Onset Of Desensitization Research Articles

Alternative splicing of the flip/flop cassette and TARP auxiliary subunits engage in a privileged relationship that fine-tunes AMPA receptor gating.

Alternative splicing of AMPA-type glutamate receptors (AMPARs) and allosteric modulation by auxiliary subunits, such as transmembrane AMPAR regulatory proteins (TARPs), are two important mechanisms that regulate the time course of glutamatergic neurotransmission. Prior work has shown that alternative splicing of the flip/flop cassette profoundly regulates TARP γ2 modulation, where flip receptor gating exhibits robust sensitivity to TARPs while flop isoforms are relatively insensitive to TARP modulation. Whether this splice variant-specific regulation extends to other auxiliary subunit families, such as cornichons (CNIHs), GSG1L, or CKAMPs, remains unknown. Here, we demonstrate that CNIH-3 modulation is unaffected by AMPAR alternative splicing due to inherent differences in how CNIH-3 and TARP γ2 modify channel gating. CNIH-3 slows receptor deactivation from the outset of current decay, consistent with structural evidence showing its point of contact at the level of the pore. In contrast, TARP γ2 acts through the KGK site of the ligand-binding domain (LBD) to slow the onset of desensitization. Although GSG1L and CKAMP44 primarily slow recovery from desensitization, their effects on channel gating are unaffected by alternative splicing, further underlining that structural events leading to the onset and recovery from desensitization are separable. Together, this work establishes that alternative splicing and TARP auxiliary subunits form a unique partnership that governs fast glutamatergic signaling at central synapses. Since proteomic studies suggest that all native AMPARs co-assemble with at least two TARPs, allosteric coupling between the flip/flop cassette and TARPs may represent a common design element in all AMPAR complexes of the mammalian brain.SIGNIFICANCE STATEMENT:All fast excitatory neurotransmission in the mammalian brain is mediated by AMPA-type glutamate receptors (AMPARs). The time course of AMPAR gating can be regulated by two distinct mechanisms: alternative splicing of the flip/flop cassette and association with auxiliary subunits. Although these regulatory mechanisms have been well studied individually, it is not clear whether alternative splicing impacts auxiliary protein modulation of AMPARs. Here, we compare the four main families of AMPAR auxiliary subunits, TARPs (γ2), Cornichons (CNIH-3), GSG1L and CKAMPs (CKAMP44), and find a privileged relationship between TARPs and the flip/flop cassette that is not shared by others. The flop cassette acts as a master switch to override TARP action, and this coupling represents a way to fine-tune AMPAR signaling.

Comparison of the efficacy and duration of desensitization of oral structures following injection of a lidocaine-bupivacaine mixture via lateral percutaneous and modified infraorbital approaches in dogs.

To compare efficacy and duration of desensitization of oral structures with a lidocaine-bupivacaine mixture administered via a lateral percutaneous or modified infraorbital approach. 6 healthy adult hound-type female dogs. In this crossover study, dogs were randomized for side (left or right) and maxillary nerve approach (lateral percutaneous or infraorbital), with a 2-week washout period. Dogs were anesthetized, and a 2-mL mixture of 2% lidocaine and 0.5% bupivacaine (50:50 [vol/vol]) was administered with a 22-gauge, 4.5-cm-long catheter inserted through the infraorbital canal (infraorbital approach) or with a shielded stimulating needle to the maxillary nerve (percutaneous approach). Reflex-evoked motor potentials were measured for the maxillary canine tooth, fourth premolar tooth, second molar tooth, and hard palate mucosa ipsilateral to the injected mixture and for the contralateral maxillary canine tooth (control) at three 10-minute intervals before injection (baseline) and at predetermined times after injection for up to 6.7 hours. For each oral structure, the proportion of dogs with desensitization (efficacy) and time to onset and duration of desensitization were compared between approaches. The proportion of dogs with successful nerve blockade did not significantly differ between infraorbital and percutaneous approaches and among the 4 oral structures. Time to onset of desensitization did not differ between approaches, but duration was significantly longer with the infraorbital approach. A modified infraorbital approach with the lidocaine-bupivacaine mixture had similar effects to a lateral percutaneous approach but provided a longer duration of desensitization. Neither approach was universally successful at desensitizing all oral structures.

Comparison of the efficacy and duration of desensitization of oral structures following injection of various volumes of a lidocaine-bupivacaine mixture via an infraorbital approach in dogs.

To compare the efficacy and duration of desensitization of oral structures following injection of various volumes of lidocaine-bupivacaine via an infraorbital approach in dogs. 6 healthy adult hound-type dogs. In a randomized crossover study, each dog received 1, 2, and 3 mL of a 2% lidocaine-0.5% bupivacaine mixture (50:50 vol/vol) injected within and near the caudal aspect of the infraorbital canal with a 14-day washout period between treatments. Dogs were anesthetized, and each treatment was administered through a 22-gauge, 4.5-cm-long catheter, which was fully inserted through and then withdrawn 2 cm to the caudal aspect of the infraorbital canal. The reflex-evoked motor potential was measured for the maxillary canine tooth (MC), fourth premolar tooth (MPM4), second molar tooth (MM2), and hard palate mucosa ipsilateral to the injected treatment and for the contralateral MC (control) at predetermined times before and for 6 hours after treatment administration or until the block was no longer effective. For each oral structure, the proportion of dogs with desensitization (efficacy) and time to onset and duration of desensitization were compared among the 3 treatments (injectate volumes). Treatment was not associated with efficacy, time to onset, or duration of desensitization. Regardless of treatment, MC and MPM4 were more frequently desensitized and mean durations of desensitization for MC and MPM4 were longer, compared with those for MM2 and the hard palate. The volume of local anesthetic used for an infraorbital nerve block had no effect on block efficacy or duration.

Combination therapy to enhance NK cell anti-tumor responses

Abstract Current cancer immunotherapies are targeted at mobilizing CD8 T cells, although numerous tumors have few or no antigens for CD8 T cells, or have lost MHC I, thereby evading CD8 T cells. There is therefore a need for improved therapies that mobilize other immune cells, such as NK cells, to the tumor microenvironment. A recent therapy candidate, cyclic dinucleotide (CDN), activates the cGAS-STING pathway of the innate immune system. Intratumoral CDN injections induce type I IFNs and other mediators that amplify the CD8 T cell response and induce regressions of primary tumors. MHC I-deficient tumors are not susceptible to CD8 T cell-mediated rejection, but data from our lab demonstrates that STING activation still leads to long-term tumor regressions, mediated by NK cells and in some cases CD4 T cells, in 30–100% of animals in 5/6 transplant models tested. In order to improve upon CDN monotherapy, we are testing combinations of CDN therapy with other therapies to target and activate NK cells in the tumor microenvironment and prevent or delay the onset of desensitization. Our results show that engineered IL-2 family cytokines synergize with CDN therapy to mobilize anti-tumor responses by NK cells and in some cases CD4 T cells. These anti-tumor responses are due to increased recruitment of NK cells to the tumor and sustained activation of these recruited NK cells. Our combination therapy regimen shows enhanced systemic effects, both in vivo in abscopal tumor models, and ex vivo by cytolysis of target cells mediated by splenocytes. Overall, our work demonstrates the impact of a novel combination therapy in mobilizing powerful NK cell-mediated anti-tumor activity, providing evidence for the potential of NK cell-targeted immunotherapeutics for cancer.

The δ-opioid receptor positive allosteric modulator BMS 986187 is a G-protein-biased allosteric agonist.

The δ-opioid receptor is an emerging target for the management of chronic pain and depression. Biased signalling, the preferential activation of one signalling pathway over another downstream of δ-receptors, may generate better therapeutic profiles. BMS 986187 is a positive allosteric modulator of δ-receptors. Here, we ask if BMS 986187 can directly activate the receptor from an allosteric site, without an orthosteric ligand, and if a signalling bias is generated. We used several clonal cell lines expressing δ-receptors, to assess effects of BMS 986187 on events downstream of δ-receptors by measuring G-protein activation, β-arrestin 2 recruitment, receptor phosphorylation, loss of surface receptor expression, ERK1/ERK2 phosphorylation, and receptor desensitization. BMS 986187 is a G protein biased allosteric agonist, relative to β-arrestin 2 recruitment. Despite showing direct and potent G protein activation, BMS 986187 has a low potency to recruit β-arrestin 2. This appears to reflect the inability of BMS 986187 to elicit any significant receptor phosphorylation, consistent with low receptor internalization and a slower onset of desensitization, compared with the full agonist SNC80. This is the first evidence of biased agonism mediated through direct binding to an allosteric site on an opioid receptor, without a ligand at the orthosteric site. Our data suggest that agonists targeting δ-receptors, or indeed any GPCR, through allosteric sites may be a novel way to promote signalling bias and thereby potentially produce a more specific pharmacology than can be observed by activation via the orthosteric site.

Brain natriuretic peptide constitutively downregulates P2X3 receptors by controlling their phosphorylation state and membrane localization.

BackgroundATP-gated P2X3 receptors are important transducers of nociceptive stimuli and are almost exclusively expressed by sensory ganglion neurons. In mouse trigeminal ganglion (TG), P2X3 receptor function is unexpectedly enhanced by pharmacological block of natriuretic peptide receptor-A (NPR-A), outlining a potential inhibitory role of endogenous natriuretic peptides in nociception mediated by P2X3 receptors. Lack of change in P2X3 protein expression indicates a complex modulation whose mechanisms for downregulating P2X3 receptor function remain unclear.ResultsTo clarify this process in mouse TG cultures, we suppressed NPR-A signaling with either siRNA of the endogenous agonist BNP, or the NPR-A blocker anantin. Thus, we investigated changes in P2X3 receptor distribution in the lipid raft membrane compartment, their phosphorylation state, as well as their function with patch clamping. Delayed onset of P2X3 desensitization was one mechanism for the anantin-induced enhancement of P2X3 activity. Anantin application caused preferential P2X3 receptor redistribution to the lipid raft compartment and decreased P2X3 serine phosphorylation, two phenomena that were not interdependent. An inhibitor of cGMP-dependent protein kinase and siRNA-mediated knockdown of BNP mimicked the effect of anantin.ConclusionsWe demonstrated that in mouse trigeminal neurons endogenous BNP acts on NPR-A receptors to determine constitutive depression of P2X3 receptor function. Tonic inhibition of P2X3 receptor activity by BNP/NPR-A/PKG pathways occurs via two distinct mechanisms: P2X3 serine phosphorylation and receptor redistribution to non-raft membrane compartments. This novel mechanism of receptor control might be a target for future studies aiming at decreasing dysregulated P2X3 receptor activity in chronic pain.

The auxiliary subunits Neto1 and Neto2 have distinct, subunit-dependent effects at recombinant GluK1- and GluK2-containing kainate receptors

The kainate-type of ionotropic glutamate receptors are assembled from a combination of five different pore-forming subunits (GluK1–5), which confer distinct functional and pharmacological properties. These receptors are also modulated by co-assembly with the auxiliary subunits Neto1 and Neto2. To determine the impact of variation in subunit composition on the functional interaction between kainate receptors and Neto subunits, the Neto subunits were combined with either GluK1 or GluK2 in HEK-293T cells and responses to glutamate examined through patch-clamp recordings. Co-expression of GluK1 with either Neto1 or Neto2 caused a substantial increase in glutamate sensitivity and a slowing of the onset of desensitization at low agonist concentrations. However, at higher glutamate concentrations the primary effect of Neto2 was to slow the onset of desensitization, while that of Neto1 was to increase recovery from desensitization. In contrast, co-expression of Neto2 with GluK2 homomeric receptors had only modest effects on glutamate sensitivity, but increased the rate of recovery from desensitization as well as slowing its onset at all agonist concentrations. The properties of chimeric Neto1/Neto2 subunits suggested that the extracellular N-terminal region including the two CUB domains was largely responsible for the distinct regulatory effects of Neto1 and Neto2 on the desensitization properties of GluK1 homomeric receptors. These results further demonstrate that the functional effects of Neto subunits depend upon the subunit identity of both the auxiliary and the pore-forming subunits.

Contributions of different kainate receptor subunits to the properties of recombinant homomeric and heteromeric receptors

The tetrameric kainate receptors can be assembled from a combination of five different subunit subtypes. While GluK1–3 subunits can form homomeric receptors, GluK4 and GluK5 require a heteromeric partner to assemble, traffic to the membrane surface, and produce a functional channel. Previous studies have shown that incorporation of a GluK4 or GluK5 subunit changes both receptor pharmacology and channel kinetics. We directly compared the functional characteristics of recombinant receptors containing either GluK4 or GluK5 in combination with the GluK1 or GluK2 subunit. In addition, we took advantage of mutations within the agonist binding sites of GluK1, GluK2, or GluK5 to isolate the response of the wild-type partner within the heteromeric receptor. Our results suggest that GluK1 and GluK2 differ primarily in their pharmacological properties, but that GluK4 and GluK5 have distinct functional characteristics. In particular, while binding of agonist to only the GluK5 subunit appears to activate the channel to a non-desensitizing state, binding to GluK4 does produce some desensitization. This suggests that GluK4 and GluK5 differ fundamentally in their contribution to receptor desensitization. In addition, mutation of the agonist binding site of GluK5 results in a heteromeric receptor with a glutamate sensitivity similar to homomeric GluK1 or GluK2 receptors, but which requires higher agonist concentrations to produce desensitization. This suggests that onset of desensitization in heteromeric receptors is determined more by the number of subunits bound to agonist than by the identity of those subunits. The distinct, concentration-dependent properties observed with heteromeric receptors in response to glutamate or kainate are consistent with a model in which either subunit can activate the channel, but in which occupancy of both subunits within a dimer is needed to allow desensitization of GluK2/K5 receptors.

Functional insight into development of positive allosteric modulators of AMPA receptors

Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) ionotropic glutamate receptors facilitate synaptic plasticity and contribute essentially to learning and memory, properties which make AMPA receptors targets for drug discovery and development. One region at which several different classes of positive allosteric modulators bind lies at the dimer interface between the ligand-binding core of the second, membrane-proximal, extracellular domain of AMPA receptors. This solvent-accessible binding pocket has been the target of drug discovery efforts, leading to the recent delineation of five “subsites” which differentially allow access to modulator moieties, and for which distinct modulator affinities and apparent efficacies are attributed. Here we use the voltage-clamp technique in conjunction with rapid drug application to study the effects of mutants lining subsites “A” and “B” of the allosteric modulator pocket to assess affinity and efficacy of allosteric modulation by cyclothiazide, CX614, CMPDA and CMPDB. A novel analysis of the decay of current produced by the onset of desensitization has allowed us to estimate both affinity and efficacy from single concentrations of modulator. Such an approach may be useful for effective high throughput screening of new target compounds.

The N-terminal Domain Modulates α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) Receptor Desensitization

AMPA receptors are tetrameric glutamate-gated ion channels that mediate fast synaptic neurotransmission in mammalian brain. Their subunits contain a two-lobed N-terminal domain (NTD) that comprises over 40% of the mature polypeptide. The NTD is not obligatory for the assembly of tetrameric receptors, and its functional role is still unclear. By analyzing full-length and NTD-deleted GluA1-4 AMPA receptors expressed in HEK 293 cells, we found that the removal of the NTD leads to a significant reduction in receptor transport to the plasma membrane, a higher steady state-to-peak current ratio of glutamate responses, and strongly increased sensitivity to glutamate toxicity in cell culture. Further analyses showed that NTD-deleted receptors display both a slower onset of desensitization and a faster recovery from desensitization of agonist responses. Our results indicate that the NTD promotes the biosynthetic maturation of AMPA receptors and, for membrane-expressed channels, enhances the stability of the desensitized state. Moreover, these findings suggest that interactions of the NTD with extracellular/synaptic ligands may be able to fine-tune AMPA receptor-mediated responses, in analogy with the allosteric regulatory role demonstrated for the NTD of NMDA receptors.

Regulatory Ions Bound at the iGluR Ligand Binding Domain Dimer Interface - A Shared Property of GluK2 and AvGluR1?

Ligand-gated ion channels activated by glutamate binding, ionotropic glutamate receptor (iGluRs), play crucial roles in our central nervous system (CNS), e.g. in learning and memory. Furthermore, iGluRs are implicated in many CNS disorders. Binding of glutamate to the iGluR ligand-binding domain (LBD) triggers opening of the transmembrane cation channel. In the overall tetrameric structure, the LBDs are organized in a dimer of dimers. As opposed to other vertebrate iGluRs, kainate-selective iGluRs (KARs) require binding of extracellular sodium and chloride ions to the LBD dimer interface in addition to agonist binding for activation. We have recently shown that the regulatory cations for the GluK2 KAR determine the onset of desensitization, with desensitization not occurring until the cation site is vacated (Nat. Struct. Mol. Biol. 2013, 20, 1054-1061). iGluRs are also found in other kingdoms of life, structurally exemplified by AvGluR1 from a primitive eukaryote (Structure 2013, 21, 1-12). Surprisingly, this LBD dimer structure shows four chloride ions bound at the dimer interface. With atomistic molecular dynamics simulations similar to our work on KARs, we have studied the binding of these chloride ions to elucidate whether they appear to have a regulatory effect on AvGluR1, and whether such a regulatory mechanism could be a shared property between KARs and AvGluR1. Furthermore, we have studied the structural changes believed to be linked to desensitization in the presence and absence of interface-bound ions for both structures. The LBD dimer interface is thought to open up around the ion binding sites in conjunction with desensitization. Interestingly, this interface is packed closer in the AvGluR1 structure than observed for LBD dimer structures of vertebrate iGluRs. Our results illustrate how this feature influences the dynamical changes of desensitization.

Modulation of homomeric and heteromeric kainate receptors by the auxiliary subunit Neto1

The ionotropic glutamate receptors are primary mediators of fast excitatory neurotransmission, and their properties are determined both by their subunit composition and their association with auxiliary subunits. The neuropilin and tolloid-like 1 and 2 proteins (Neto1 and Neto2) have been recently identified as auxiliary subunits for kainate-type glutamate receptors. Heteromeric kainate receptors (KARs) can be assembled from varying combinations of low-affinity (GluK1-GluK3) and high-affinity (GluK4-GluK5) subunits. To better understand the functional impact of auxiliary subunits on KARs, we examined the effect of Neto1 on the responses of recombinant homomeric and heteromeric KARs to varying concentrations of glutamate. We found that co-expression of Neto1 with homomeric GluK2 receptors had a small effect on sensitivity of the receptors to glutamate, but decreased the onset of desensitization while speeding recovery from desensitization. In the absence of Neto1, addition of GluK5 subunits to form GluK2/GluK5 heteromeric receptors slowed the onset of desensitization at low glutamate concentrations, compared with GluK2 homomers. Co-expression of Neto1 with GluK2/GluK5 receptors further enhanced these effects, essentially eliminating desensitization at μm glutamate concentrations without altering the EC50 for activation by glutamate. In addition, a prominent rebound current was observed upon removal of the agonist. The rate of recovery from desensitization was increased to the same degree by Neto1 for both homomeric GluK2 and heteromeric GluK2/GluK5 receptors. Expression of Neto1 with GluK1/GluK5, GluK3/GluK5 or GluK2/GluK4 receptors produced qualitatively similar effects on whole-cell currents, suggesting that the impact of Neto1 on the desensitization properties of heteromeric receptors was not subunit dependent. These results provide greater insight into the functional effects of the auxiliary subunit Neto1 on both homomeric and heteromeric KARs. Alteration of the characteristics of desensitization at both sub-maximal and saturating glutamate concentrations could influence the responsiveness of these receptors to repeated stimuli. As a result, assembly of KARs with the Neto auxiliary subunits could change the kinetic properties of the neuronal response to glutamatergic input.

Desensitization properties of P2X3 receptors shaping pain signaling

ATP-gated P2X3 receptors are mostly expressed by nociceptive sensory neurons and participate in transduction of pain signals. P2X3 receptors show a combination of fast desensitization onset and slow recovery. Moreover, even low nanomolar agonist concentrations unable to evoke a response, can induce desensitization via a phenomenon called “high affinity desensitization.” We have also observed that recovery from desensitization is agonist-specific and can range from seconds to minutes. The recovery process displays unusually high temperature dependence. Likewise, recycling of P2X3 receptors in peri-membrane regions shows unexpectedly large temperature sensitivity. By applying kinetic modeling, we have previously shown that desensitization characteristics of P2X3 receptor are best explained with a cyclic model of receptor operation involving three agonist molecules binding a single receptor and that desensitization is primarily developing from the open receptor state. Mutagenesis experiments suggested that desensitization depends on a certain conformation of the ATP binding pocket and on the structure of the transmembrane domains forming the ion pore. Further molecular determinants of desensitization have been identified by mutating the intracellular N- and C-termini of P2X3 receptor. Unlike other P2X receptors, the P2X3 subtype is facilitated by extracellular calcium that acts via specific sites in the ectodomain neighboring the ATP binding pocket. Thus, substitution of serine275 in this region (called “left flipper”) converts the natural facilitation induced by extracellular calcium to receptor inhibition. Given their strategic location in nociceptive neurons and unique desensitization properties, P2X3 receptors represent an attractive target for development of new analgesic drugs via promotion of desensitization aimed at suppressing chronic pain.

Functional differences between ATP‐gated human and rat P2X3 receptors are caused by critical residues of the intracellular C‐terminal domain

ATP-activated P2X3 receptors of sensory ganglion neurons contribute to pain transduction and are involved in chronic pain signaling. Although highly homologous (97%) in rat and human species, it is unclear whether P2X3 receptors have identical function. Studying human and rat P2X3 receptors expressed in patch-clamped human embryonic kidney (HEK) cells, we investigated the role of non-conserved tyrosine residues in the C-terminal domain (rat tyrosine-393 and human tyrosine-376) as key determinants of receptor function. In comparison with rat P2X3 receptors, human P2X3 receptors were more expressed and produced larger responses with slower desensitization and faster recovery. In general, desensitization was closely related to peak current amplitude for rat and human receptors. Downsizing human receptor expression to the same level of the rat one still yielded larger responses retaining slower desensitization and faster recovery. Mutating phenylalanine-376 into tyrosine in the rat receptor did not change current amplitude; yet, it retarded desensitization onset, demonstrating how this residue was important to functionally link these two receptor states. Conversely, removing tyrosine from position 376 strongly down-regulated human receptor function. The different topology of tyrosine residues in the C-terminal domain has contrasting functional consequences and is sufficient to account for species-specific properties of this pain-transducing channel.

Sensitization and Intergranular Corrosion Behavior of High Nitrogen Type 304LN Stainless Steels for Reprocessing and Waste Management Applications

High nitrogen 304LN stainless steels (SS) intended for chloride and nitric acid environments in spent nuclear fuel reprocessing and waste management applications were evaluated for their sensitization and intergranular corrosion (IGC) resistance. For this purpose, high nitrogen (0.132 pct, 0.193 pct and 0.406 pct) containing, impurity-controlled, vanadium-added 304LN SS alloys were developed. For comparison, 304L SS, which is currently used in reprocessing plants, was also studied. These stainless steels were subjected to heat treatment at 948 K (675 °C) for various durations ranging from 1 to 1000 hours and tested for susceptibility to IGC as per ASTM A262 Practice A and E tests. The degree of sensitization was estimated with the double loop electrochemical potentiokinetic reactivation technique. The increase in nitrogen content resulted in higher hardness and finer grain size. Based on the detailed microstructural and corrosion studies, it was determined that an addition of 0.132 pct and 0.193 pct nitrogen showed better IGC resistance and an additional increase in nitrogen resulted in deterioration resulting from chromium nitride precipitation, which was confirmed by electrochemical phase separation and X-ray diffraction studies. The onset of desensitization was faster for the alloy with 0.132 pct nitrogen as well as 0.406 pct nitrogen because of the lower nitrogen content in the former case and the finer grain size in the latter case. The higher hardness and superior IGC resistance of 0.132 pct and 0.193 pct nitrogen containing Type 304LN SS suggests the suitability of this alloy for nitric acid- and chloride-containing environments of reprocessing and waste management plants.

Distinct Functional Roles of Subunits within the Heteromeric Kainate Receptor

Kainate receptors (KARs) have been implicated in a number of neurological disorders, including epilepsy. KARs are tetrameric, composed of a combination of GluK1-GluK5 subunits. We examined the contribution of GluK2 and GluK5 subunits to activation and desensitization of the heteromeric receptor. Heteromeric GluK2/K5 receptors expressed in HEK-293T cells showed markedly higher glutamate sensitivity than GluK2 homomers and did not desensitize at low glutamate concentrations. Mutation of residue E738 in GluK2 substantially lowered its glutamate sensitivity. However, heteromeric KARs containing this mutant GluK2 [GluK2(E738D)] assembled with wild-type GluK5 showed no change in glutamate EC(50) compared with wild-type heteromeric KARs. Instead, higher concentrations of glutamate were required to produce desensitization. This suggested that, within the heteromeric receptor, glutamate binding to the high-affinity GluK5 subunit alone was sufficient for channel activation but not desensitization, whereas agonist binding to the low-affinity GluK2 subunit was not necessary to open the channel but instead caused the channel to enter a closed, desensitized state. To test this hypothesis in wild-type receptors, we used the competitive antagonist kynurenate, which has higher affinity for the GluK2 than the GluK5 subunit. Coapplication of kynurenate with glutamate to heteromeric receptors reduced the onset of desensitization without affecting the peak current response, consistent with our hypothesis. Our results suggest that GluK2 and GluK5 subunits can be individually activated within the heteromeric receptor and that these subunits serve dramatically different functional roles.

Role of the Ectodomain Serine 275 in Shaping the Binding Pocket of the ATP-Gated P2X3 Receptor

ATP-activated P2X3 receptors expressed in nociceptive sensory neurons play an important role in pain signaling. Basic properties of this receptor subtype, including very strong desensitization, depend on the rate of dissociation of the agonist from the binding site. Even though the rough structure of the ATP binding site has been proposed on the basis of the X-ray structure of the zebrafish P2X4 receptor and mutagenesis studies, the fine subunit-specific structural properties predisposing the receptor to tight capture of the agonist inside the binding pocket have not been elucidated. In this work, by exploring in silico the functional role for the left flipper located in the ectodomain region, we identified within this loop a candidate residue S275, which could contribute to the closure of the agonist-binding pocket. Testing of the S275 mutants using the patch-clamp technique revealed a crucial role for S275 in agonist binding and receptor desensitization. The S275A mutant showed a reduced rate of onset of desensitization and accelerated resensitization and was weakly inhibited by nanomolar agonist. Extracellular calcium application produced inhibition instead of facilitation of membrane currents. Moreover, some full agonists became only partial agonists when applied to the S275A receptor. These effects were stronger with the more hydrophobic mutants S275C and S275V. Taken together, our data suggest that S275 contributes to the closure of the agonist-binding pocket and that effective capture of the agonist provided by the left flipper in calcium-dependent manner determines the high rate of desensitization, slow recovery, and sensitivity to nanomolar agonist of the P2X3 receptor.

Kinetics of quasi-isoenergetic transition processes in biological macromolecules

A master equation describing the evolution of averaged molecular state occupancies in molecular systems where alternation of molecular energy levels is caused by discrete dichotomous and trichotomous stochastic fields, is derived. This study is focused on the kinetics of quasi-isoenergetic transition processes in the presence of moderately high frequency stochastic field. A novel physical mechanism for temperature-independent transitions in flexible molecular systems is proposed. This mechanism becomes effective when the conformation transitions between quasi-isoenergetic molecular states take place. At room temperatures, stochastic broadening of molecular energy levels predominates the energy of low-frequency vibrations accompanying the transition. This leads to a cancellation of the temperature dependence in the stochastically averaged rate constants. As examples, physical interpretations of the temperature-independent onset of P2X3 receptor desensitization in neuronal membranes, as well as degradation of PER2 protein in embrionic fibroblasts, are provided.

Desensitization and models of receptor‐channel activation

Desensitization and models of receptor‐channel activation

Subunit-specific desensitization of heteromeric kainate receptors

Kainate receptor subunits can form functional channels as homomers of GluK1, GluK2 or GluK3, or as heteromeric combinations with each other or incorporating GluK4 or GluK5 subunits. However, GluK4 and GluK5 cannot form functional channels by themselves. Incorporation of GluK4 or GluK5 into a heteromeric complex increases glutamate apparent affinity and also enables receptor activation by the agonist AMPA. Utilizing two-electrode voltage clamp of Xenopus oocytes injected with cRNA encoding kainate receptor subunits, we have observed that heteromeric channels composed of GluK2/GluK4 and GluK2/GluK5 have steady state concentration-response curves that were bell-shaped in response to either glutamate or AMPA. By contrast, homomeric GluK2 channels exhibited a monophasic steady state concentration-response curve that simply plateaued at high glutamate concentrations. By fitting several specific Markov models to GluK2/GluK4 heteromeric and GluK2 homomeric concentration-response data, we have determined that: (a) two strikingly different agonist binding affinities exist; (b) the high-affinity binding site leads to channel opening; and (c) the low-affinity agonist binding site leads to strong desensitization after agonist binding. Model parameters also approximate the onset and recovery kinetics of desensitization observed for macroscopic currents measured from HEK-293 cells expressing GluK2 and GluK4 subunits. The GluK2(E738D) mutation lowers the steady state apparent affinity for glutamate by 9000-fold in comparison to GluK2 homomeric wildtype receptors. When this mutant subunit was expressed with GluK4, the rising phase of the glutamate steady state concentration-response curve overlapped with the wildtype curve, whereas the declining phase was right-shifted toward lower affinity. Taken together, these data are consistent with a scheme whereby high-affinity agonist binding to a non-desensitizing GluK4 subunit opens the heteromeric channel, whereas low-affinity agonist binding to GluK2 desensitizes the whole channel complex.