Rate Of Desensitization Research Articles

Desformylflustrabromine Potentiates High-Sensitivity α4β2 Receptors by Increasing Channel Opening Rate

Desformylflustrabromine (dFBr) is a positive allosteric modulator of α4β2 neuronal nicotinic receptors (nAChRs). Investigations of mechanisms of nAChR modulators may facilitate the development of new therapeutic drugs that offset or correct errors in signaling associated with Alzheimer's disease, nicotine addiction, and other disorders of cholinergic signaling. We made steady-state, cell-attached patch-clamp recordings of 20 pS, high-sensitivity (HS) human α4β2 receptors stably expressed in HEK-293 cells. We developed 6-state gating models from data acquired with 1 μM ACh with and without 1 μM dFBr. Models included two sequential ligand-binding reactions leading to a desensitized state (C1↔C2↔C3↔D), and mono- and diliganded openings (O1 and O2) from C2 and C3, respectively. Rate constants governing transitions between connected states were obtained by fitting single-channel data to the model (constraints: C1→C2 = 2⋅C2→C3 and C3→C2=2⋅C2→C1) using a maximum likelihood method implemented in QUB. dFBr increased binding and unbinding rates for ACh (2.6- and 2-fold, respectively), the opening rate (C3→O2) for diliganded receptors (4.8-fold), and the (microscopic) desensitization rate (5.3-fold). Simulations of macroscopic responses to concentration-jumps (range: 0.1 μM - 100 μM) revealed that the enhanced rate into O2 yielded approximately 2 to 3-fold potentiation of peak currents (maximal Popen in 100 μM ACh = 0.29) despite the fact that potentiation was blunted by the enhanced rate of desensitization. Moreover, at ACh concentrations above 10 μM, our data are consistent with the interpretation that potentiation of HS receptor responses to dFBr arises not from modulation of ligand binding rates, but from an increase in the rate of channel opening, corresponding to a decrease in the latency to first opening and a reduction in the time to peak current (ACh 23 ms; dFBr 8 ms) of simulated macroscopic responses.

Band-pass processing in a GPCR signaling pathway selects for NFAT transcription factor activation.

Many biological processes are rhythmic and proper timing is increasingly appreciated as being critical for development and maintenance of physiological functions. To understand how temporal modulation of an input signal influences downstream responses, we employ microfluidic pulsatile stimulation of a G-protein coupled receptor, the muscarinic M3 receptor, in single cells with simultaneous real-time imaging of both intracellular calcium and NFAT nuclear localization. Interestingly, we find that reduced stimulation with pulses of ligand can give more efficient transcription factor activation, if stimuli are timed appropriately. Our experiments and computational analyses show that M3 receptor-induced calcium oscillations form a low pass filter while calcium-induced NFAT translocation forms a high pass filter. The combination acts as a band-pass filter optimized for intermediate frequencies of stimulation. We demonstrate that receptor desensitization and NFAT translocation rates determine critical features of the band-pass filter and that the band-pass may be shifted for different receptors or NFAT dynamics. As an example, we show that the two NFAT isoforms (NFAT4 and NFAT1) have shifted band-pass windows for the same receptor. While we focus specifically on the M3 muscarinic receptor and NFAT translocation, band-pass processing is expected to be a general theme that applies to multiple signaling pathways.

Characteristics of the Desensitization of Glycine-Mediated Ion Currents in Frog Spinal Cord Neurons

Whole-cell patch clamp studies of isolated spinal cord neurons from the frog Rana temporaria in voltage-clamped conditions addressed the kinetics of ion channel desensitization evoked by glycine application. The ion current desensitization phase (decay from 90% to 10%), induced by periodic applications of glycine (1 mM), approximated an exponential function. Approximation results identified three types of glycine-mediated response. The decay constant of type 1 responses in the first minute of the experiment was τ1 = 1693 ± 135 msec (n = 8) and decreased from application to application during further recording. Type 2 responses had a decay constant τ2 = 364 ± 42 msec (n = 9) at the beginning of recording and showed virtually no change with time. Type 3 responses had a biexponential decay consisting of a rapid (τ2) component and a slow (τ1) component, acceleration of the desensitization phase of this response from application to application occurring as a result of a decrease in the larger decay constant τ1. These data suggest that the frog spinal cord membrane has at least two varieties of glycine receptor with different rates of desensitization and different dynamics of changes in properties during patch-clamp recording. Different neurons can evidently have these receptor varieties simultaneously or separately.

Enhanced desensitization followed by unusual resensitization in GABAA receptors in phospholipase C-related catalytically inactive protein-1/2 double-knockout mice.

Phospholipase C-related catalytically inactive proteins (PRIP-1/2) are previously reported to be involved in the membrane trafficking of GABAA receptor (GABAAR) and the regulation of intracellular Ca(2+) stores. GABAAR-mediated currents can be regulated by the intracellular Ca(2+). However, in PRIP-1/2 double-knockout (PRIP-DKO) mice, it remains unclear whether the kinetic properties of GABAARs are modulated by the altered regulation of intracellular Ca(2+) stores. Here, we investigated whether GABAAR currents (IGABA) evoked by GABA puff in layer 3 (L3) pyramidal cells (PCs) of the barrel cortex are altered in PRIP-DKO mice. The deletion of PRIP-1/2 enhanced the desensitization of IGABA but induced a hump-like tail current (tail-I) at the GABA puff offset. IGABA and the hump-like tail-I were suppressed by GABAAR antagonists. The enhanced desensitization of IGABA and the hump-like tail-I in PRIP-DKO PCs were mediated by increases in the intracellular Ca(2+) concentration and were largely abolished by a calcineurin inhibitor and ruthenium red. Calcium imaging revealed that Ca(2+)-induced Ca(2+) release (CICR) and subsequent store-operated Ca(2+) entry (SOCE) are more potent in PRIP-DKO PCs than in wild-type PCs. A mathematical model revealed that a slowdown of GABA-unbinding rate and an acceleration of fast desensitization rate by enhancing its GABA concentration dependency are involved in the generation of hump-like tail-Is. These results suggest that in L3 PCs of the barrel cortex in PRIP-DKO mice, the increased calcineurin activity due to the potentiated CICR and SOCE enhances the desensitization of GABAARs and slows the GABA-unbinding rate, resulting in their unusual resensitization following removal of GABA.

Loose Packing of the Extracellular Domain Core of Pentameric Ligand-Gated Ion Channels is Important for Fast Activation and Desensitization

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission. pLGIC activation is initiated by agonist binding to the orthosteric site in the extracellular domain (ECD), which promotes rapid opening of a membrane-embedded ion-conducting pore. Prolonged agonist exposure leads to pLGIC desensitization, which results in channel closure. Eukaryotic pLGICs open in the sub-millisecond to millisecond timescale, and transition into desensitized closed states in tens of milliseconds. To date, we have limited knowledge of the structural elements that underlie the rapid kinetics of activation and desensitization in pLGICs. We previously showed that eukaryotic pLGICs are characterized by loose packing of the ECD hydrophobic core, due to cavities created at two conserved sites by buried side-chains of either polar residues (threonine and serine in nicotinic receptors, nAChRs) or alanines (in GABA-A receptors), and that increasing the packing of the ECD core at these two sites slowed agonist-induced current activation and desensitization rates (Dellisanti et al., Nat Neurosci 2007; Dellisanti and Czajkowski, J Biol Chem 2011). Interestingly, prokaryotic pLGICs, like proton-activated GLIC, activate and desensitize slowly and have a tightly packed ECD core. Here, we generated loose packing mutants of GLIC by mutating V38 and V109 (aligned to nAChR-α1 T52 and S126) to either alanines or threonine and serine, respectively. We expressed the GLIC mutants in Xenopus oocytes and monitored the effects of these mutations on proton-elicited currents using two-electrode voltage clamp. The loose packing GLIC mutants had 2- to 3-fold faster current activation and desensitization rates compared to wild-type GLIC. The effects were larger when both valines (V38AV109A and V38TV109S mutants) were mutated. Our data suggest loose packing of the ECD core is directly implicated in fast activation and desensitization of eukaryotic pLGICs.

Inhibition of P2X4 function by P2Y6 UDP receptors in microglia

ATP-gated P2X4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. P2Y6 metabotropic Gq -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by UDP. It has been reported recently that expression of both P2X4 and P2Y6 is upregulated in activated microglia following nerve injury. We show here, in resting as well as LPS-activated primary microglia, that P2Y6 decreases P2X4-mediated calcium entry and inhibits the dilation of P2X4 channels into a large-conductance pore measured with a YO-PRO-1 uptake assay. Furthermore, P2Y6 activation modulates the ATP-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype. Reconstituting the P2X4-P2Y6 interaction in recombinant systems shows that P2Y6 activation decreases P2X4 current amplitude, activation and desensitization rates, and reduces P2X4 channel permeability to the large cation NMDG(+) . Phospholipase C-mediated hydrolysis of the phosphoinositide PI(4,5)P2 , a necessary cofactor for P2X4 channel function, underlies this inhibitory crosstalk. As extracellular levels of both ATP and UDP are increased in the spinal cord following nerve injury, the control of P2X4 activity by P2Y6 might play a critical role in regulating neuropathic pain-inducing microglial responses.

Real‐time characterization of cannabinoid receptor 1 (CB1) allosteric modulators reveals novel mechanism of action

The cannabinoid receptor type 1 (CB1 ) has an allosteric binding site. The drugs ORG27569 {5-chloro-3-ethyl-N-[2-[4-(1-piperidinyl)phenyl]ethyl]-1H-indole-2-carboxamide} and PSNCBAM-1 {1-(4-chlorophenyl)-3-[3-(6-pyrrolidin-1-ylpyridin-2-yl)phenyl]urea} have been extensively characterized with regard to their effects on signalling of the orthosteric ligand CP55,940 {(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol}, and studies have suggested that these allosteric modulators increase binding affinity but act as non-competitive antagonists in functional assays. To gain a deeper understanding of allosteric modulation of CB1 , we examined real-time signalling and trafficking responses of the receptor in the presence of allosteric modulators. Studies of CB1 signalling were carried out in HEK 293 and AtT20 cells expressing haemagglutinin-tagged human and rat CB1 . We measured real-time accumulation of cAMP, activation and desensitization of potassium channel-mediated cellular hyperpolarization and CB1 internalization. ORG27569 and PSNCBAM-1 produce a complex, concentration and time-dependent modulation of agonist-mediated regulation of cAMP levels, as well as an increased rate of desensitization of CB1 -mediated cellular hyperpolarization and a decrease in agonist-induced receptor internalization. Contrary to previous studies characterizing allosteric modulators at CB1, this study suggests that the mechanism of action is not non-competitive antagonism of signalling, but rather that enhanced binding results in an increased rate of receptor desensitization and reduced internalization, which results in time-dependent modulation of cAMP signalling. The observed effect of the allosteric modulators is therefore dependent on the time frame over which the signalling response occurs. This finding may have important consequences for the potential therapeutic application of these compounds.

Reply: Adverse Effects of Long-Acting Beta-Agonists on Airway Hyperresponsiveness

From the Authors: In their letter, Lipworth and colleagues raise an interesting point and we welcome their suggestion that our work in murine asthma models may help understand a clinically important point (1). We fully concur with the idea that a patient’s β2-adrenoceptor (β2AR) genotype may increase their susceptibility to adverse effects of β2AR agonists. Lipworth and colleagues suggest our data are consistent with their finding that individuals with the arginine-16 polymorphism have enhanced airway hyperresponsiveness following exposure to the high-efficacy β2AR agonist formoterol (but not the low-efficacy agonist salmeterol) (1); they also raise concerns with the recently developed high-efficacy ultra-long-acting β2AR agonists, like indacaterol and vilanterol. Lipworth and colleagues conclude by suggesting that the likely explanation for this observation is that the arginine-16 polymorphism enhances the higher rates of desensitization and down-regulation that have been shown to be produced by high-efficacy β2AR agonists (2). We fully concur that this pharmacological principle, of a significant correlation between ligand efficacy and desensitization and down-regulation, is the Occam’s razor explanation for the clinical findings and must be ruled out before introducing complex alternatives. However, we wish to point out that in wider studies, the uniqueness of formoterol over salmeterol in producing adverse effects has not been observed, and the FDA guidelines have labeled them with identical precautions and warnings. A more inclusive, albeit more speculative, explanation may be that the arginine-16 polymorphism results in preferential trafficking of the formoterol occupied β2AR to an alternate pathway than the canonical Gs-cAMP-PKA signaling. In asthma we suggest β2AR signaling via β-arrestin is the alternate pathway promoting airway hyperresponsiveness and inflammation (3, 4). Preferential pathway activation resulting from a specific ligand (ligand bias) is well documented as an ability of the ligand to form a binary complex with the receptor producing a conformation that results in biased pathway activation (4). Here we simply suggest that a structural change in the other component of the binary complex, the receptor, is now responsible for conferring the bias. Finally, we would like to add that there is an intrinsic weakness to the desensitization/down-regulation hypothesis. While it is true that high-efficacy ligands produce greater loss of receptor function and number in comparison to low-efficacy ligands that produce minimal alterations in receptor function and numbers, it is also true that high-efficacy ligands require less occupied receptors to produce their effects. Thus, from a receptor theory point of view, these effects tend to cancel each other out, and that is why both strategies have been employed and rationalized by different pharmaceutical companies.

Arrestin‐dependent but G‐protein coupled receptor kinase‐independent uncoupling of D2‐dopamine receptors

We reconstituted D2 like dopamine receptor (D2R) and the delta opioid receptor (DOR) coupling to G-protein gated inwardly rectifying potassium channels (K(ir)3) and directly compared the effects of co-expression of G-protein coupled receptor kinase (GRK) and arrestin on agonist-dependent desensitization of the receptor response. We found, as described previously, that co-expression of a GRK and an arrestin synergistically increased the rate of agonist-dependent desensitization of DOR. In contrast, only arrestin expression was required to produce desensitization of D2R responses. Furthermore, arrestin-dependent GRK-independent desensitization of D2R-K(ir)3 coupling could be transferred to DOR by substituting the third cytoplasmic loop of DOR with that of D2R. The arrestin-dependent GRK-independent desensitization of D2R desensitization was inhibited by staurosporine treatment, and blocked by alanine substitution of putative protein kinase C phosphorylation sites in the third cytoplasmic loop of D2R. Finally, the D2R construct in which putative protein kinase C phosphorylation sites were mutated did not undergo significant agonist-dependent desensitization even after GRK co-expression, suggesting that GRK phosphorylation of D2R does not play an important role in uncoupling of the receptor.

Are dopamine D2 receptors out of control in psychosis?

It is known that schizophrenia patients are behaviorally supersensitive to dopamine-like drugs (amphetamine, methylphenidate). There is evidence for an increased release of dopamine, a slight increase of dopamine D2 receptors and an increase of dopamine D2High receptors in schizophrenia, all possibly explaining the clinical supersensitivity to dopamine. The elevation in apparent D2High receptors in vivo in schizophrenia matches the elevation in D2High receptors in many animal models of psychosis. The increased amounts of D2High receptors in psychotic-like behavior in animals may result from a loss of control of D2 by various factors. These factors include the rate of phosphorylation and desensitization of D2 receptors by kinases, the attachment of arrestin to D2 receptors, internalization of D2 receptors, the rate of receptor de-phosphorylation, formation of D2 receptor dimers, and GTP regulation by various GTPases. While at present there are no statistically significant associations of any of these controlling factors and their genes with schizophrenia, investigation of D2High receptors in schizophrenia will require a new radioligand in order to selectively label D2High receptors in vivo in patients. Finally, haloperidol reduces the number of D2High receptors that are elevated by amphetamine, indicating that this therapeutic effect may occur clinically.

Desensitization of human CRF2(a) receptor signaling governed by agonist potency and βarrestin2 recruitment

The primary goal was to determine agonist-specific regulation of CRF2(a) receptor function. Exposure of human retinoblastoma Y79 cells to selective (UCN2, UCN3 or stresscopins) and non-selective (UCN1 or sauvagine) agonists prominently desensitized CRF2(a) receptors in a rapid, concentration-dependent manner. A considerably slower rate and smaller magnitude of desensitization developed in response to the weak agonist CRF. CRF1 receptor desensitization stimulated by CRF, cortagine or stressin1-A had no effect on CRF2(a) receptor cyclic AMP signaling. Conversely, desensitization of CRF2(a) receptors by UCN2 or UCN3 did not cross-desensitize Gs-coupled CRF1 receptor signaling. In transfected HEK293 cells, activation of CRF2(a) receptors by UCN2, UCN3 or CRF resulted in receptor phosphorylation and internalization proportional to agonist potency. Neither protein kinase A nor casein kinases mediated CRF2(a) receptor phosphorylation or desensitization. Exposure of HEK293 or U2OS cells to UCN2 or UCN3 (100nM) produced strong βarrestin2 translocation and colocalization with membrane CRF2(a) receptors while CRF (1μM) generated only weak βarrestin2 recruitment. βarrestin2 did not internalize with the receptor, however, indicating that transient CRF2(a) receptor–arrestin complexes dissociate at or near the cell membrane. Since deletion of the βarrestin2 gene upregulated Gs-coupled CRF2(a) receptor signaling in MEF cells, a βarrestin2 mechanism restrains Gs-coupled CRF2(a) receptor signaling activated by urocortins. We further conclude that the rate and extent of homologous CRF2(a) receptor desensitization are governed by agonist-specific mechanisms affecting GRK phosphorylation, βarrestin2 recruitment, and internalization thereby producing unique signal transduction profiles that differentially affect the stress response.

Three distinct motifs within the C-terminus of acid-sensing ion channel 1a regulate its surface trafficking

Various protein motifs play a key role in regulating protein biogenesis and trafficking. Here, we discovered that three distinct motifs regulate the trafficking of acid-sensing ion channel 1a (ASIC1a), the primary neuronal proton receptor which plays critical roles in neurological diseases including stroke, multiple sclerosis and seizures. Mutating the PDZ binding motif of ASIC1a increased its surface expression and current density. In contrast, mutating either a RRGK motif or a KEAKR motif reduced ASIC1a surface expression and acid-activated current density. Mutating or deleting the RRGK motif also reduced pH sensitivity and the rate of desensitization of ASIC1a. These changes were likely due to a change in ASIC1a biogenesis; mutating either the RRGK or KEAKR motif reduced N-glycosylation of ASIC1a while mutating the PDZ binding motif had the opposite effect. Our results demonstrate that these C-terminal motifs are important for ASIC1a trafficking and channel function. In addition, in contrast to multiple previous studies, which all show that K/R containing motifs lead to endoplasmic reticulum (ER) retention, our findings indicate that these motifs can also be required for efficient trafficking.

Correlating efficacy and desensitization with GluK2 ligand-binding domain movements

Gating of AMPA- and kainate-selective ionotropic glutamate receptors can be defined in terms of ligand affinity, efficacy and the rate and extent of desensitization. Crucial insights into all three elements have come from structural studies of the ligand-binding domain (LBD). In particular, binding-cleft closure is associated with efficacy, whereas dissociation of the dimer formed by neighbouring LBDs is linked with desensitization. We have explored these relationships in the kainate-selective subunit GluK2 by studying the effects of mutating two residues (K531 and R775) that form key contacts within the LBD dimer interface, but whose truncation unexpectedly attenuates desensitization. One mutation (K531A) also switches the relative efficacies of glutamate and kainate. LBD crystal structures incorporating these mutations revealed several conformational changes that together explain their phenotypes. K531 truncation results in new dimer contacts, consistent with slower desensitization and sideways movement in the ligand-binding cleft correlating with efficacy. The tested mutants also disrupted anion binding; no chloride was detected in the dimer-interface site, including in R775A where absence of chloride was the only structural change evident. From this, we propose that the charge balance in the GluK2 LBD dimer interface maintains a degree of instability, necessary for rapid and complete desensitization.

Anesthetic Effects on γ-Aminobutyric Acid A Receptors

Anesthetic Effects on γ-Aminobutyric Acid A Receptors

Differences in β‐arrestin dependent signaling by PAR2 and NK1R are directed by their C terminal tails

PAR2 and NK1R are 7 transmembrane receptors (7TMRs), which signal by Gαq leading to Ca2+ release and protein kinase C (PKC) activation. Both receptors are desensitized by β‐arrestin binding to their C termini. They can also activate ERK1/2 through β‐arrestin scaffolding complexes. They differ in ERK1/2 mediated physiological outcomes: cell migration versus proliferation. Using β‐arrestins, PAR2 activates cofilin, to promote chemotaxis. We hypothesized that the differences in β‐arrestin dependent signaling by 7TMRs can be attributed to how the molecular scaffolds bind the C‐tail of the receptors. Using wild type and C‐tail chimeras, we showed that the rate of desensitization, internalization, subcellular localization post endocytosis and ERK1/2 mediated responses depend on the nature of interaction of β‐arrestins with the C‐termini. BRET assays showed that PAR2 recruits both β‐arrestin 1/2 faster and with greater affinity than NK1R. We further show that initial Gαq signaling is necessary for β‐arrestin 1/2 recruitment to NK1R. PAR2 recruits β‐arrestin 1/2 even when Gαq pathway is blocked. Assays with C terminal phosphorylation mutants of PAR2 indicated that, phosphorylation of certain residues are necessary for β‐arrestin 1/2 recruitment. Phosphorylation at putative PKC sites determine stability of PAR2‐β‐arrestin 1/2 complex. This in turn ensures downstream cofilin activation and cell migration

Desensitization of the P2Y1 receptor in platelets

Understanding thrombosis is critical for effective treatment of patients at risk for heart attack and stroke. Simultaneous activation of the Gq‐coupled P2Y1 receptor and the Gi‐coupled P2Y12 receptor is required for ADP‐promoted platelet aggregation— deletion or inhibition of either receptor results in the loss of ADP‐induced aggregation. Unlike the P2Y12 receptor, the P2Y1 receptor in both mouse and human platelets undergoes profound and rapid desensitization in response to sub‐aggregating levels of ADP. We hypothesize that this rapid inactivation is specific to the P2Y1 receptor and is physiologically relevant for proper regulation of platelet hemostasis. To test the specificity of desensitization, we compared the rate of desensitization after agonist addition of the 5HT2A and TxA2 receptors, which are also Gq‐coupled, to that of the P2Y1 receptor. These data reveal that only the P2Y1 receptor rapidly desensitizes in response to its cognate agonist. To determine the residue(s) responsible for P2Y1 desensitization, we transplanted retrovirally infected bone marrow cells expressing P2Y1 receptor Ser/Thr mutants into P2Y1−/− mice to examine the desensitization of the P2Y1 receptor ex vivo. These mice will also be used to examine the physiological consequences of expressing a non‐desensitizing P2Y1 receptor by measuring thrombus formation in vivo in response to arterial vessel wall injury.(Supported by NIH grant HL54889)

Rebuttal from David M. MacLean

The main effects of the transmembrane AMPA receptor (AMPAR) auxiliary proteins (TARPs) on AMPAR gating are slowing of deactivation and desensitization, increasing peak Popen and speeding of recovery from desensitization (Priel et al. 2005; Tomita et al. 2005). All of these observations can be reproduced by increasing the rate of recovery from desensitization, γ, and by one of two additional means: either increasing the open rate, β (as proposed by Dr Howe, 2013), or reducing the cleft opening rate, CO, and desensitization rate, δ (as suggested by myself, MacLean, 2013). Moreover, increasing the channel opening rate or decreasing the cleft open rate both result in the reported substantial left-shift in steady-state glutamate EC50 values produced by TARPs (Priel et al. 2005; Tomita et al. 2005; Kott et al. 2007) as well as the smaller left-shift in peak EC50 values (Morimoto-Tomita et al. 2009). Therefore, such concentration–response data are not helpful in distinguishing an effect on gating transitions from an effect on pre-gating transitions such as stabilizing closed-cleft states. However, peak inhibition curves using NBQX and CNQX may provide some support for the hypothesis that TARPs affect pre-gating transitions by promoting states with closed ligand-binding domains (LBDs). The LBDs of AMPARs exist in multiple closed-cleft conformations, both when the agonist is bound and in the apo state (Landes et al. 2011). If the presence of TARPs causes AMPAR LBDs to adopt more closed conformations, then even in the apo state the binding cleft would be, on average, slightly more shut. Consequently, larger ligands such as NBQX or CNQX would have slower binding rates since there would be fewer favourable collisions between these compounds and the more closed LBDs. Such slowed binding rates of larger antagonists would result in right-shifts of peak inhibition curves and this is precisely what is observed experimentally. The peak response IC50 values obtained using rapid perfusion on outside-out patches for both CNQX and NBQX are right-shifted approximately 2-fold by stargazin (MacLean & Bowie, 2011). While this observation is consistent with TARPs stabilizing more closed LBD conformations, more compelling evidence is needed. Ultimately, to discriminate between these mechanisms, direct measurements of both the degree of LBD closure and coupling between LBD closure and channel opening will be required.

Unravelling the mechanism of action of NS9283, a positive allosteric modulator of (α4)3(β2)2 nicotinic ACh receptors

Strong implications in major neurological diseases make the neuronal α4β2 nicotinic ACh receptor (nAChR) a highly interesting drug target. In this study, we present a detailed electrophysiological characterization of NS9283, a potent positive allosteric modulator acting selectively at 3α:2β stoichiometry of α2* and α4* nAChRs. The whole-cell patch-clamp technique equipped with an ultra-fast drug application system was used to perform electrophysiological characterization of NS9283 modulatory actions on human α4β2 nAChRs stably expressed in HEK293 cells (HEK293-hα4β2). NS9283 was demonstrated to increase the potency of ACh-evoked currents in HEK293-hα4β2 cells by left-shifting the concentration-response curve ~60-fold. Interestingly, this modulation did not significantly alter maximal efficacy levels of ACh. Further, NS9283 did not affect the rate of desensitization of ACh-evoked currents, was incapable of reactivating desensitized receptors and only moderately slowed recovery from desensitization. However, NS9283 strongly decreased the rate of deactivation kinetics and also modestly decreased the rate of activation. This resulted in a left-shift of the ACh window current of (α4)3(β2)2 nAChRs in the presence of NS9283. This study demonstrates that NS9283 increases responsiveness of human (α4)3(β2)2 nAChR to ACh with no change in maximum efficacy. We propose that this potentiation is due to a significant slowing of deactivation kinetics. In summary, the mechanism of action of NS9283 bears high resemblance to that of benzodiazepines at the GABAA receptor and to our knowledge, NS9283 constitutes the first nAChR compound of this class.

Molecular weight of different angiotensin II polymers directly determines: Density of endothelial membrane AT1 receptors and coronary vasoconstriction

We have shown that angiotensin II (Ang II) does not diffuse across the vessel wall, remaining intravascularly confined and acting solely on the coronary endothelial luminal membrane (CELM) receptors. A sustained intracoronary infusion of Ang II causes transient coronary vasoconstriction (desensitization) due to membrane internalization of CELM Ang II type 1 receptors (CELM-AT1R). In contrast, sustained intracoronary infusion of a non-diffusible polymer of Ang II (Ang II-Pol, 15,000kDa) causes a sustained vasoconstriction by preventing CELM-AT1R internalization. In addition, a sustained intracoronary infusion of Ang II leads to a depressed response following a secondary Ang II administration (tachyphylaxis) that is reversed by Ang II-Pol. These findings led us to hypothesize that the rate of desensitization, tachyphylaxis, and AT1R internalization were dependent on Ang II-Pol molecular weight. To test this hypothesis, we synthesized Ang II-Pols of the following molecular weights (in kDa): 1.3, 2.7, 11, 47, 527, 3270 and 15,000. Vasoconstriction was measured following intracoronary infusion of Ang II-Pols in Langendorff-perfused guinea pig hearts at constant flow. The CELM protein fraction was extracted using the silica pellicle technique at different time points in order to determine the rate of AT1R internalization following each Ang II-Pol infusion. CELM-AT1R density was quantified by Western blot. We found that the rate of desensitization and the tachyphylaxis effect varied inversely with the molecular weight of the Ang II-Pols. Inversely proportional to the molecular weight of Ang II-Pol the CELM-AT1R density decreases over time. These results indicate that the mechanism responsible for the decreased rate of desensitization and tachyphylaxis by higher molecular weight Ang II polymers is due to reduction in the rate of CELM-AT1R internalization. These Ang II polymers would be valuable tools for studying the relationship between AT1R internalization and physiological effects.

The Pyrrole Etomidate Analog Carboetomidate Potently Inhibits Human 5-HT3A Receptor Function

5-Hydroxytryptamine type 3 (5-HT(3)) receptors are excitatory ion channels belonging to the cys-loop family of ligand-gated ion channels. They are involved in nausea and vomiting and their antagonists are used clinically as antiemetic drugs. We previously reported the development of a novel pyrrole analog of etomidate, (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate), which retains etomidate's desirable anesthetic and hemodynamic properties, but lacks its potent inhibitory effect on adrenocorticotropic hormone-stimulated steroid synthesis. Also in contrast to etomidate, carboetomidate potently inhibits nicotinic acetylcholine receptors. Because nicotinic acetylcholine and 5-HT(3) receptors are highly homologous, we hypothesized that carboetomidate might also potently inhibit 5-HT(3) receptors with potentially important implications for the drug's emetogenic activity. In the current studies, we investigated and compared modulation of 5-HT(3A) receptors by carboetomidate and etomidate. 5-HT(3) receptors were heterologously expressed in human embryonic kidney cells. Drugs were applied with a multichannel superfusion pipette coupled to piezoelectric elements, and currents were recorded from cells in either the whole-cell or excised outside-out patch configuration of patch-clamp recordings. Carboetomidate and etomidate inhibited integrated 5-HT(3A) receptor-mediated currents with respective half-inhibitory concentrations of 1.9 μM (95% confidence interval [CI] = 1.4-2.7 μM) and 25 μM (95% CI = 17-37 μM). These values may be compared with respective hypnotic concentrations of 5.4 and 2.3 µM. This inhibition reflected hypnotic effects on peak current amplitudes and desensitization rates. Half-inhibitory concentrations for reducing peak current amplitudes were 34 μM (95% CI = 24-48 µM) for carboetomidate and 171 μM (95% CI = 128-228 µM) for etomidate. Half-inhibitory concentrations for reducing the desensitization time constant were 3.5 μM (95% CI = 2.4-5.1 µM) for carboetomidate and 36 μM (95% CI = 21-59 µM) for etomidate. In contrast to etomidate, carboetomidate inhibits 5-HT(3A) receptor-mediated currents at hypnotic concentrations. This inhibition is primarily the result of enhancing the rate of desensitization. Because carboetomidate potently inhibits 5-HT(3A) receptors, it may be less emetogenic than etomidate.