Lipid-protein interactions in reconstituted membranes containing acetylcholine receptor

Functional membranes containing purified Torpedo californica acetylcholine receptor and dioleoylphosphatidylcholine (DOPC) were prepared by a cholate dialysis procedure with lipid to protein ratios of 100-400 to 1 (mol/mol). Spin-labeled lipids were incorporated into the reconstituted membranes and into native membranes prepared from Torpedo electroplax, and electron paramagnetic resonance (EPR) spectra were recorded between 0 and 20 degrees C. The spin-labels included nitroxide derivatives of stearic acid (16-doxylstearic acid), androstane, phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). The phospholipid spin-labels had 16-doxylstearic acid in the sn-2 position. All the spectra showed two components corresponding to a relatively mobile bilayer component and a motionally restricted "protein-perturbed" component. The relative amounts of mobile and perturbed components were quantitated by spectral subtraction and integration techniques. The mobile/perturbed ratio was somewhat temperature dependent, and the results are discussed in terms of exchange between mobile and perturbed environments. Plots of the mobile/perturbed ratios vs. lipid/protein ratios at 1 degree C gave straight lines from which the relative binding affinity of each spin-label and the number of perturbed lipids per receptor protein could be calculated. All the spin-labels gave similar values for the number of perturbed lipids (40 +/- 7), a number close to the number of lipids that will fit around the intramembranous perimeter of the receptor. The affinities of the spin-labeled lipids for the receptor relative to DOPC were androstane (K = 4.3) congruent to 16-doxylstearic acid (4.1) greater than PA (2.7) greater than PE (1.1) approximately PC (1.0) approximately PS (0.7). The lipids having the highest affinity for the acetylcholine receptor were also those that have the largest effects on the ion flux functional properties of the receptor, and the results are discussed in terms of lipid effects on receptor function.     

Internal dynamics of the nicotinic acetylcholine receptor in reconstituted membranes

The structure and 1H/2H exchange kinetics of affinity-purified nAChR reconstituted into egg phosphatidylcholine membranes with increasing levels of either dioleoylphosphatidic acid (DOPA) or cholesterol (Chol) have been examined using infrared spectroscopy. All spectra of the reconstituted nAChR membranes recorded after 72 h in 2H2O exhibit comparable amide I band shapes, suggesting a similar secondary structure for the nAChR in each lipid environment. Increasing levels of either DOPA or Chol, however, lead to an increasing intensity of the amide II band, indicating a decreasing proportion of nAChR peptide hydrogens that have exchanged for deuterium. Spectra recorded as a function of time after exposure of the nAChR to 2H2O show that the presence of either lipid slows down the 1H/2H exchange of those peptide hydrogens that normally exchange on the minutes to hours time scale. The slowing of peptide 1H/2H exchange correlates with both an increasing ability of the nAChR to undergo agonist-induced conformational change [Baenziger, J. E., Morris, M.-L., Darsaut, T. E., and Ryan, S. E. (1999) in preparation] and possibly a decreasing membrane fluidity. Our data suggest that lipid composition dependent changes in nAChR peptide 1H/2H exchange kinetics reflect altered internal dynamics of the nAChR. Lipids may influence protein function by changing the internal dynamics of integral membrane proteins.     

Mechanisms of muscarinic receptor action on Go in reconstituted phospholipid vesicles

Purified muscarinic receptors (0.5-10 nmol of L-[3H]quinuclidinyl benzilate-binding sites/mg of protein) from bovine brain and the GTP-dependent regulatory protein, Go, were reconstituted with a lipid mixture of phosphatidylcholine and cholesterol. Essentially all of the receptors could interact with Go as evinced by increases in affinity for agonist as large as 800-fold. Both the alpha and beta gamma subunits of Go were required for this effect. Similarly, both subunits were required for the stimulation of guanine nucleotide exchange by agonists. This latter action of the receptor on Go was catalytic and potentiated markedly by prior treatment with dithiothreitol. Initially, agonist stimulation of association of GTP and guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) to Go was small and variable due to high basal rates. Prior addition of excess GDP inhibited the basal rate of exchange but allowed stimulation by agonists. Under these conditions, oxotremorine stimulated the rates of association of GTP gamma S up to 10-fold. This selective effect was not mimicked by GTP which inhibited both the basal and hormone-dependent rates. Direct examination of the association of GTP and GDP to Go demonstrated that agonist caused either stimulation or marked inhibition, respectively. These results indicate that receptors stimulate guanine nucleotide exchange on G proteins by both increasing the rates of dissociation of nucleotides and altering their relative affinities such that binding of GTP becomes highly favored over GDP. This would ensure the activation of G proteins by receptors in the presence of both nucleotides.     

Segregation of phosphatidic acid-rich domains in reconstituted acetylcholine receptor membranes

Purified Acetylcholine Receptor (AcChR) from Torpedo has been reconstituted at low (approximately 1:3500) and high (approximately 1:560) protein to phospholipid molar ratios into vesicles containing egg phosphatidylcholine, cholesterol, and different dimyristoyl phospholipids (dimyristoyl phosphatidylcholine, phosphatidylserine, phosphatidylglycerol and phosphatidic acid) as probes to explore the effects of the protein on phospholipid organization by differential scanning calorimetry, infrared, and fluorescence spectroscopy. All the experimental results indicate that the presence of the AcChR protein, even at the lower protein to phospholipid molar ratio, directs lateral phase separation of the monoanionic phosphoryl form of the phosphatidic acid probe, causing the formation of specific phosphatidic acid-rich lipid domains that become segregated from the bulk lipids and whose extent (phosphatidic acid sequestered into the domain, out of the total population in the vesicle) is protein-dependent. Furthermore, fluorescence energy transfer using the protein tryptophan residues as energy donors and the fluorescence probes trans-parinaric acid or diphenylhexatriene as acceptors, establishes that the AcChR is included in the domain. Other dimyristoyl phospholipid probes (phosphatidylcholine, phosphatidylserine, phosphatidylglycerol) under identical conditions could not mimic the protein-induced domain formation observed with the phosphatidic acid probe and result in ideal mixing of all lipid components in the reconstituted vesicles. Likewise, in the absence of protein, all the phospholipid probes, including phosphatidic acid, exhibit ideal mixing behavior. Since phosphatidic acid and cholesterol have been implicated in functional modulation of the reconstituted AcChR, it is suggested that such a specific modulatory role could be mediated by domain segregation of the relevant lipid classes.     

The lipid environment of the nicotinic acetylcholine receptor in native and reconstituted membranes

Detailed knowledge of the membrane framework surrounding the nicotinic acetylcholine receptor (AChR) is key to an understanding of its structure, dynamics, and function. Recent theoretical models discuss the structural relationship between the AChR and the lipid bilayer. Independent experimental data on the composition, metabolism, and dynamics of the AChR lipid environment are analyzed in the first part of the review. The composition of the lipids in which the transmembrane AChR chains are inserted bears considerable resemblance among species, perhaps providing this evolutionarily conserved protein with an adequate milieu for its optimal functioning. The effects of lipids on the latter are discussed in the second part of the review. The third part focuses on the information gained on the dynamics of AChR and lipids in the membrane, a section that also covers the physical properties and interactions between the protein, its immediate annulus, and the bulk lipid bilayer.     

Multiple binding sites for local anesthetics on reconstituted acetylcholine receptor membranes

Using electron spin resonance spectroscopy and a spin-labeled analog of a tertiary amine local anesthetic, we have identified several populations of the local anesthetic within reconstituted lipid membranes containing purified acetylcholine receptors. These populations represent the local anesthetic interacting with membrane lipid and with the acetylcholine receptor. The data also suggest the existence of at least two classes of binding sites for the local anesthetic on the acetylcholine receptor.     

Calcitonin modifies ligand binding to muscarinic receptor in CNS membranes

Calcitonin (CT) is a peptide produced by the thyroid gland, whose best described role is to prevent bone reabsorption, though it also participates in other biological functions through both central and peripheral mechanisms. CT is able to inhibit brain Na(+), K(+)-ATPase activity (Rodríguez de Lores Arnaiz, López Ordieres, Peptides 1997;18:613-5) and a relationship between such enzyme activity and cholinergic function has been suggested. Accordingly, we tested CT effect on [(3)H]-quinuclidinyl benzilate ([(3)H]-QNB) binding to rat CNS membranes to determine whether the peptide is able to modify the cholinergic muscarinic receptor as well. It was found that 1x10(-7)-1x10(-5) M CT decreased 20-70% ligand binding to hippocampal, cerebellar, cortical and striatal membranes. Scatchard analysis of saturation curves showed that 5x10(-6) M CT significantly modified binding kinetic constants, thus it increased roughly 220% K(d) values and decreased 20-36% B(max) values in cerebral cortical and cerebellar membranes. Since the peptide decreases affinity ligand binding and reduces the number of binding sites, CT may well be acting as a cholinergic modulator through a decrease in muscarinic receptor functionality.     

Functional properties of acetylcholine receptor monomeric and dimeric forms in reconstituted membranes

The dimeric and monomeric forms of the acetylcholine receptor from $\text{Torpedo californica}$ electroplax have been purified in the presence of lipids and reconstituted. A spectroscopic method was applied to study the rapid kinetics of cation transport mediated by each of the reconstituted AcChR oligomers. Both the AcChR dimer and monomer responded to carbamylcholine by mediating cation transport on the time scale of a few milliseconds. The responses to carbamylcholine were blocked by histrionicotoxin and by desensitization, demonstrating that both forms manifest pharmacological properties observed $\text{in vivo}$. Analysis of the fast ion transport produced by various agonist concentrations yielded estimated rates of transport through a single receptor channel. These were comparable for the monomer and dimer and in agreement with those obtained for a preparation containing a mixture of both oligomers.     

Activation and inactivation kinetics of Torpedo californica acetylcholine receptor in reconstituted membranes

By use of a quench-flow technique to measure tracer ion flux rates in a physiologically significant time domain, the kinetics of activation and inactivation of purified reconstituted acetylcholine receptor (AChR) were investigated. After solubilization in sodium cholate, purification by affinity chromatography, and reconstitution into soybean lipids, the AChR from Torpedo californica displayed a characteristically fast rate of ion influx measured with 86Rb+. At 4 degrees C 1 mM carbamoylcholine (Carb) stimulated a fast (t1/2 = 7 ms) first-order filling of vesicle internal volume that presented a 10(4)-fold stimulation of ion flux rate by Carb. The concentration dependence of activation was sigmoidal with a half-maximal value at 3 X 10(-4) M Carb. In the presence of Carb, the purified AChR also underwent a two-step inactivation (desensitization) process. Inactivation was measured by preincubating AChR with Carb for various times (milliseconds to minutes) and then measuring the 86Rb+ influx rate. The two inactivation processes were each characterized by a distinct maximum rate (5.3 and 0.10 s-1) and by a different dependence on Carb concentration. The slow phase of inactivation gave a half-maximal rate at 2.5 X 10(-4) M Carb, and the fast inactivation was half-maximal at 1.3 X 10(-3) M Carb. The concentration dependence curves for both inactivation processes were approximately hyperbolic. The results are discussed in terms of models that describe the relationship between ligand binding and the processes of channel activation and desensitization.     

Ultrastructure of reconstituted rat liver microsomal membranes and cytochrome b5- or P-450-containing proteoliposomes

Thin sectioning and freeze-fracture electron microscopy have been used to show that it is possible to obtain topologically closed vesicles by means of reconstitution of rat liver microsomal membrane "ghosts." The reconstitution by 15 hr dialysis resulted in the formation of vesicles with intramembrane particles (IMP) while after 40 hr dialysis no IMP were observed in the membranes. The protein/lipid ratio and functional activity of NADPH- and NADH-linked enzyme systems were similar in both cases. Cytochrome P-450 (LM2) was incorporated into liposomes of different composition (protein: lipid ratio--1:200). IMP were observed only when the incorporation of cytochrome P-450 was performed in the presence of detergent Emulgen 913 as specific additive to the initial protein-lipid-sodium cholate mixture or in the course of incubation of proteoliposomal suspensions at 37 degrees C. After the incorporation of cytochrome b5 into azolectin liposomes vesicular membranes contain IMP if the incorporated membrane protein: lipid ratio is at least 1:50. Pronase-induced splitting off of a 11 kDa heme-containing fragment of cytochrome b5 did not affect IMP content. The conditions of IMP formation in reconstituted membranes and in microsomal ghosts are discussed.     

The effect of amantadine on nicotinic acetylcholine receptor (nAchR) in reconstituted membranes

The antiviral drug amantadine is also a potent neuromuscular blocking agent. When the nicotinic receptor from a Torpedinidae species is reconstituted into soybean liposomes, the binding of α-bungarotoxin is not altered although the carbamylcholine induced radioactive cation influx is blocked.By studying cation fluxes in amantadine preincubated membranes previously exposed to different concentrations of carbamylcholine for different periods of time, we have shown that the drug accelerates the conversion of the nicotinic acetylcholine receptor from a state of low affinity to a state of high affinity for carbamyalcholine, a phenomenon correlated with receptor desensitization. The drug did not induce such a shift by itself.The present data and those by Earnest et al. (Biochemistry22, 5523–5535, 1984) show that the nicotinic acetylcholine receptor reconstituted into liposomes is a good model for studying the effects of noncompetitive blockers of nicotinic acetylcholine receptor function.     

Recovery of oligomers and cooperativity when monomers of the M2 muscarinic cholinergic receptor are reconstituted into phospholipid vesicles

FLAG- and HA-tagged M2 muscarinic receptors from coinfected Sf9 cells have been purified in digitonin-cholate and reconstituted into phospholipid vesicles. The purified receptor was predominantly monomeric: it showed no detectable coimmunoprecipitation; it migrated as a monomer during electrophoresis before or after cross-linking with bis(sulfosuccinimidyl)suberate; and it bound agonists and antagonists in a manner indicative of identical and mutually independent sites. Receptor cross-linked after reconstitution or after reconstitution and subsequent solubilization in digitonin-cholate migrated almost exclusively as a tetramer. The binding properties of the reconstituted receptor mimicked those reported previously for cardiac muscarinic receptors. The apparent capacity for N-[3H]methylscopolamine (NMS) was only 60% of that for [3H]quinuclidinylbenzilate (QNB), yet binding at saturating concentrations of [3H]QNB was inhibited fully and in a noncompetitive manner at comparatively low concentrations of unlabeled NMS. Reconstitution of the receptor with a saturating quantity of functional G proteins led to the appearance of three classes of sites for the agonist oxotremorine-M in assays with [3H]QNB; GMP-PNP caused an apparent interconversion from highest to lowest affinity and the concomitant emergence of a fourth class of intermediate affinity. All of the data can be described quantitatively in terms of cooperativity among four interacting sites, presumably within a tetramer; the effect of GMP-PNP can be accommodated as a shift in the distribution of tetramers between two states that differ in their cooperative properties. Monomers of the M2 receptor therefore can be assembled into tetramers with binding properties that closely resemble those of the muscarinic receptor in myocardial preparations.     

Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranes

The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing.     

Nicotinic acetylcholine receptor induces lateral segregation of phosphatidic acid and phosphatidylcholine in reconstituted membranes

Purified nicotinic acetylcholine receptor (AChR) protein was reconstituted into synthetic lipid membranes having known effects on receptor function in the presence and absence of cholesterol (Chol). The phase behavior of a lipid system (DPPC/DOPC) possessing a known lipid phase profile and favoring nonfunctional, desensitized AChR was compared with that of a lipid system (POPA/POPC) containing the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the AChR. Fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and AChR intrinsic fluorescence by a nitroxide spin-labeled phospholipid showed that the AChR diminishes the degree of DPH quenching and promotes DPPC lateral segregation into an ordered lipid domain, an effect that was potentiated by Chol. Fluorescence anisotropy of the probe DPH increased in the presence of AChR or Chol and also made apparent shifts to higher values in the transition temperature of the lipid system in the presence of Chol and/or AChR. The values were highest when both Chol and AChR were present, further reinforcing the view that their effect on lipid segregation is additive. These results can be accounted for by the increase in the size of quencher-free, ordered lipid domains induced by AChR and/or Chol. Pyrene phosphatidylcholine (PyPC) excimer (E) formation was strongly reduced owing to the restricted diffusion of the probe induced by the AChR protein. The analysis of Forster energy transfer (FRET) from the protein to DPH further indicates that AChR partitions preferentially into these ordered lipid microdomains, enriched in saturated lipid (DPPC or POPA), which segregate from liquid phase-enriched DOPC or POPC domains. Taken together, the results suggest that the AChR organizes its immediate microenvironment in the form of microdomains with higher lateral packing density and rigidity. The relative size of such microdomains depends not only on the phospholipid polar headgroup and fatty acyl chain saturation but also on AChR protein-lipid interactions. Additional evidence suggests a possible competition between Chol and POPA for the same binding sites on the AChR protein.     

Carbamylcholine-induced rapid cation efflux from reconstituted membrane vesicles containing purified acetylcholine receptor.

Membrane preparations containing essentially only the four polypeptides considered to constitute the acetylcholine receptor are purified from $\text{Torpedo}\text{californica}$ electroplax. Treatment of these membranes with 2% ($\text{w}\text{v}$ aqueous sodium cholate followed by removal of all insoluble matter results in a solubilized purified receptor preparation that can be reassociated with phospholipids during dialysis to remove the detergent. Such reconstituted receptor is shown to retain the capability of translocating ²²Na⁺ across the membrane in response to carbamylcholine binding in a highly reproducible manner. The dose response for this effect is similar to that observed for the original electroplax membrane preparation and the carbamylcholine induced signal is completely blocked by α-bungarotoxin.     

Lipid rafts reconstituted in model membranes

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.     

Reconstitution of the muscarinic acetylcholine receptor. Guanine nucleotide-sensitive high affinity binding of agonists to purified muscarinic receptors reconstituted with GTP-binding proteins (Gi and Go)

Muscarinic acetylcholine receptors purified from porcine brain were reconstituted with two kinds of GTP-binding proteins (Gi and Go). The binding of agonists was affected by guanine nucleotides when the receptor was reconstituted with either Gi or Go, but not in the absence of one of the GTP-binding proteins. The displacement curves with agonists for the [3H]quinuclidinyl benzylate [( 3H]QNB) binding were explained by assuming there are two sites with different affinities for a given agonist. The proportion of the high affinity site increased with increasing concentrations of the GTP-binding proteins, and the maximum value represented 50-70% of the total [3H]QNB-binding sites. Reconstitution of the receptor with both Gi and Go did not increase the proportion any further. These results indicate that Gi and Go interact with the same site, which rules out the possibility that there are two kinds of muscarinic receptors, one interacting with Gi and the other with Go. GDP as well as GTP decreased the affinity for the agonists of the muscarinic receptors reconstituted with Gi or Go. The conversion of GDP to GTP during the incubation was less than 1%, indicating that the effect of GDP is not due to its conversion to GTP, and that the binding of either GTP or GDP with the GTP-binding proteins suppresses their interaction with the receptor.     

Electron spin resonance studies of acyl chain motion in reconstituted nicotinic acetylcholine receptor membranes.

The electron spin resonance spectra of spin-label positional isomers of stearic acid (n-SASL) incorporated into nicotinic acetylcholine receptors (nAcChoR) reconstituted into dioleoylphosphatidylcholine (DOPC) were deconvoluted into bilayer- and protein-associated components by subtraction under conditions of slow exchange. The selectivity of n-SASL (n = 6, 9, 12, and 14) for the lipid-protein interface of the nAcChoR was threefold greater than that of DOPC and independent of the spin label position. The temperature at which exchange became apparent as judged from lineshape broadening of the mobile lipid component spectrum was dependent upon the position of the spin-label moiety; near the bilayer center, exchange broadening occurred at lower temperatures than it did closer to the lipid headgroup. This suggests that the lipid headgroup region of boundary lipids is relatively fixed, whereas its acyl chain whips on and off the protein with increasing frequency near the bilayer center. Motions on the microsecond time scale were examined by microwave power saturation. Each n-SASL saturated more readily when incorporated into vesicles containing the nAcChoR than when in pure DOPC liposomes. Therefore, lipid mobility is perturbed by the nAcChoR on the microsecond time scale with an apparent magnitude that is relatively modest, probably due to exchange on this time scale.     

Increased muscarinic receptor binding in heart membranes by an inhibitor of protein kinase C

The number of muscarinic acetylcholine receptors (MAChRs), as detected by binding of [3H]quinuclidinyl benzilate (QNB), was investigated under conditions which promote protein phosphorylation. Incubation of a crude heart membrane preparation in the presence of ATP/Mg2+ reduced MAChR number by 50%. Incubation with polymyxin B, an inhibitor of protein kinase C, blocked the effect of ATP/Mg2+ and increased MAChR number by 74%.     

Binding of radioiodinated SPECT ligands to transfected cell membranes expressing single muscarinic receptor subtypes

The equilibrium dissociation constant and the kinetic rate constants were determined for the binding of (R)-[3H]3-quinuclidinyl benzilate ([3H]QNB) and [125I]3-quinuclidinyl-4-iodobenzilate ((R,R)- and (R,S)-[125I]IQNB) to transfected cell membranes expressing one single muscarinic acetylcholine receptor (mAChR) subtype. The association and dissociation kinetics for the m2 subtype were more rapid than for the m1 and m3 subtypes. The differential kinetic properties may be useful for the single photon emission computed tomographic (SPECT) evaluation of regional mAChR subtype alterations in disease states.