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The role of protein sulfhydryl groups and protein disulfides of the platelet surface in aggregation processes involving thiol exchange reactions

Blood platelets are central to haemostasis and platelet aggregation is considered to be a direct index of platelet function. Although protein disulfides (PSSP) are structural components of most proteins, current evidence suggests that PSSP work together with protein SH groups (PSH) to activate various platelet functions in dynamic processes involving thiol/disulfide exchange reactions. Based on these assumptions, we performed experiments to demonstrate how PSH and PSSP are involved in platelet aggregation and how modifications of PSH and PSSP concentrations on the platelet surface by N-ethylmaleimide (NEM) (a PSH-blocking reagent) and dithiothreitol (DTT) (a PSSP-reducing reagent), respectively, may condition platelet susceptibility in protein rich plasma and washed platelets and integrin αIIbβ3 conformation. Our data strongly suggest that the PSH blockage and the PSSP reduction of the platelet surface are deeply involved in aggregation processes evoked in protein rich plasma and washed platelets by ADP and collagen; that endogenous thiols (e.g. GSH) may interfere with NEM actions; that NEM and DTT, acting on preexisting PSH and PSSP of active platelets have opposite conformational changes on integrin αIIbβ3 conformation. Although the precise mechanism and the populations of specific PSH and PSSP involved remain unresolved, our data support the notion that PSH and PSSP of the platelet surface are involved in platelet activation by thiol exchange reactions. A plausible molecular mechanism of PSH and PSSP recruitment during thiol exchange reactions is here proposed.

Contribution of a tyrosine-based motif to cellular trafficking of wild-type and truncated NPY Y 1 receptors

The human NPY Y 1 receptor undergoes fast agonist-induced internalization via clathrin-coated pits then recycles back to the cell membrane. In an attempt to identify the molecular determinants involved in this process, we studied several C-terminal truncation mutants tagged with EFGP. In the absence of agonist, Y 1 receptors lacking the last 32 C-terminal amino acids (Y 1Δ32) are constitutively internalized, unlike full-length Y 1 receptors. At steady state, internalized Y 1Δ32 receptors co-localize with transferrin, a marker of early and recycling endosomes. Inhibition of constitutive internalization of Y 1Δ32 receptors by hypertonic sucrose or by co-expression of Rab5aS34N, a dominant negative form of the small GTPase Rab5a or depletion of all three isoforms of Rab5 indicates the involvement of clathrin-coated pits. In contrast, a truncated receptor lacking the last 42 C-terminal amino acids (Y 1Δ42) does not constitutively internalize, consistent with the possibility that there is a molecular determinant responsible for constitutive internalization located in the last 10 amino acids of Y 1Δ32 receptors. We show that the agonist-independent internalization of Y 1Δ32 receptors involves a tyrosine-based motif YXXΦ. The potential role of this motif in the behaviour of full-length Y 1 receptors has also been explored. Our results indicate that a C-terminal tyrosine-based motif is critical for the constitutive internalization of truncated Y 1Δ32 receptors. We suggest that this motif is masked in full-length Y 1 receptors which do not constitutively internalize in the absence of agonist.

Lactadherin functions as a probe for phosphatidylserine exposure and as an anticoagulant in the study of stored platelets

Annexin V, the long established standard method of measuring phosphatidylserine (PS) exposure, is not the most suitable probe for the study of stored platelets because of calcium dependence and low sensitivity under 8% PS exposure. The aim of this study was to show lactadherin as a sensitive probe for PS exposure and an effective anticoagulant for stored platelets. PS exposure and the associated procoagulant activity of platelets in 20 platelet concentrate units were investigated by flow cytometry, confocal microscopy, coagulation time analysis and enzymatic assays. Annexin V and lactadherin were utilized separately in this study. Using lactadherin, we identified higher levels of PS exposure on the platelets and microparticles compared to detection using annexin V by flow cytometry. Lactadherin staining showed earlier PS exposure localized to platelets membrane in the earlier stage of storage. Subsequently, PS was distributed on the narrow rim of the plasma membrane and blebbing vesicles. Lactadherin or annexin V(32 nm) prolonged coagulation time 2·4 fold versus twofold. The production of thrombin and intrinsic/extrinsic factor Xase were inhibited approximately 85∼90% and 65∼70% by lactadherin and annexin V, respectively. Lactadherin can provide the quantification and location of PS exposure on banked platelets in the absence of agonists. Furthermore, lactadherin is a more effective anticoagulant for inhibiting the procoagulant activity of stored platelets compared with annexin V.

Mechanistic Insights into Xenon Inhibition of NMDA Receptors from MD Simulations

Inhibition of N-methyl-D-aspartate (NMDA) receptors has been viewed as a primary cause of xenon anesthesia, yet the mechanism is unclear. Here, we investigated interactions between xenon and the ligand-binding domain (LBD) of a NMDA receptor and examined xenon-induced structural and dynamical changes that are relevant to functional changes of the NMDA receptor. Several comparative molecular dynamics simulations were performed on two X-ray structures representing the open- and closed-cleft LBD of the NMDA receptor. We identified plausible xenon action sites in the LBD, including those nearby agonist sites, in the hinge region, and at the interface between two subunits. The xenon-binding energy varies from -5.3 to -0.7 kcal/mol. Xenon's effect on the NMDA receptor is conformation-dependent and is produced through both competitive and noncompetitive mechanisms. Xenon can promote cleft opening in the absence of agonists and consequently stabilizes the closed channel. Xenon can also bind at the interface of two subunits, alter the intersubunit interaction, and lead to a reduction of the distance between two GT linkers. This reduction corresponds to a rearrangement of the channel toward a direction of pore size decreasing, implying a closed or desensitized channel. In addition to these noncompetitive actions, xenon was found to weaken the glutamate binding, which could lead to low agonist efficacy and appear as competitive inhibition.

Direct voltage control of endogenous lysophosphatidic acid G-protein-coupled receptors inXenopusoocytes

Lysophosphatidic acid (LPA) G-protein-coupled receptors (GPCRs) play important roles in a variety of physiological and pathophysiological processes, including cell proliferation, angiogenesis, central nervous system development and carcinogenesis. Whilst many ion channels and transporters are recognized to be controlled by a change in cell membrane potential, little is known about the voltage dependence of other proteins involved in cell signalling. Here, we show that the InsP(3)-mediated Ca(2+) response stimulated by the endogenous LPA GPCR in Xenopus oocytes is potentiated by membrane depolarization. Depolarization was able to repetitively stimulate transient [Ca(2+)](i) increases after the initial agonist-evoked response. In addition, the initial rate and amplitude of the LPA-dependent Ca(2+) response were significantly modulated by the steady holding potential over the physiological range, such that the response to LPA was potentiated at depolarized potentials and inhibited at hyperpolarized potentials. Enhancement of LPA receptor-evoked Ca(2+) mobilization by membrane depolarization was observed over a wide range of agonist concentrations. Importantly, the amplitude of the depolarization-evoked intracellular Ca(2+) increase displayed an inverse relationship with agonist concentration such that the greatest effect of voltage was observed at near-threshold levels of agonist. Voltage-dependent Ca(2+) release was not induced by direct elevation of InsP(3) or by activation of heterotrimeric G-proteins in the absence of agonist, indicating that the LPA GPCR itself represents the primary site of action of membrane voltage. This novel modulation of LPA signalling by membrane potential may have important consequences for control of Ca(2+) signals both in excitable and non-excitable tissues.

Agonism with the omega-3 fatty acids α-linolenic acid and docosahexaenoic acid mediates phosphorylation of both the short and long isoforms of the human GPR120 receptor

The newly discovered G protein-coupled receptor GPR120 has recently been shown to stimulate secretion of the gut hormones glucagon-like peptide-1 and cholecystokinin upon binding of free fatty acids, thrusting it to the forefront of drug discovery efforts for treatment of type 2 diabetes as well as satiety and obesity. Although sequences for two alternative splice variants of the human GPR120 receptor have been reported, there have been no studies which directly compare the signaling of these isoforms. We have identified an additional 16 amino acid gap containing four phospho-labile serine/threonine residues which is localized to the third intracellular loop of the GPR120-long (GPR120-L) isoform. Based on this finding, we hypothesized that the agonist-stimulated phosphorylation profiles of this isoform would be distinct from that of the short isoform (GPR120-S). Using a clonal HEK293 cell model, we examined agonist-mediated phosphorylation of GPR120-S and GPR120-L with the omega-3 fatty acids α-linolenic acid (ALA) and docosahexaenoic acid (DHA). Our results show rapid phosphorylation of both isoforms following agonism by either ALA or DHA. Moreover, we show no significant difference in the degree or rate of phosphorylation of both isoforms upon agonism with either ALA or DHA, suggesting that the additional gap in the longer variant is not phosphorylated. Importantly, our results demonstrate that the shorter variant exhibits significantly more pronounced basal phosphorylation in the absence of agonist, suggesting that the additional gap in the long variant may contribute to masking of constitutive phosphorylation sites. These are the first results which demonstrate specific phosphorylation of GPR120 isoforms upon agonism by free fatty acids and the first which distinguish the phosphorylation profiles of the two GPR120 isoforms.

Modulation of silent and constitutively active nociceptin/orphanin FQ receptors by potent receptor antagonists and Na+ ions in rat sympathetic neurons.

The pharmacology of G protein-coupled receptors can be influenced by factors such as constitutive receptor activation and Na(+) ions. In this study, we examined the coupling of natively and heterologously expressed nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptors with voltage-dependent Ca(2+) channels after exposure to four high-affinity NOP receptor blockers [[Nphe(1)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-101), 1-[1-(cyclooctylmethyl)-1,2,3,6-tetrahydro-5-(hydroxymethyl)-4-pyridinyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one (Trap-101), 1-benzyl-N-{3-[spiroisobenzofuran-1(3H),4'-piperidin-1-yl]propyl}pyrrolidine-2-carboxamide (compound 24), and N-(4-amino-2-methylquinolin-6-yl)-2-(4-ethylphenoxymethyl)benzamide hydrochloride (JTC-801)] in sympathetic neurons. The enhanced tonic inhibition of Ca(2+) currents in the absence of agonists, indicative of constitutively active NOP receptors in transfected neurons, was abolished after pretreatment with pertussis toxin. In control neurons, the four antagonists did not exert any effects when applied alone but significantly blocked the N/OFQ-mediated Ca(2+) current inhibition. Exposure of transfected neurons to UFP-101 resulted in partial agonist effects. In contrast, Trap-101, compound 24, and JTC-801 exerted inverse agonism, as measured by the loss of tonic Ca(2+) current inhibition. In experiments designed to measure the N/OFQ concentration-response relationship under varying Na(+) concentrations, a leftward shift of IC(50) values was observed after Na(+) exposure. Although similar N/OFQ efficacies were measured with all solutions, a significant decrease of Hill coefficient values was obtained with increasing Na(+) concentrations. Examination of the allosteric effects of Na(+) on heterologously overexpressed NOP receptors showed that the tonic Ca(2+) current inhibition was abolished in the presence of the monovalent cation. These results demonstrate that constitutively active NOP receptors exhibit differential blocker pharmacology and allosteric regulation by Na(+). Data are also presented demonstrating that heterologously expressed mu opioid receptors in sympathetic neurons are similarly modulated.

Single-Channel Recording of Muscle and Neuronal Nicotinic Acetylcholine Receptors: Implications for Allosteric Transitions

The muscle and neuronal nicotinic acetylcholine receptors (nAChRs) are pentameric ligand gated ion channels. In most cases these receptors open when two molecules of an agonist, endogenously acetylcholine, bind to the receptor at binding sites located in the extracellular domain. Agonist binding causes a conformational change in the channel pore that produces channel isomerization from the nonconducting to the conducting form. Whole-cell studies revealed two distinct subunit combinations of the α4β2 neuronal receptor: 3 α subunits and 2 β subunits (A3B2) and 2 α subunits and 3 β subunits (A2B3). The A2B3 stoichiometry has higher sensitivity for nicotine than the A3B2 receptor. Based on recent whole-cell work that distinguishes the two stoichiometries by their rectification behaviors, we are developing a single-channel recording-based voltage ramp method to distinguish different stoichiometries of the α4β2 receptors. We also seek to quantify the different types of openings and conductances of the 2 stoichiometries.In our studies of the muscle nAChR, our manipulations include mutations in the pore region that facilitate gating. We are evaluating a recently proposed model that invokes multiple open states of the nAChR, even in the absence of agonist.

The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation

The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

AMPA Receptor Ligand Binding Domain Mobility Revealed by Functional Cross Linking

Glutamate receptors mediate the majority of excitatory synaptic transmission in the CNS. The AMPA-subtype has rapid kinetics, with activation, deactivation and desensitization proceeding on the millisecond timescale or faster. Crystallographic, biochemical, and functional studies suggest that GluR2 Cys mutants which form intermolecular disulfide cross-links between the lower D2 lobes of the ligand binding cores can be trapped in a conformation that represents the desensitized state. We used multi-channel rapid perfusion techniques to examine the state dependence of cross-linking in these mutants. Under reducing conditions, both wild-type GluR2 and the G725C and S729C mutants have normal activation and desensitization kinetics, but the Cys mutants can be efficiently trapped in nonconducting states when oxidized. In contrast the I664C mutant is only partially inactivated under oxidizing conditions. For S729C, disulfide cross-links form rapidly when receptors are desensitized in the presence of glutamate, but receptors also become trapped at rest, in the absence of agonist. We assessed such spontaneous trapping in various conditions, including CNQX, a competitive antagonist; kainate, a weak partial agonist; or when desensitization was blocked by the L483Y mutation that stabilizes the D1 dimer interface. These experiments suggest that trapping in the absence of glutamate is due to two motions: Spontaneous breaking of the D1 dimer interface and hyperextension of the lower lobes of the ligand binding core. These data show that the glutamate binding domains are surprisingly mobile in the absence of ligand, which could influence receptor activity in the brain.

G Protein-coupled Receptor Kinase-mediated Phosphorylation Regulates Post-endocytic Trafficking of the D2 Dopamine Receptor

We investigated the role of G protein-coupled receptor kinase (GRK)-mediated phosphorylation in agonist-induced desensitization, arrestin association, endocytosis, and intracellular trafficking of the D(2) dopamine receptor (DAR). Agonist activation of D(2) DARs results in rapid and sustained receptor phosphorylation that is solely mediated by GRKs. A survey of GRKs revealed that only GRK2 or GRK3 promotes D(2) DAR phosphorylation. Mutational analyses resulted in the identification of eight serine/threonine residues within the third cytoplasmic loop of the receptor that are phosphorylated by GRK2/3. Simultaneous mutation of these eight residues results in a receptor construct, GRK(-), that is completely devoid of agonist-promoted GRK-mediated receptor phosphorylation. We found that both wild-type (WT) and GRK(-) receptors underwent a similar degree of agonist-induced desensitization as assessed using [(35)S]GTPgammaS binding assays. Similarly, both receptor constructs internalized to the same extent in response to agonist treatment. Furthermore, using bioluminescence resonance energy transfer assays to directly assess receptor association with arrestin3, we found no differences between the WT and GRK(-) receptors. Thus, phosphorylation is not required for arrestin-receptor association or agonist-induced desensitization or internalization. In contrast, when we examined recycling of the D(2) DARs to the cell surface, subsequent to agonist-induced endocytosis, the GRK(-) construct exhibited less recycling in comparison with the WT receptor. This impairment appears to be due to a greater propensity of the GRK(-) receptors to down-regulate once internalized. In contrast, if the receptor is highly phosphorylated, then receptor recycling is promoted. These results reveal a novel role for GRK-mediated phosphorylation in regulating the post-endocytic trafficking of a G protein-coupled receptor.

Mechanism of bis(7)-tacrine inhibition of GABA-activated current in cultured rat hippocampal neurons

Bis(7)-tacrine is a novel dimeric acetylcholinesterase inhibitor derived from tacrine that shows promise for the treatment of Alzheimer's disease. We have previously reported that bis(7)-tacrine inhibits GABA A receptors. In the present study we investigated the mechanism of bis(7)-tacrine inhibition of GABA A receptor function using whole-cell patch-clamp recording in cultured rat hippocampal neurons. Bis(7)-tacrine produced a gradual decline of GABA-activated current to a steady-state, but this was not an indication of use-dependence, as the gradually declining component could be eliminated by exposure to bis(7)-tacrine prior to GABA application. In addition, bis(7)-tacrine inhibition did not require the presence of agonist, and GABA-activated current recovered completely from inhibition by bis(7)-tacrine in the absence of agonist. The slow onset of inhibition by bis(7)-tacrine was not apparently due to an action at an intracellular site, as inclusion of 25 μM bis(7)-tacrine in the recording pipette did not alter inhibition by bis(7)-tacrine applied externally. Bis(7)-tacrine shifted the GABA concentration–response curve to the right in a parallel manner and the pA 2 value estimated from a Schild plot was 5.7. Bis(7)-tacrine increased the time constant of activation of GABA-gated ion channels without affecting the time constants of deactivation or desensitization. These results suggest that bis(7)-tacrine is a competitive GABA A receptor antagonist with slow onset and offset kinetics. The competitive inhibition of GABA receptors by bis(7)-tacrine could contribute to its ability to enhance memory.

Possible involvement of PPARγ in the regulation of basal channel opening of P2X7 receptor in cultured mouse astrocytes

Aims Recently, we demonstrated that cultured mouse astrocytes exhibited basal channel opening of P2X7 receptor (P2X7R) in the absence of any exogenous ligand, but the regulatory mechanism involved was not elucidated. Since our preliminary experiments suggested possible involvement of peroxisome proliferator-activated receptor (PPAR) γ in the regulation, we examined whether PPARγ regulated P2X7R basal channel opening in mouse astrocytes. Main methods P2X7R channel opening was assessed as to the uptake of a marker dye, YO-PRO-1 ® (YP), in the presence or absence of agonists and antagonists for PPARγ under a fluorescence microscope. Expression of PPARγ was evaluated by Western blotting and immunocytochemistry. Key findings NSAIDs such as flufenamic acid (FFA) and indomethacin, which are a cyclooxygenase inhibitor and a PPARγ agonist, showed enhancing and inhibiting effects on YP uptake at low and high concentrations, respectively, and the enhanced uptake was abolished by periodate-oxidized ATP (oxATP), a selective P2X7R antagonist. The PPARγ agonists 15-deoxy- Δ 12,14-prostaglandin J 2 and ciglitazone decreased the basal and FFA-enhanced YP uptake, while the antagonist GW9662 increased YP uptake, this effect being blocked by the agonists and also by oxATP. PPARγ was distributed in the nucleus and cytosolic/membrane fraction of cultured mouse astrocytes. Significance These findings indicate that basal channel opening of P2X7R in mouse astrocytes is at least in part regulated by PPARγ.

The contribution of Ca2+ signaling and Ca2+ sensitivity to the regulation of airway smooth muscle contraction is different in rats and mice

To determine the relative contributions of Ca(2+) signaling and Ca(2+) sensitivity to the contractility of airway smooth muscle cells (SMCs), we compared the contractile responses of mouse and rat airways with the lung slice technique. Airway contraction was measured by monitoring changes in airway lumen area with phase-contrast microscopy, whereas changes in intracellular calcium concentration ([Ca(2+)](i)) of the SMCs were recorded with laser scanning microscopy. In mice and rats, methacholine (MCh) or serotonin induced concentration-dependent airway contraction and Ca(2+) oscillations in the SMCs. However, rat airways demonstrated greater contraction compared with mice, in response to agonist-induced Ca(2+) oscillations of a similar frequency. Because this indicates that rat airway SMCs have a higher Ca(2+) sensitivity compared with mice, we examined Ca(2+) sensitivity with Ca(2+)-permeabilized airway SMCs in which the [Ca(2+)](i) was experimentally controlled. In the absence of agonists, high [Ca(2+)](i) induced a sustained contraction in rat airways but only a transient contraction in mouse airways. This sustained contraction of rat airways was relaxed by Y-23672, a Rho kinase inhibitor, but not affected by GF-109203X, a PKC inhibitor. The subsequent exposure of Ca(2+)-permeabilized airway SMCs, with high [Ca(2+)](i), to MCh elicited a further contraction of rat airways and initiated a sustained contraction of mouse airways, without changing the [Ca(2+)](i) of the SMCs. Collectively, these results indicate that airway SMCs of rats have a substantially higher innate Ca(2+) sensitivity than mice and that this strongly influences the transduction of the frequency of Ca(2+) oscillations into the contractility of airway SMCs.

Association Between Increased Platelet P-Selectin Expression and Obesity in Patients With Type 2 Diabetes

OBJECTIVETo determine whether obesity increases platelet reactivity and thrombin activity in patients with type 2 diabetes plus stable coronary artery disease.RESEARCH DESIGN AND METHODSWe assessed platelet reactivity and markers of thrombin generation and activity in 193 patients from nine clinical sites of the Bypass Angioplasty Revascularization Investigation 2 Diabetes (BARI 2D). Blood taken at the time of enrollment was used for assay of the concentration of prothrombin fragment 1.2 (PT1.2, released when prothrombin is activated) and fibrinopeptide A (FPA, released when fibrinogen is cleaved). Platelet activation was identified with the use of flow cytometry in response to 0, 0.2, and 1 μmol/l adenosine diphosphate (ADP).RESULTSConcentrations of FPA, PT1.2, and platelet activation in the absence of agonist were low. Greater BMI was associated with higher platelet reactivity in response to 1 μm ADP as assessed by surface expression of P-selectin (r = 0.29, P < 0.0001) but not reflected by the binding of fibrinogen to activated glycoprotein IIb-IIIa. BMI was not associated with concentrations of FPA or PT1.2. Platelet reactivity correlated negatively with A1C (P < 0.04), was not related to the concentration of triglycerides in blood, and did not correlate with the concentration of C-reactive peptide.CONCLUSIONSAmong patients enrolled in this substudy of BARI 2D, a greater BMI was associated with higher platelet reactivity at the time of enrollment. Our results suggest that obesity and insulin resistance that accompanies obesity may influence platelet reactivity in patients with type 2 diabetes.

Constitutive Internalization of G Protein-coupled Receptors and G Proteins via Clathrin-independent Endocytosis

Although agonist-dependent endocytosis of G protein-coupled receptors (GPCRs) as a means to modulate receptor signaling has been widely studied, the constitutive endocytosis of GPCRs has received little attention. Here we show that two prototypical class I GPCRs, the beta2 adrenergic and M3 muscarinic receptors, enter cells constitutively by clathrin-independent endocytosis and colocalize with markers of this endosomal pathway on recycling tubular endosomes, indicating that these receptors can subsequently recycle back to the plasma membrane (PM). This constitutive endocytosis of these receptors was not blocked by antagonists, indicating that receptor signaling was not required. Interestingly, the G proteins that these receptors couple to, Galpha(s) and Galpha(q), localized together with their receptors at the plasma membrane and on tubular recycling endosomes. Upon agonist stimulation, Galpha(s) and Galpha(q) remained associated with the PM and these endosomal membranes, whereas beta2 and M3 receptors now entered cells via clathrin-dependent endocytosis. Deletion of the third intracellular loop (i3 loop), which is thought to play a role in agonist-dependent endocytosis of the M3 receptor, had no effect on the constitutive internalization of the receptor. Surprisingly, with agonist, the mutated M3 receptor still internalized and accumulated in cells but through clathrin-independent and not clathrin-dependent endocytosis. These findings demonstrate that GPCRs are versatile PM proteins that can utilize different mechanisms of internalization depending upon ligand activation.

Modulation Of Nicotinic Acetylcholine Receptor Conformational States By Free Fatty Acids And Steroids

Steroids and free fatty acids (FFA) are non-competitive antagonists of the nicotinic acetylcholine receptor (AChR). Their site of action is purportedly located at the lipid-AChR interface, but their exact mechanism of action is still unknown. Here we studied the effect of structurally different free fatty acids and steroids on the conformational equilibrium of the AChR in T. californica receptor-rich membranes. We took advantage of the higher affinity of the fluorescent AChR open channel blocker, crystal violet (CrV), for the desensitized state than for the resting state. Increasing concentrations of steroids and certain cis-unsaturated free fatty acids decreased the KD of CrV in the absence of agonist. The position of the double bond at the hydrocarbon chain of cis-monounsaturated fatty acids appears to be critical for their effect on the AChR resting conformation state. All cis-unsaturated fatty acids tested, but not trans-unsaturated or saturated fatty acids, caused an increase of the KD value in the presence of agonist. This latter effect was also observed with membrane treatments that caused opposite effects on membrane polarity (phospholipase A2 treatment or temperature increase, which decreased or increased the membrane polarity, respectively). Quenching by spin-labeled fatty acids of pyrene-labeled AChR reconstituted into model membranes, with the label located at the γM4 transmembrane segment, disclosed the occurrence of conformational changes induced by steroids and cis-FFA. These results suggest that the direct contact between exogenous lipids and the AChR transmembrane segments removes the AChR from its resting state and that membrane polarity modulates the AChR activation equilibrium by an independent mechanism.

α7 and non‐α7 nicotinic acetylcholine receptors modulate dopamine release in vitro and in vivo in the rat prefrontal cortex

Nicotine enhances attentional and working memory aspects of executive function in the prefrontal cortex (PFC) where dopamine plays a major role. Here, we have determined the nicotinic acetylcholine receptor (nAChR) subtypes that can modulate dopamine release in rat PFC using subtype-selective drugs. Nicotine and 5-Iodo-A-85380 (beta2* selective) elicited [(3)H]dopamine release from both PFC and striatal prisms in vitro and dopamine overflow from medial PFC in vivo. Blockade by dihydro-beta-erythroidine supports the participation of beta2* nAChRs. However, insensitivity of nicotine-evoked [(3)H]dopamine release to alpha-conotoxin-MII in PFC prisms suggests no involvement of alpha6beta2* nAChRs, in contrast to the striatum, and this distinction is supported by immunoprecipitation of nAChR subunits from these tissues. The alpha7 nAChR-selective agonists choline and Compound A also promoted dopamine release from PFC in vitro and in vivo, and their effects were enhanced by the alpha7 nAChR-selective allosteric potentiator PNU-120596 and blocked by specific antagonists. DNQX and MK801 inhibited [(3)H]dopamine release evoked by choline and PNU-120596, suggesting crosstalk between alpha7 nAChRs, glutamate and dopamine in the PFC. In vivo, systemic (but not local) administration of PNU-120596, in the absence of agonist, facilitated dopamine overflow in the medial PFC, consistent with the activation of extracortical alpha7 nAChRs by endogenous acetylcholine or choline. These data establish that both beta2* and alpha7 nAChRs can modulate dopamine release in the PFC in vitro and in vivo. Through their distinct actions on dopamine release, these nAChR subtypes could contribute to executive function, making them specific therapeutic targets for conditions such as schizophrenia and attention deficit hyperactivity disorder.

Detection and Trapping of Elusive Priming Intermediates Towards Open Nicotinic Receptor Channel

Acetylcholine receptors (AChRs) mediate rapid synaptic transmission by transducing a chemical signal into an electrical impulse. Transduction comprises binding of agonist followed by opening of the AChR ion channel, and in the classical view both processes depend on the agonist. However previous studies suggest the ultimate channel opening step is agonist-independent1,2, and is preceded by a priming step facilitated by the agonist3. Here, by studying mutant AChRs, we detect two such priming steps; the first generates a closed state that elicits brief openings, and the second generates a closed state that elicits long-lived openings. Long-lived openings and the associated priming step are detected in the absence of agonist and in its presence, and show identical kinetics under each condition. By covalently locking the agonist binding sites in the bound conformation, we show that each site initiates a priming step. Thus a change in binding site conformation primes the AChR for channel opening in a process that determines the maximum response to agonist and functional consequences of disease-causing mutations.

The Unliganded Gating Mechanism Of Nicotinic Acetylcholine Receptors

The nicotinic acetylcholine receptor (AChR) switches between C (low agonist affinity and low conductance) and O (high agonist affinity and high conductance) conformations (‘gating’). The probability of channel opening is very low in the absence of agonist, but when agonists are present at the two transmitter-binding-sites opening increases rapidly (∼20μs), transiently to a high probability (∼0.95). We observe that ‘gain-of-function’ mutants that increase the diliganded gating equilibrium constant (without affecting agonist binding to C) also increase the frequency of spontaneous openings. Unliganded openings occur in clusters in AChRs having several of such mutations. We analyzed the intra-cluster interval durations to estimate that the unliganded gating equilibrium constant is ∼1.15 × 10-7 (mouse, α2βδε, -100 mV). The agonist affinity ratios (C vs. O) for acetylcholine, carbamylcholine, tetramethylammonium and choline are ∼15,600, ∼6700, ∼6700 and ∼600. The monoliganded (with ACh) gating equilibrium constant is ∼1.7 × 10-3. Acetylcholine provides only ∼0.9 kBT more binding energy per site than tetramethylammonium, but ∼3.1 kBT more than choline. Mutations of binding site residue αW149 increase unliganded gating, and the mutation αW149F reduces the ACh affinity of C only by 13-fold, but of O by 190-fold. Rate-equilibrium free energy relationships for different regions of the protein show similar slopes (Φ-values) for un- vs. diliganded gating. The mechanisms of the gating conformational change and of desensitization are similar with and without ligands at the transmitter binding sites.