximum after the treatment was not different from the value
of the control.
To evaluate the environments surrounding the tryptophan residues in
rhodopsin, experiments were performed with a water-soluble quencher,
acrylamide. Acrylamide weakly quenched tryptophan fluorescence for
bleached RDM and regenerated RDM (17% and 36%, respectively, at 0.8 M) in the absence of halothane (Fig. 2A).
Acrylamide also caused the emission maximum to shift from 329.6 ± 0.2 nm to 326.8 ± 0.1 nm in the bleached RDM, and from 330.8 ± 0.2 nm to 326.1 ± 0.2 nm in the regenerated RDM. Halothane had
no significant effect on this acrylamide-sensitive component (Fig. 2B),
indicating that halothane does not bind near the more exposed
tryptophan residue accessible to acrylamide. The recently reported
crystal structure of bovine rhodopsin identifies Trp35 as the most
solvent exposed tryptophan residue (Palczewski et al., 2000), and thus Trp35 is a reasonable candidate for quenching by acrylamide.
|
1.4 (95% CI, 2.1 ~ 0.8) (Fig.
3). In regenerated isorhodopsin, although the IC50 was essentially unaltered (1.1 mM),
label incorporation was significantly decreased. The calculated maximum
stoichiometry was 1.08 (halothane/rhodopsin molar ratio) in opsin and
0.28 in isorhodopsin.
|
Halothane Binds to the Ligand Binding Site.
The fluorescence
and photoaffinity labeling data both suggest that halothane binds close
to the ligand binding site in the transmembrane core of rhodopsin.
However, hydrophobic volatile anesthetics have been shown to
preferentially bind to the lipid-protein interface as well (Tang et
al., 2000), which might alter fluorescence and ligand binding
allosterically. Accordingly, we tested this possibility by performing
proteolysis and radiosequence analysis of
[14C]halothane-photolabeled rhodopsin. A small
fragment from enzymatic digestion with V8 protease, previously shown to
include the site where photoreactive retinal analogs are incorporated
(Zhang et al., 1994), was used (Fig. 4A,
V8-S). Release of 14C was observed in cycle 26, indicating halothane photoincorporation at Trp265 (Fig. 4B). In the
same fragment from regenerated isorhodopsin (retinal-bound),
14C release at Trp265 was significantly smaller
than that released from opsin. Because the efficiency of Edman
degradation is not 100%, the repetitive yield results in a well-known
lag of the residue release (and therefore cpm), which grows in
magnitude with the number of cycles. Therefore, most of the cpm release measured at cycle 27 and the cycles thereafter represents lag from
cycle 26 and is unlikely to represent labeling of Leu266.
|
-helical bundle (Palczewski et al., 2000). Trp265 is
known to interact with retinal (Lin and Sakmar, 1996;
Kochendoerfer et al., 1997; Palczewski et al., 2000) and to play an
important role in rhodopsin regeneration (Reeves et al., 1999). To
illustrate the relationship between the retinal cavity formed in the
interhelical space of rhodopsin and the photolabeled residues, the
Gravitational Radiation Analysis and Simulation Package (GRASP;
http://www.lsc-group.phys.uwm.edu/~ballen/grasp-distribution/) was used to render the cavity surface on the crystal structure of
rhodopsin with retinal removed (Fig. 5)
(Nicholls et al., 1991). This cavity is measured to be 428 Å3, sufficient volume to accommodate at least
two halothane molecules (volume of ~130 Å3)
but it is likely to at least partially collapse on removal of retinal.
No other suitable cavity exists in the protein structure.
|
Competition between Halothane and the Ligand.
Finally, to
confirm the predicted competitive interaction between halothane and the
ligand retinal, retinal binding kinetics was monitored by the specific
absorption wavelength for isorhodopsin after the addition of
9-cis-retinal. Halothane significantly prolonged isorhodopsin formation in a concentration-dependent manner, indicating that halothane inhibits retinal binding
to opsin (Fig. 6; Table 1). The kinetics
of the fluorescence decrease produced by retinal binding was also
prolonged in the presence of halothane (Table 1). These data not only
confirm competition between halothane and retinal at the retinal
binding site in rhodopsin but also predict specific in vivo functional
changes (see Discussion). Although we noted that the
increase in the absorbance was slower than the fluorescence decrease,
the spectroscopic probes report different endpoints of the regeneration
process of isorhodopsin, and the data suggest that quenching of
tryptophan fluorescence is established at an earlier stage of
chromophore regeneration.
|
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Using several different approaches, we have shown here that a
general inhaled anesthetic binds to the endogenous ligand binding site
and competitively inhibits the ligand binding in a prototypical GPCR.
Photoincorporation of [14C]halothane at Trp265
indicates unambiguous location of halothane in the interhelical core in
opsin, which is consistent with the photoaffinity stoichiometry of 1 for halothane in opsin. The crystal structure of bovine rhodopsin has
confirmed that the ligand-binding cavity can accommodate halothane in
the core formed by the -helical bundle.
Competition between halothane and the ligand retinal predicts that the
anesthetic may interfere with retinal binding in a functionally
apparent manner. Indeed, the functional importance of this binding
interaction is shown by a recent in vivo finding that rhodopsin
regeneration in mice and rats was significantly inhibited under
halothane anesthesia at 1.5-1.8% (v/v), corresponding to about 0.4 mM
at 37°C (Keller et al., 2001). Dark adaptation of vision, in which
retinal chromophore regeneration plays an important role, was also
reported to be retarded in humans and monkeys under halothane
anesthesia (van Norren and Padmos, 1975). Our data are consistent with
other studies showing functional effects of volatile anesthetics on
GPCRs in vitro as well. For example, halothane competitively inhibited
muscarinic and thromboxane A2 signaling monitored
with Ca2+-activated Cl
currents in Xenopus laevis oocytes, but it had no effect on
intracellular signaling pathways, indicating that halothane interacts
with the receptor and/or receptor-G protein coupling (Durieux, 1995;
Honemann et al., 1998). Halothane also decreased ligand binding
affinity for the 
-adrenergic receptor in rat myocardium, and reduced
positive inotropic potency of its agonist (Schotten et al., 1998).
Although the effect of inhaled anesthetics on protein kinase C (PKC) or on PKC phosphorylation sites of the receptor was suggested in the
metabotropic glutamate receptor, direct interactions of anesthetics with the hydrophobic domain of the receptor cannot be eliminated because of the lack of the concentration-effect relationships between
PKC inhibitors/activators and anesthetics (Minami et al., 1998).
Further studies may need to define anesthetic actions at the downstream
of a variety of GPCR signaling cascades.
It is interesting that the effective concentrations of halothane in
these previous in vivo and in vitro studies seem to be lower than those
describing the binding relationship in this study (1.1-2.3 mM,
equivalent to the gaseous concentration of 4 to 9%). Dark adaptation
was significantly altered at the lowest halothane concentration of
0.2% (van Norren and Padmos, 1975). Halothane inhibited muscarinic
signaling in oocytes with an IC50 of 0.3 mM
(Durieux, 1995). These differences probably reflect the enormous signal
amplification and the well-known displacement of concentration-effect curves from receptor-occupancy profiles in the GPCR systems (Ross, 1996). In this context, it is particularly interesting that halothane almost completely inhibited retinal regeneration in mice at 1.8% (Keller et al., 2001), the same phenomenon that we observed using membrane preparations at higher halothane concentrations. The explanation for this difference in halothane sensitivity is not clear,
but could be caused by species differences or by altered functioning of
this receptor in the in vitro membrane preparation.
The site of halothane binding identified in this study may represent a
common anesthetic binding motif in many GPCRs. First, the residue
identified as most contributing to the site for halothane, Trp265, is
one of the most conserved residues in more than 199 unique sequences of
the GPCRs in the rhodopsin family (Baldwin et al., 1997). Second, a
similar interhelical domain is created with a sufficient number of
conserved residues in the transmembrane portion in other GPCRs (Baldwin
et al., 1997). It is interesting that most of these highly conserved
residues face the interior of the molecule in the crystal structure of
rhodopsin (Palczewski et al., 2000), confirming a similar molecular
environment in the core in these GPCRs. Furthermore, with the use of
designed four--helical bundles (Johansson et al., 1998, 2000), and
other soluble proteins with hydrophobic cavities (Eckenhoff et al.,
2000; Eriksson et al., 1992), it has been shown that preformed
accessible cavities are important features to facilitate anesthetic
binding. Although inhaled anesthetics have divergent chemical
structures, the molecules are relatively small (90-160
Å3) and have few interactive atoms or groups to
provide much selectivity, compared with other molecules that interact
with GPCRs, such as biogenic amines and retinal. All these data support
the notion that inhaled anesthetic binding in the interhelical core is
a feature of general significance in the GPCR family.
A common binding motif in this class of receptor may also suggest a
common mechanism of action of inhaled anesthetics. Although several
distinct modes of ligand-receptor interactions are reported for GPCRs,
small ligands, such as biogenic amines, eicosanoids, enter and bind in
the transmembrane core of their GPCRs as retinal does (Strader et al.,
1994; Ji et al., 1998). Covalent attachment of the ligand is indeed
unique to rhodopsin, but the binding site for catecholamine in the
-adrenergic receptor is thought to be remarkably similar to the
binding site for retinal in rhodopsin (Strader et al., 1994; Sakmar,
1998). Cholinergic ligands have also been reported to interact with
aromatic as well as polar residues, such as tyrosine, in the core of
the muscarinic receptor (Hulme et al., 1999). Our results thus suggest
that disruption of signal transduction through competition with
endogenous ligands in the core may be a common mechanism of the action
of an inhaled anesthetic like halothane at these GPCRs. Moreover, it
has been reported that the ligands that bind to the transmembrane core of the metabotropic glutamate receptor, another family of the GPCRs,
allosterically inhibit receptor signaling after agonist binding to the
extracellular domain (Litschig et al., 1999; Pagano et al., 2000). This
suggests that anesthetic occupancy of this core region may have both
competitive and allosteric effects on receptor function. Key features
of general anesthesia, such as loss of consciousness and
antinociception, as well as anesthetic "side" effects, which
include a multitude of cardiovascular and autonomic features, could be
explained by a wide spectrum of the roles of GPCR signaling in the
central and peripheral nervous systems.
It is important to note that there are striking differences in the
effective concentrations between inhaled anesthetics and other
pharmacological agents. For example, in contrast to the aqueous
EC50 of inhaled anesthetics, ranging between 0.2 and 1.0 mM, opioids and benzodiazepines are effective in nanomolar
concentrations. This has led to the interpretation that the inhaled
anesthetics may interact with multiple protein sites to produce
anesthetic state. Our present data also suggest possible interactions
of inhaled anesthetics with other neuronal membrane proteins as well as
GPCRs. Because -helical bundles are a commonly found motif in many
membrane proteins, occupancy of interhelical cavities could cause
changes in helical arrangement, disruption of oligomerization equilibrium, or alteration in dynamic behavior, all of which may further explain divergent physiological effects of these widely used drugs.
| |
Acknowledgments |
|---|
We thank Dr. Jonas S. Johansson and Prof. David E. Longnecker for helpful discussions and continuous encouragement, Dr. David C. Chiara and Prof. Jonathan B. Cohen for the help of sequence analysis, and Kin Chan, Christina Reilly, and Robert Sharp for expert technical assistance.
| |
Footnotes |
|---|
Received November 16, 2001; Accepted January 31, 2002
This work was supported by National Institutes of Health grants GM51595 and EY00012.
Address correspondence to: Dr. Yumiko Ishizawa, Department of Anesthesia, University of Pennsylvania Medical Center, 3400 Spruce Street, 7 Dulles, Philadelphia, PA 19104-4283. E-mail: ishizawa{at}mail.med.upenn.edu
| |
Abbreviations |
|---|
GPCR, G protein-coupled receptor; RDM, rod disk membranes; MOPS, 3-(N-morpholino)propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PTH, phenylthiohydantoin; NATA, N-acetyl-tryptophan-amide; PKC, protein kinase C.
| |
References |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
This article has been cited by other articles:
|
|
 |
|
  M. M. Sedensky, J. M. Siefker, J. Y. Koh, D. M. Miller III, and P. G. Morgan A stomatin and a degenerin interact in lipid rafts of the nervous system of Caenorhabditis elegans Am J Physiol Cell Physiol, August 1, 2004; 287(2): C468 - C474. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Zhang and J. S. Johansson An Isothermal Titration Calorimetry Study on the Binding of Four Volatile General Anesthetics to the Hydrophobic Core of a Four-{alpha}-Helix Bundle Protein Biophys. J., November 1, 2003; 85(5): 3279 - 3285. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Ouyang, G. Wang, and H. C. Hemmings Jr Isoflurane and Propofol Inhibit Voltage-Gated Sodium Channels in Isolated Rat Neurohypophysial Nerve Terminals Mol. Pharmacol., August 1, 2003; 64(2): 373 - 381. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Okamoto, K. Minami, Y. Uezono, J. Ogata, M. Shiraishi, A. Shigematsu, and Y. Ueta The Inhibitory Effects of Ketamine and Pentobarbital on Substance P Receptors Expressed in Xenopus Oocytes Anesth. Analg., July 1, 2003; 97(1): 104 - 110. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
