The voltage-gated K+ channel Kv1.3 modulates platelet motility and α2ß1 integrin-dependent adhesion to collagen.

Kv1.3 is a voltage-gated K+-selective channel with roles in immunity, insulin-sensitivity, neuronal excitability and olfaction. Despite being one of the largest ionic conductances of the platelet surface membrane, its contribution to platelet function is poorly understood. Here we show that Kv1.3-deficient platelets display enhanced ADP-evoked platelet aggregation and secretion, and an increased surface expression of platelet integrin αIIb. In contrast, platelet adhesion and thrombus formation in vitro under arterial shear conditions on surfaces coated with collagen were reduced for samples from Kv1.3-/- compared to wild type mice. Use of collagen-mimetic peptides revealed a specific defect in the engagement with α2ß1. Kv1.3-/- platelets developed significantly fewer, and shorter, filopodia than wild type platelets during adhesion to collagen fibrils. Kv1.3-/- mice displayed no significant difference in thrombus formation within cremaster muscle arterioles using a laser-induced injury model, thus other pro-thrombotic pathways compensate in vivo for the adhesion defect observed in vitro. This may include the increased platelet counts of Kv1.3-/- mice, due in part to a prolonged lifespan. The ability of Kv1.3 to modulate integrin-dependent platelet adhesion has important implications for understanding its contribution to normal physiological platelet function in addition to its reported roles in auto-immune diseases and thromboinflammatory models of stroke.

Why do platelets express K+ channels?

Potassium ions have widespread roles in cellular homeostasis and activation as a consequence of their large outward concentration gradient across the surface membrane and ability to rapidly move through K+-selective ion channels. In platelets, the predominant K+ channels include the voltage-gated K+ channel Kv1.3, and the intermediate conductance Ca2+-activated K+ channel KCa3.1, also known as the Gardos channel. Inwardly rectifying potassium GIRK channels and KCa1.1 large conductance Ca2+-activated K+ channels have also been reported in the platelet, although they remain to be demonstrated using electrophysiological techniques. Whole-cell patch clamp and fluorescent indicator measurements in the platelet or their precursor cell reveal that Kv1.3 sets the resting membrane potential and KCa3.1 can further hyperpolarize the cell during activation, thereby controlling Ca2+ influx. Kv1.3-/- mice exhibit an increased platelet count, which may result from an increased splenic megakaryocyte development and longer platelet lifespan. This review discusses the evidence in the literature that Kv1.3, KCa3.1. GIRK and KCa1.1 channels contribute to a number of platelet functional responses, particularly collagen-evoked adhesion, procoagulant activity and GPCR function. Putative roles for other K+ channels and known accessory proteins which to date have only been detected in transcriptomic or proteomic studies, are also discussed.

Ca2+ waves coordinate purinergic receptor-evoked integrin activation and polarization.

Cells sense extracellular nucleotides through the P2Y class of purinergic G protein-coupled receptors (GPCRs), which stimulate integrin activation through signaling events, including intracellular Ca2+ mobilization. We investigated the relationship between P2Y-stimulated repetitive Ca2+ waves and fibrinogen binding to the platelet integrin αIIbß3 (GPIIb/IIIa) through confocal fluorescence imaging of primary rat megakaryocytes. Costimulation of the receptors P2Y1 and P2Y12 generated a series of Ca2+ transients that each induced a rapid, discrete increase in fibrinogen binding. The peak and net increase of individual fibrinogen binding events correlated with the Ca2+ transient amplitude and frequency, respectively. Using BAPTA loading and selective receptor antagonists, we found that Ca2+ mobilization downstream of P2Y1 was essential for ADP-evoked fibrinogen binding, whereas P2Y12 and the kinase PI3K were also required for αIIbß3 activation and enhanced the number of Ca2+ transients. ADP-evoked fibrinogen binding was initially uniform over the cell periphery but subsequently redistributed with a polarity that correlated with the direction of the Ca2+ waves. Polarization of αIIbß3 may be mediated by the actin cytoskeleton, because surface-bound fibrinogen is highly immobile, and its motility was enhanced by cytoskeletal disruption. In conclusion, spatial and temporal patterns of Ca2+ increase enable fine control of αIIbß3 activation after cellular stimulation. P2Y1-stimulated Ca2+ transients coupled to αIIbß3 activation only in the context of P2Y12 coactivation, thereby providing an additional temporal mechanism of synergy between these Gq- and Gi-coupled GPCRs.

Development of a P2X1-eYFP receptor knock-in mouse to track receptors in real time.

A P2X1-eYFP knock-in mouse was generated to study receptor expression and mobility in smooth muscle and blood cells. eYFP was added to the C-terminus of the P2X1R and replaced the native P2X1R. Fluorescence corresponding to P2X1-eYFPR was detected in urinary bladder smooth muscle, platelets and megakaryocytes. ATP-evoked currents from wild type and P2X1-eYFP isolated urinary bladder smooth muscle cells had the same peak current amplitude and time-course showing that the eYFP addition had no obvious effect on properties. Fluorescence recovery after photobleaching (FRAP) in bladder smooth muscle cells demonstrated that surface P2X1Rs are mobile and their movement is reduced following cholesterol depletion. Compared to the platelet and megakaryocyte, P2X1-eYFP fluorescence was negligible in red blood cells and the majority of smaller marrow cells. The spatial pattern of P2X1-eYFP fluorescence in the megakaryocyte along with FRAP assessment of mobility suggested that P2X1Rs are expressed extensively throughout the membrane invagination system of this cell type. The current study highlights that the spatiotemporal properties of P2X1R expression can be monitored in real time in smooth muscle cells and megakaryocytes/platelets using the eYFP knock-in mouse model.

A major interspecies difference in the ionic selectivity of megakaryocyte Ca2+-activated channels sensitive to the TMEM16F inhibitor CaCCinh-A01.

TMEM16F is a surface membrane protein critical for platelet procoagulant activity, which exhibits both phospholipid scramblase and ion channel activities following sustained elevation of cytosolic Ca2+. The extent to which the ionic permeability of TMEM16F is important for platelet scramblase responses remains controversial. To date, only one study has reported the electrophysiological properties of TMEM16F in cells of platelet/megakaryocyte lineage, which observed cation-selectivity within excised patch recordings from murine marrow-derived megakaryocytes. This contrasts with reports using whole-cell recordings that describe this channel as displaying either selectivity for anions or being relatively non-selective amongst the major physiological monovalent ions. We have studied TMEM16F expression and channel activity in primary rat and mouse megakaryocytes and the human erythroleukemic (HEL) cell line that exhibits megakaryocytic surface markers. Immunocytochemical analysis was consistent with surface TMEM16F expression in cells from all three species. Whole-cell recordings in the absence of K+-selective currents revealed an outwardly rectifying conductance activated by a high intracellular Ca2+ concentration in all three species. These currents appeared after 5-6 minutes and were blocked by CaCCinh-A01, properties typical of TMEM16F. Ion substitution experiments showed that the underlying conductance was predominantly Cl--permeable in rat megakaryocytes and HEL cells, yet non-selective between monovalent anions and cations in mouse megakaryocytes. In conclusion, the present study further highlights the difference in ionic selectivity of TMEM16F in platelet lineage cells of the mouse compared to other mammalian species. This provides additional support for the ionic "leak" hypothesis that the scramblase activity of TMEM16F does not rely upon its ability to conduct ions of a specific type.

P2X1 Receptors Amplify FcγRIIa-Induced Ca2+ Increases and Functional Responses in Human Platelets.

Platelets express key receptors of the innate immune system such as FcγRIIa and Toll-like receptors (TLR). P2X1 cation channels amplify the platelet responses to several major platelet stimuli, particularly glycoprotein (GP)VI and TLR2/1, whereas their contribution to Src tyrosine kinase-dependent FcγRIIa receptors remains unknown. We investigated the role of P2X1 receptors during activation of FcγRIIa in human platelets, following stimulation by cross-linking of an anti-FcγRIIa monoclonal antibody (mAb) IV.3, or bacterial stimulation with Streptococcus sanguinis. Activation was assessed in washed platelet suspensions via measurement of intracellular Ca2+ ([Ca2+]i) increases, ATP release and aggregation. P2X1 activity was abolished by pre-addition of α,ß-meATP, exclusion of apyrase or the antagonist NF449. FcγRIIa activation evoked a robust increase in [Ca2+]i (441 ± 33 nM at 30 µg/mL mAb), which was reduced to a similar extent (to 66-70% of control) by NF449, pre-exposure to α,ß-meATP or apyrase omission, demonstrating a significant P2X1 receptor contribution. FcγRIIa activation-dependent P2X1 responses were partially resistant to nitric oxide (NO), but abrogated by 500 nM prostacyclin (PGI2). Aggregation responses to bacteria and FcγRIIa activation were also inhibited by P2X1 receptor desensitization (to 66 and 42% of control, respectively). However, FcγRIIa-mediated tyrosine phosphorylation and ATP release were not significantly altered by the loss of P2X1 activity. In conclusion, we show that P2X1 receptors enhance platelet FcγRIIa receptor-evoked aggregation through an increase in [Ca2+]i downstream of the initial tyrosine phosphorylation events and early dense granule release. This represents a further route whereby ATP-gated cation channels can contribute to platelet-dependent immune responses in vivo.

Differential integrin activity mediated by platelet collagen receptor engagement under flow conditions.

The platelet receptors glycoprotein (Gp)VI, integrin α2ß1 and GpIb/V/IX mediate platelet adhesion and activation during thrombogenesis. Increases of intracellular Ca2+ ([Ca2+]i) are key signals during platelet activation; however, their relative importance in coupling different collagen receptors to functional responses under shear conditions remains unclear. To study shear-dependent, receptor-specific platelet responses, we used collagen or combinations of receptor-specific collagen-mimetic peptides as substrates for platelet adhesion and activation in whole human blood under arterial flow conditions and compared real-time and endpoint parameters of thrombus formation alongside [Ca2+]i measurements using confocal imaging. All three collagen receptors coupled to [Ca2+]i signals, but these varied in amplitude and temporal pattern alongside variable integrin activation. GpVI engagement produced large, sustained [Ca2+]i signals leading to real-time increases in integrins α2ß1- and αIIbß3-mediated platelet adhesion. αIIbß3-dependent platelet aggregation was dependent on P2Y12 signalling. Co-engagement of α2ß1 and GpIb/V/IX generated transient [Ca2+]i spikes and low amplitude [Ca2+]i responses that potentiated GpVI-dependent [Ca2+]i signalling. Therefore α2ß1, GpIb/V/IX and GpVI synergise to generate [Ca2+]i signals that regulate platelet behaviour and thrombus formation. Antagonism of secondary signalling pathways reveals distinct, separate roles for αIIbß3 in stable platelet adhesion and aggregation.

Evidence for shear-mediated Ca2+ entry through mechanosensitive cation channels in human platelets and a megakaryocytic cell line.

The role of mechanosensitive (MS) Ca2+-permeable ion channels in platelets is unclear, despite the importance of shear stress in platelet function and life-threatening thrombus formation. We therefore sought to investigate the expression and functional relevance of MS channels in human platelets. The effect of shear stress on Ca2+ entry in human platelets and Meg-01 megakaryocytic cells loaded with Fluo-3 was examined by confocal microscopy. Cells were attached to glass coverslips within flow chambers that allowed applications of physiological and pathological shear stress. Arterial shear (1002.6 s-1) induced a sustained increase in [Ca2+] i in Meg-01 cells and enhanced the frequency of repetitive Ca2+ transients by 80% in platelets. These Ca2+ increases were abrogated by the MS channel inhibitor Grammostola spatulata mechanotoxin 4 (GsMTx-4) or by chelation of extracellular Ca2+ Thrombus formation was studied on collagen-coated surfaces using DiOC6-stained platelets. In addition, [Ca2+] i and functional responses of washed platelet suspensions were studied with Fura-2 and light transmission aggregometry, respectively. Thrombus size was reduced 50% by GsMTx-4, independently of P2X1 receptors. In contrast, GsMTx-4 had no effect on collagen-induced aggregation or on Ca2+ influx via TRPC6 or Orai1 channels and caused only a minor inhibition of P2X1-dependent Ca2+ entry. The Piezo1 agonist, Yoda1, potentiated shear-dependent platelet Ca2+ transients by 170%. Piezo1 mRNA transcripts and protein were detected with quantitative RT-PCR and Western blotting, respectively, in both platelets and Meg-01 cells. We conclude that platelets and Meg-01 cells express the MS cation channel Piezo1, which may contribute to Ca2+ entry and thrombus formation under arterial shear.

Transcriptomic analysis of the ion channelome of human platelets and megakaryocytic cell lines.

Ion channels have crucial roles in all cell types and represent important therapeutic targets. Approximately 20 ion channels have been reported in human platelets; however, no systematic study has been undertaken to define the platelet channelome. These membrane proteins need only be expressed at low copy number to influence function and may not be detected using proteomic or transcriptomic microarray approaches. In our recent work, quantitative real-time PCR (qPCR) provided key evidence that Kv1.3 is responsible for the voltage-dependent K+ conductance of platelets and megakaryocytes. The present study has expanded this approach to assess relative expression of 402 ion channels and channel regulatory genes in human platelets and three megakaryoblastic/erythroleukaemic cell lines. mRNA levels in platelets are low compared to other blood cells, therefore an improved method of isolating platelets was developed. This used a cocktail of inhibitors to prevent formation of leukocyte-platelet aggregates, and a combination of positive and negative immunomagnetic cell separation, followed by rapid extraction of mRNA. Expression of 34 channel-related transcripts was quantified in platelets, including 24 with unknown roles in platelet function, but that were detected at levels comparable to ion channels with established roles in haemostasis or thrombosis. Trace expression of a further 50 ion channel genes was also detected. More extensive channelomes were detected in MEG-01, CHRF-288-11 and HEL cells (195, 185 and 197 transcripts, respectively), but lacked several channels observed in the platelet. These "channelome" datasets provide an important resource for further studies of ion channel function in the platelet and megakaryocyte.

Calcium Signalling through Ligand-Gated Ion Channels such as P2X1 Receptors in the Platelet and other Non-Excitable Cells.

Ligand-gated ion channels on the cell surface are directly activated by the binding of an agonist to their extracellular domain and often referred to as ionotropic receptors. P2X receptors are ligand-gated non-selective cation channels with significant permeability to Ca(2+) whose principal physiological agonist is ATP. This chapter focuses on the mechanisms by which P2X1 receptors, a ubiquitously expressed member of the family of ATP-gated channels, can contribute to cellular responses in non-excitable cells. Much of the detailed information on the contribution of P2X1 to Ca(2+) signalling and downstream functional events has been derived from the platelet. The underlying primary P2X1-generated signalling event in non-excitable cells is principally due to Ca(2+) influx, although Na(+) entry will also occur along with membrane depolarization. P2X1 receptor stimulation can lead to additional Ca(2+) mobilization via a range of routes such as amplification of G-protein-coupled receptor-dependent Ca(2+) responses. This chapter also considers the mechanism by which cells generate extracellular ATP for autocrine or paracrine activation of P2X1 receptors. For example cytosolic ATP efflux can result from opening of pannexin anion-permeable channels or following damage to the cell membrane. Alternatively, ATP stored in specialised secretory vesicles can undergo quantal release via the process of exocytosis. Examples of physiological or pathophysiological roles of P2X1-dependent signalling in non-excitable cells are also discussed, such as thrombosis and immune responses.

Detachment of surface membrane invagination systems by cationic amphiphilic drugs.

Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulin inhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs also caused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of human platelets. Anionic amphiphiles, U73343 (a less electrophilic U73122 analogue) and a range of kinase inhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cell membrane where they bind to anionic lipids, especially PI(4,5)P2. We therefore propose that surface detachment of membrane invaginations results from an ability of CADs to interfere with PI(4,5)P2 interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a large class of pharmaceutical compounds.

Regulation of Pannexin-1 channel activity.

Pannexin-1 (Panx1) forms anion-selective channels with a permeability up to 1 kDa and represents a pathway for the release of cytosolic ATP. Several structurally similar connexin (Cx) proteins have been identified in platelets and shown to play roles in haemostasis and thrombosis. More recently, functional Panx1 channels have been demonstrated on the surface of human platelets [Taylor et al. (2014) J. Thromb. Haemost. 12, 987-998]. Since their identification in the year 2000, several mechanisms have been reported to activate Panx1 channels, including mechanical stimulation, oxygen-glucose deprivation, a rise of [Ca2+]i, caspase cleavage and phosphorylation. Within this review, the regulation of Panx1 channels is discussed, with a focus on how they may contribute to platelet function.

Ca2+ influx through P2X1 receptors amplifies P2Y1 receptor-evoked Ca2+ signaling and ADP-evoked platelet aggregation.

Many cells express both P2X cation channels and P2Y G-protein-coupled receptors that are costimulated by nucleotides released during physiologic or pathophysiologic responses. For example, during hemostasis and thrombosis, ATP-gated P2X1 channels and ADP-stimulated P2Y1 and P2Y12 G-protein coupled receptors play important roles in platelet activation. It has previously been reported that P2X1 receptors amplify P2Y1-evoked Ca(2+) responses in platelets, but the underlying mechanism and influence on function is unknown. In human platelets, we show that maximally activated P2X1 receptors failed to stimulate significant aggregation but could amplify the aggregation response to a submaximal concentration of ADP. Costimulation of P2X1 and P2Y1 receptors generated a superadditive Ca(2+) increase in both human platelets and human embryonic kidney 293 (HEK293) cells via a mechanism dependent on Ca(2+) influx rather than Na(+) influx or membrane depolarization. The potentiation, due to an enhanced P2Y1 response, was observed if ADP was added up to 60 seconds after P2X1 activation. P2X1 receptors also enhanced Ca(2+) responses when costimulated with type 1 protease activated and M1 muscarinic acetylcholine receptors. The P2X1-dependent amplification of Gq-coupled [Ca(2+)]i increase was mimicked by ionomycin and was not affected by inhibition of protein kinase C, Rho-kinase, or extracellular signal-regulated protein kinase 1/2, which suggests that it results from potentiation of inositol 1,4,5-trisphosphate receptors and/or phospholipase C. We conclude that Ca(2+) influx through P2X1 receptors amplifies Ca(2+) signaling through P2Y1 and other Gq-coupled receptors. This represents a general form of co-incidence detection of ATP and coreleased agonists, such as ADP at sites of vascular injury or synaptic transmitters acting at metabotropic Gq-coupled receptors.

Connexin40 regulates platelet function.

The presence of multiple connexins was recently demonstrated in platelets, with notable expression of Cx37. Studies with Cx37-deficient mice and connexin inhibitors established roles for hemichannels and gap junctions in platelet function. It was uncertain, however, whether Cx37 functions alone or in collaboration with other family members through heteromeric interactions in regulation of platelet function. Here we report the presence and functions of an additional platelet connexin, Cx40. Inhibition of Cx40 in human platelets or its deletion in mice reduces platelet aggregation, fibrinogen binding, granule secretion and clot retraction. The effects of the Cx37 inhibitor (37,43)Gap27 on Cx40(-/-) mouse platelets and of the Cx40 inhibitor (40)Gap27 on Cx37(-/-) mouse platelets revealed that each connexin is able to function independently. Inhibition or deletion of Cx40 reduces haemostatic responses in mice, indicating the physiological importance of this protein in platelets. We conclude that multiple connexins are involved in regulating platelet function, thereby contributing to haemostasis and thrombosis.

A role for platelet TRPC channels in the Ca2+ response that induces procoagulant activity.

After vascular injury, platelets are rapidly activated by collagen and other agonists, causing them to adhere and aggregate to prevent blood loss. In addition, phosphatidylserine (PS) exposure on the platelet surface accelerates thrombin formation by the coagulation pathway. Thrombin is a potent platelet agonist and converts fibrinogen to fibrin, thereby stabilizing the platelet plug. PS exposure during hemostasis and thrombosis results from a sustained cytosolic Ca(2+) increase; however, the underlying Ca(2+) mobilization pathways have remained unclear. Store-operated Orai1 channels provide substantial, prolonged Ca(2+) influx after inositol trisphosphate-dependent release, and anoctamin 6 (TMEM16F) may operate as a Ca(2+)-activated, Ca(2+)-permeable channel in addition to its scramblase activity that exteriorizes PS. A new study shows that Na(+) entry, resulting from coactivation of the transient receptor potential (TRP) nonselective cation channels TRPC3 and TRPC6, followed by reverse-mode operation of Na(+)/Ca(2+) exchangers, is an important mechanism for the increase in cytosolic Ca(2+) that triggers PS exposure, particularly during combined thrombin and collagen stimulation.

Heat shock protein 90 inhibitors reduce trafficking of ATP-gated P2X1 receptors and human platelet responsiveness.

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70-85% by the selective HSP90 inhibitor geldanamycin (2 µM, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40-45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.

Gap junctions and connexin hemichannels underpin hemostasis and thrombosis.

BACKGROUND: Connexins are a widespread family of membrane proteins that assemble into hexameric hemichannels, also known as connexons. Connexons regulate membrane permeability in individual cells or couple between adjacent cells to form gap junctions and thereby provide a pathway for regulated intercellular communication. We have examined the role of connexins in platelets, blood cells that circulate in isolation but on tissue injury adhere to each other and the vessel wall to prevent blood loss and to facilitate wound repair. METHODS AND RESULTS: We report the presence of connexins in platelets, notably connexin37, and that the formation of gap junctions within platelet thrombi is required for the control of clot retraction. Inhibition of connexin function modulated a range of platelet functional responses before platelet-platelet contact and reduced laser-induced thrombosis in vivo in mice. Deletion of the Cx37 gene (Gja4) in transgenic mice reduced platelet aggregation, fibrinogen binding, granule secretion, and clot retraction, indicating an important role for connexin37 hemichannels and gap junctions in platelet thrombus function. CONCLUSIONS: Together, these data demonstrate that platelet gap junctions and hemichannels underpin the control of hemostasis and thrombosis and represent potential therapeutic targets.

Platelet Ca(2+) responses coupled to glycoprotein VI and Toll-like receptors persist in the presence of endothelial-derived inhibitors: roles for secondary activation of P2X1 receptors and release from intracellular Ca(2+) stores.

Inhibition of Ca(2+) mobilization by cyclic nucleotides is central to the mechanism whereby endothelial-derived prostacyclin and nitric oxide limit platelet activation in the intact circulation. However, we show that ∼ 50% of the Ca(2+) response after stimulation of glycoprotein VI (GPVI) by collagen, or of Toll-like 2/1 receptors by Pam(3)Cys-Ser-(Lys)(4) (Pam(3)CSK(4)), is resistant to prostacyclin. At low agonist concentrations, the prostacyclin-resistant Ca(2+) response was predominantly because of P2X1 receptors activated by ATP release via a phospholipase-C-coupled secretory pathway requiring both protein kinase C and cytosolic Ca(2+) elevation. At higher agonist concentrations, an additional pathway was observed because of intracellular Ca(2+) release that also depended on activation of phospholipase C and, for TLR 2/1, PI3-kinase. Secondary activation of P2X1-dependent Ca(2+) influx also persisted in the presence of nitric oxide, delivered from spermine NONOate, or increased ectonucleotidase levels (apyrase). Surprisingly, apyrase was more effective than prostacyclin and NO at limiting secondary P2X1 activation. Dilution of platelets reduced the average extracellular ATP level without affecting the percentage contribution of P2X1 receptors to collagen-evoked Ca(2+) responses, indicating a highly efficient activation mechanism by local ATP. In conclusion, platelets possess inhibitor-resistant Ca(2+) mobilization pathways, including P2X1 receptors, that may be particularly important during early thrombotic or immune-dependent platelet activation.

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists.

BACKGROUND AND OBJECTIVE: Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M(1) mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET). METHODS: Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M(1) mAChR, respectively. The optimized FRET receptor construct (M(1)-cam5) was expressed stably in HEK293 cells. RESULTS: The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα(q/11) proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M(1) FRET (F(EYFP)/F(ECFP)) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine. CONCLUSION: The M(1) FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M(1) mAChR activation.