Rate Of Desensitization Research Articles

The Aminoglycosides Modulate the Acid-Sensing Ionic Channel Currents in Dorsal Root Ganglion Neurons from the Rat

Acid-sensing ionic channels (ASICs) have been shown to have a significant role in a growing number of physiological and pathological processes, such as nociception, synaptic transmission and plasticity, mechanosensation, and acidosis-induced neuronal injury. The discovery of pharmacological agents targeting ASICs has significant therapeutic potential and use as a research tool. In our work, we studied the action of transient perfusion (5-15 s) of aminoglycosides (AGs) (streptomycin and neomycin) on the proton-gated ionic currents in dorsal root ganglion (DRG) neurons of the rat and in human embryonic kidney (HEK)-293 cells. In DRG neurons, streptomycin and neomycin (30 microM) produced a significant, concentration-dependent, and reversible reduction in the amplitude of the proton-gated current, and a slowing of the desensitization rate of the ASIC current. Gentamycin (30 microM) also showed a significant reversible action on the ASIC currents. The curves of the pH effect for streptomycin and neomycin indicated that their effect was not significantly affected by pH. In HEK-293 cells, streptomycin (30 microM) produced a significant reduction in the amplitude of the proton-gated current. Neomycin and gentamycin had no significant action. Reduction of extracellular Ca(2+) concentration produced a significant increase in the action of streptomycin and neomycin on the desensitization time course of ASIC currents. These results indicate that ASICs are molecular targets for AGs, which may contribute to the understanding of their actions on excitable cells. Moreover, AGs may constitute a source to develop novel molecules with a greater affinity, specificity, and selectivity for the different ASIC subunits.

The role of GABA and glutamate receptors in susceptibility and resistance to chloride channel blocker insecticides

Despite a point mutation in the pore-forming segment of the Rdl GABA receptor subunit that is widespread and persistent in insect populations and confers high levels of resistance to dieldrin and other polychlorocycloalkane (PCCA) insecticides, the phenylpyrazole insecticide fipronil, which binds at same site, has proven to be effective in controlling many insects, including dieldrin-resistant populations. Fipronil and its major sulfone metabolite are unique among chloride channel blocking insecticides in that they also potently block GluCls. We present here a patch clamp study of the action of fipronil sulfone on native GABA receptors and GluCl receptors from susceptible and dieldrin-resistant German cockroaches, to provide a better understanding of the effect of the Rdl mutation on the function and insecticide sensitivity of these two targets, and its role in resistance. Dieldrin blocked GABA currents with an IC 50 of 3 nM in wild-type cockroaches, and 383 nM in resistant insects, yielding a resistance ratio of 128. Fipronil sulfone blocked GABA currents with an IC 50 of 0.8 nM in susceptible insects and 12.1 nM, or 15-fold higher, in resistant insects. While both GluClD (desensitizing) and GluClN (non-desensitizing) receptors were found in German cockroach neurons, GluClN receptors were rare and could not be included in this study. GluClD receptors from resistant insects had reduced sensitivity to glutamate and a lower rate of desensitization than those from susceptible insects, but their sensitivity to block by fipronil sulfone was not significantly changed, with an IC 50 of 38.5 ± 2.4 nM ( n = 8) in the susceptible strain and 40.3 ± 1.0 nM ( n = 7) in the resistant strain. Fipronil sulfone also slowed the decay time course of GluClD currents. These results suggest that GluClD receptors contain the Rdl subunit, but their sensitivity to fipronil sulfone is not altered in resistant insects.

Influence of Y151F mutation in loop B on the agonist potency in insect nicotinic acetylcholine receptor

Abstract Nicotinic acetylcholine receptors (nAChRs) are ligand‐gated ion channels, which mediate fast cholinergic synaptic transmission in insect and vertebrate nervous systems. The nAChR agonist‐binding site is present at the interface of adjacent subunits and is formed by loops A–C present in α subunits together with loops D–F present in either non‐α subunits or homomer‐forming α subunits. Although Y151 in loop B has been identified as important in agonist binding, various residues at the 151‐site are found among vertebrate and invertebrate nAChR α subunits, such as F151. In Xenopus oocytes expressing Nlα1 or Nlα1Y151F plus rat β2, Y151F mutation was found to significantly change the rate of receptor desensitization and altered the pharmacological properties of acetylcholine, but not imidacloprid, including the decrease of Imax, the increase of EC50 (the concentration causing 50% of the maximum response) and the fast time‐constant of decay (τf). By comparisons of residue structure, the hydroxyl group in the side chain of Y151 was thought to be important in the interaction between Nlα1/β2 nAChRs and acetylcholine, and the phenyl group to be important between Nlα1/β2 nAChRs and imidacloprid.

Tricks of Perspective: Insights and Limitations to the Study of Macroscopic Currents for the Analysis of nAChR Activation and Desensitization

Activation, inactivation, and desensitization are key features of ion channel behavior. We endeavor to understand these processes at the level of the single molecules and extrapolate from such microscopic models the behavior of ion channels in contexts of cellular physiology and therapeutics. In the case of ligand-gated ion channels, such as nicotinic acetylcholine receptors (nAChRs), it is also important to consider the nature of the dynamic changes in the chemical stimulus required for activation. The amplitude and time course of the agonist pulse provided to nAChR at a fast synapse will be vastly different from those of the ACh stimulus presented to presynaptic receptors in the brain and neither of these physiological processes will resemble the stimuli presented by nicotine self-administration or with systemic delivery of a therapeutic agent. Likewise, specific experimental protocols will provide unique stimulus profiles, which will impact the relationship between the macroscopic data and the underlying molecular processes. In this work, ion channel simulations intended to model heteromeric neuronal nAChR are conducted under varying conditions of agonist presentation, and the impact of a key microscopic process, desensitization, is studied on the macroscopic responses. With instantaneous jumps in agonist concentrations, the microscopic desensitization rate impacts essentially all aspects of the macroscopic responses, rise rates, decay rates, and both peak and steady-state currents. In contrast, with an agonist pulse like that used in Xenopus oocyte experiments, microscopic desensitization rates have a profound impact on peak current amplitude and very little effect on the kinetics of the macroscopic responses.

Isomerization of the proline in the M2–M3 linker is not required for activation of the human 5‐HT3A receptor

Each subunit of the cation-selective members of the Cys-loop family of ligand-gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5-hydroxytryptamine type 3A (5-HT(3)A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans-cis isomerization of the protein backbone at this position is integral to agonist-induced channel opening [Nature (2005) vol. 438, pp. 248-252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5-HT(3)A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist-mediated currents, compromise channel gating by 5-HT or inhibition of 5-HT-induced currents by either picrotoxin or d-tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca(2+) concentration and a 10-fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5-HT(3)A receptor function but indicate that trans-cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.

Puberty, steroids and GABA A receptor plasticity

GABA(A) receptors (GABAR) mediate most inhibition in the CNS and are also a target for neuroactive steroids such as 3alpha,5[alpha]beta-THP (3alphaOH-5[alpha]beta-OH-pregnan-20-one or [allo]pregnanolone). Although these steroids robustly enhance current gated by alpha1beta2delta GABAR, we have shown that 3alpha,5[alpha]beta-THP effects at recombinant alpha4beta2delta GABAR depend on the direction of Cl(-) flux, where the steroid increases outward flux, but decreases inward flux through the receptor. This polarity-dependent inhibition of alpha4beta2delta GABAR resulted from an increase in the rate and extent of rapid desensitization of the receptor, recorded from recombinant receptors expressed in HEK-293 cells with whole cell voltage clamp techniques. This inhibitory effect of 3alpha,5[alpha]beta-THP was not observed at other receptor subtypes, suggesting it was selective for alpha4beta2delta GABAR. Furthermore, it was prevented by a selective mutation of basic residue arginine 353 in the intracellular loop of the receptor, suggesting that this might be a putative chloride modulatory site. Expression of alpha4betadelta GABAR increases markedly at extrasynaptic sites at the onset of puberty in female mice. At this time, 3alpha,5[alpha]beta-THP decreased the inhibitory tonic current, recorded with perforated patch techniques to maintain the physiological Cl(-) gradient. By decreasing this shunting inhibition, 3alpha,5[alpha]beta-THP increased the excitability of CA1 hippocampal pyramidal cells at puberty. These effects of the steroid were opposite to those observed before puberty when 3alpha,5[alpha]beta-THP reduced neuronal excitability as a pre-synaptic effect. Behaviorally, the excitatory effect of 3alpha,5[alpha]beta-THP was reflected as an increase in anxiety at the onset of puberty in female mice. Taken together, these findings suggest that the emergence of alpha4beta2delta GABAR at the onset of puberty reverses the effect of a stress steroid. These findings may be relevant for the mood swings and increased response to stressful events reported in adolescence.

Ethanol increases desensitization of recombinant GluR-D AMPA receptor and TARP combinations

Glutamate receptors are important target molecules of the acute effect of ethanol. We studied ethanol sensitivity of homomeric GluR-D receptors expressed in human embryonic kidney 293 cells and examined whether recently discovered transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory proteins (TARPs) affect ethanol sensitivity. Coexpression of the TARPs, stargazin, and γ4 increased the time constant (τ-value) of current decay in the presence of agonist, thus slowing the onset of desensitization and increasing the steady-state current. Ethanol produced less inhibition of the peak current than the steady-state current for all types of the GluR-D receptors. In addition, ethanol concentration-dependently accelerated the rate of desensitization, measured as the τ-value of fast decay of peak current. This effect was enhanced with coexpression of TARPs. The recovery from desensitization was slowed down by coexpression of γ4 but ethanol did not affect this process in any GluR-D combination. The results support the idea that increased desensitization is an important mechanism in the ethanol inhibition of AMPA receptors and indicate that coexpression of TARPs can alter this effect of ethanol.

Flip and Flop: A Molecular Determinant for AMPA Receptor Channel Opening

Alternative splicing in the extracellular ligand binding domain of the AMPA receptors generates two variants, i.e., flip and flop. The flop variant of the GluR2 AMPA receptor is known to desensitize faster than the flip counterpart, whereas the GluR1 flip and flop variants exhibit the same rate of desensitization. However, whether the alternative splicing affects the channel opening kinetic properties of these receptors is unknown. Using a laser-pulse photolysis technique, we have characterized the channel opening kinetic mechanism for the flip and flop channels of GluR1 and GluR2, respectively. We find that the flop variant of GluR2 opens the channel, following the binding of glutamate, with the same rate as the flip channel, but closes its channel more rapidly. The difference in the kinetic properties between the two receptor isoforms can be described by a model we proposed previously in which the channel closing rate, a measure of the stability of the open channel state, controls an apparent tendency of the channel to desensitize, most likely, through the closed channel state. Specifically, the flop sequence of GluR2 promotes the channel to close more rapidly and consequently to desensitize with a faster rate than the flip sequence. For GluR1, the alternative splicing does not seem to affect the channel opening kinetics, since the flip and flop variants of GluR1 have the same channel opening rate, and the same channel closing rate. As expected and indeed observed, the flop variant desensitizes with the same rate as the flip variant does. On the basis of these results, we hypothesize that the flip/flop sequence cassette of AMPA receptors, in a sequence-dependent manner, regulates the rate of the channel closing process, in the microsecond time domain, through which it further regulates the channel desensitization in the millisecond time region.

Stability of ligand-binding domain dimer assembly controls kainate receptor desensitization

AMPA and kainate receptors mediate fast synaptic transmission. AMPA receptor ligand-binding domains form dimers, which are key functional units controlling ion-channel activation and desensitization. Dimer stability is inversely related to the rate and extent of desensitization. Kainate and AMPA receptors share common structural elements, but functional measurements suggest that subunit assembly and gating differs between these subtypes. To investigate this, we constructed a library of GluR6 kainate receptor mutants and directly measured changes in kainate receptor dimer stability by analytical ultracentrifugation, which, combined with electrophysiological experiments, revealed an inverse correlation between dimer stability and the rate of desensitization. We solved crystal structures for a series of five GluR6 mutants, to understand the molecular mechanisms for dimer stabilization. We demonstrate that the desensitized state of kainate receptors acts as a deep energy well offsetting the stabilizing effects of dimer interface mutants, and that the deactivation of kainate receptor responses is dominated by entry into desensitized states. Our results show how neurotransmitter receptors with similar structures and gating mechanisms can exhibit strikingly different functional properties.

Two Distinct Mechanisms Mediate Acute μ-Opioid Receptor Desensitization in Native Neurons

Sustained stimulation of G-protein coupled receptors (GPCRs) leads to rapid loss of receptor function (acute desensitization). For many GPCRs including the mu-opioid receptor (MOR), an accepted mechanism for acute desensitization is through G-protein coupled receptor kinase (GRKs) mediated phosphorylation of the receptor, which facilitates the binding of beta-arrestins (betaarrs) to the receptor and then promotes endocytosis. However, the mechanism(s) that mediate acute desensitization have not yet been well defined in native neurons. This study used whole-cell patch clamp recording of G-protein coupled inward-rectifying potassium (GIRK) currents to assay MOR function and identify mechanisms of acute MOR desensitization in locus ceruleus (LC) neurons. The rate and extent of MOR desensitization were unaffected by beta(arr)-2 knock-out. Disruption of GRK2 function via inhibitory peptide introduced directly into neurons also failed to affect desensitization in wild type or beta(arr)-2 knock-outs. Inhibition of ERK1/2 activation alone had little effect on acute desensitization. However, when both GRK2-beta(arr)-2 and ERK1/2 functions were disrupted simultaneously, desensitization of MOR was nearly abolished. Together, these results suggest that acute desensitization of MOR in native LC neurons is determined by at least two molecular pathways, one involving GRK2 and beta(arr)2, and a parallel pathway mediated by activated ERK1/2.

Triton x-100 inhibits agonist -induced currents and suppresses benzodiazepine modulation of GABA A receptors in Xenopus oocytes

Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na + and L-type Ca 2+ channels and GABA A receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA A receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA A α 1β 3γ 2S receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 μM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA A receptor currents and suppressed allosteric modulation by flunitrazepam at α 1β 3γ 2S receptors. All effects were independent of the presence of a γ 2S subunit in the GABA A receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA A receptor through changes in lipid bilayer elasticity.

Mechanisms of Short-Term Desensitization of GIRK Channel Activity

G protein-coupled receptors (GPCR) signaling is precisely timed. This can be achieved at the level of the G protein activation and/or at the receptor level. At the receptor level, classical desensitization is believed to be controlled by a decrease in receptors number in the membrane: Activated receptors are phosphorylated by GPCR-kinase (GRK), tagging them for arrestin binding. This in-turn leads to clathrin-mediated receptor endocytosis. This multisteps process is extended in time (min), and is dependent on initial GPCR phosphorylation by GRK. In contrast, effector regulation is controlled at much faster time scale (s). Here we used the G protein-coupled inwardly rectifying K+ channels (GIRK), as an effector model for GPCR activation, to achieve real-time readout, in intact cells, of GPCR-mediated effector regulation. Upon GIRK activation, currents were desensitized within seconds. This regulation may serve as fast negative feedback for GPCR activation. Here we show that GIRK desensitization rates are dependent on GRK2 activity. GRK2 accelerates desensitization of currents induced by some Gαi/o coupled receptors, but not all. In contrast, silencing endogenous GRK2, results in a decreased in the desensitization rates. A dominant-negative mutant lacking kinase activity did not affect the ability of GRK2 to accelerate currents desensitization, suggesting the lack of GPCR classical desensitization in this process. In contrast, GRK2 mutant displaying limited Gβγ binding affinity, failed to accelerate current desensitization.Measuring simultaneously membrane fluorescence signals from cells expressing GFP-GRK2 and GIRK activity under TIRF microscopy and electrophysiological recordings, respectively, show that GFP-GRK2 membrane recruitment and GIRK desensitization occurs simultaneously.We suggest that GPCR activation induces GRK2 recruitment to the membrane, where it competes with GIRK for Gβγ, to result in short-term current desensitization. The results will be discussed in terms of a novel mechanism of selective regulation of effectors by GPCRs.

α-7 Nicotinic Receptor Positive Allosteric Modulators have Varying Kinetic Effects on Desensitization and Current Amplitude

The α-7 nicotinic receptor is an important neuronal subtype of ligand gated ion channels. It forms a pentameric homomer that is activated by acetylcholine or nicotine to evoke rapidly activating and desensitizing currents. Activation of α-7 receptors has been implicated as a therapeutic strategy in schizophrenia and Alzheimer disease. Small molecule positive allosteric modulators (PAM's) have been shown to enhance α-7 currents, and are classified as type 1 or type 2 PAMs. The type 1 PAMs enhance the current amplitude but do not alter desensitization, while the type 2 PAMs enhance the current amplitude but also slow the desensitization of the receptor. In order to understand the mechanism of action of the type 1 and type 2 PAM's, we tested three modulators on the QPatch automated patch-clamp system using GH4C1 cells stably expressing the rat α-7 receptor. We also explored the effect of repeated applications of these agents on their modulatory activity. Consistent with previously published data, PNU-120596 showed type 2 PAM activity accompanied with a decreasing magnitude of potentiation with repeated applications. Estimated EC50 values for PNU-120596 were stable with repeated compound application. NS-1738 produced a type 1 PAM activity, and a cumulatively increased potentiation following repeated applications, accompanied by an approximate 3-fold increase in EC50. In contrast, SB-206553 had similar potency and effects on the potentiation with repeated application. These results indicate that the different α-7 receptor PAMs have different rates of activation and desensitization, in addition to their type 1 or 2 effects on receptor desensitization.

The effect of the neuroactive steroid 5β-pregnane-3β, 20(R)-diol on the time course of GABA evoked currents is different to that of pregnenolone sulphate

The endogenous progesterone metabolite allopregnanolone has a number of properties including anesthetic, sedative, antiepileptic, anxiolytic, impaired memory function and negative mood symptoms. Allopregnanolone is a potent positive GABA A receptor function modulators. In contrast, 3β-hydroxy-steroids (3β-steroids) usually modulate the GABA A receptor negatively. They have attracted some interest for their possible use as therapeutic agents that could counteract the negative symptoms induced by allopregnanolone. Two hypotheses for the action of 3β-steroids have been proposed: 1) 3β-steroids act in a similar way to pregnenolone sulphate, which non-competitively reduces GABA A receptor activity. 2) 3β-steroids specifically antagonize the effect of allopregnanolone. We have therefore tried to clarify this issue by comparing the effect of pregnenolone sulphate and 5β-pregnane-3β, 20(R)-diol on the GABA-evoked currents by the patch clamp technique on neurons from the medial preoptic nucleus. Both pregnenolone sulphate and 5β-pregnane-3β, 20(R)-diol increase the desensitization rate of the current response evoked by a 2 s GABA application. However, their effects on other parameters of the GABA evoked currents differed in degree and sometimes even in direction. The actions of pregnenolone sulphate and 5β-pregnane-3β, 20(R)-diol were not altered in the presence of allopregnanolone, which indicates that they do not directly interact with allopregnanolone. In addition, when 5β-pregnane-3β, 20(R)-diol was tested on spontaneous inhibitory postsynaptic currents (sIPSCs), it dramatically reduced the allopregnanolone-induced prolongation of the decay time constant but it had no effect on the decay under control conditions. In conclusion, the effect of 5β-pregnane-3β, 20(R)-diol on GABA-evoked currents is different to that of pregnenolone sulphate in medial preoptic nucleus neurons.

The Cytoskeletal Protein α-Actinin Regulates Acid-sensing Ion Channel 1a through a C-terminal Interaction

The acid-sensing ion channel 1a (ASIC1a) is widely expressed in central and peripheral neurons where it generates transient cation currents when extracellular pH falls. ASIC1a confers pH-dependent modulation on postsynaptic dendritic spines and has critical effects in neurological diseases associated with a reduced pH. However, knowledge of the proteins that interact with ASIC1a and influence its function is limited. Here, we show that α-actinin, which links membrane proteins to the actin cytoskeleton, associates with ASIC1a in brain and in cultured cells. The interaction depended on an α-actinin-binding site in the ASIC1a C terminus that was specific for ASIC1a versus other ASICs and for α-actinin-1 and -4. Co-expressing α-actinin-4 altered ASIC1a current density, pH sensitivity, desensitization rate, and recovery from desensitization. Moreover, reducing α-actinin expression altered acid-activated currents in hippocampal neurons. These findings suggest that α-actinins may link ASIC1a to a macromolecular complex in the postsynaptic membrane where it regulates ASIC1a activity.

Modeling the Role of Homologous Receptor Desensitization in Cell Gradient Sensing

G-protein-coupled chemoattractant receptors signal transiently upon ligand binding to effect cell orientation and motility but then are rapidly desensitized. The importance of desensitization has been unclear, because mutated nondesensitizable receptors mediate efficient chemotaxis. We hypothesized that homologous receptor desensitization is required for cellular navigation in fields of competing attractants. Modeling of receptor-mediated orientation shows that desensitization allows integration of attractant signals. Cells expressing normal receptors are predicted to 1) orient preferentially to distant gradients; 2) seek an intermediate position between balanced agonist sources; 3) and can be repositioned between chemoattractant-defined microenvironmental domains by modest changes in receptor number. In contrast, in the absence of desensitization, orientation is dominated by local agonist sources, precluding continued navigation. Furthermore, cell orientation in competing ligand gradients depends on the relative kinetic rates of receptor desensitization and recycling. We propose that homologous receptor desensitization is critical for cellular navigation in complex chemoattractant fields.

The Membrane Lateral Pressure-Perturbing Capacity of Parabens and Their Effects on the Mechanosensitive Channel Directly Correlate with Hydrophobicity

Lipid bilayers provide a natural anisotropic environment for membrane proteins and can serve as apolar reservoirs for lipid-derived second messengers or lipophilic drugs. Partitioning of lipophilic agents changes the lateral pressure distribution in the bilayer, affecting integral proteins. p-Hydroxybenzoic acid esters (parabens) are amphipathic compounds widely used as food and cosmetics preservatives, but the mechanisms of their broad antibacterial action are unknown. Here we describe effects of ethyl, propyl, and butyl parabens on the gating of the bacterial mechanosensitive channel of small conductance (MscS) and compare them with the surface activity and lateral pressure changes measured in lipid monolayers in the presence of these substances. Near the bilayer-monolayer equivalence pressure of 35 mN/m, ethyl, propyl, or butyl paraben present in the subphase at 1 mM increased the surface pressure of the monolayer by 5, 12.5, or 20%, respectively. No spontaneous activation of MscS channels was observed in patch-clamp experiments with parabens added from either the cytoplasmic or periplasmic side. Increasing concentrations of parabens on the cytoplasmic side of excised patches shifted activation curves of MscS toward higher tensions. A good correlation between the pressure increases in monolayers and shifts in activation midpoints in patch-clamp experiments suggested that the more hydrophobic parabens partition more strongly into the lipid and exert larger effects on channel gating through changes in lateral pressure. We show that cytoplasmically presented ethyl or butyl parabens both hasten the process of desensitization of MscS and influence inactivation differently. The higher rate of desensitization is likely due to increased lateral pressure in the cytoplasmic leaflet surrounding the gate. Neither of the parabens strongly affects the rate of recovery and does not seem to penetrate the TM2-TM3 interhelical clefts in MscS. We conclude that the bacterial mechanosensitive channel MscS provides a sensitive readout of lateral membrane pressure exerted by amphipathic molecules but may not be the primary target for the parabens in their antimicrobial activity.

AMPA Receptor Subunit-Specific Regulation by a Distinct Family of Type II TARPs

AMPA-type glutamate receptors (GluRs) play major roles in excitatory synaptic transmission. Neuronal AMPA receptors comprise GluR subunits and transmembrane AMPA receptor regulatory proteins (TARPs). Previous studies identified five mammalian TARPs, gamma-2 (or stargazin), gamma-3, gamma-4, gamma-7, and gamma-8, that enhance AMPA receptor function. Here, we classify gamma-5 as a distinct class of TARP that modulates specific GluR2-containing AMPA receptors and displays properties entirely dissimilar from canonical TARPs. Gamma-5 increases peak currents and decreases the steady-state currents selectively from GluR2-containing AMPA receptors. Furthermore, gamma-5 increases rates of GluR2 deactivation and desensitization and decreases glutamate potency. Remarkably, all effects of gamma-5 require editing of GluR2 mRNA. Unlike other TARPs, gamma-5 modulates GluR2 without promoting receptor trafficking. We also find that gamma-7 regulation of GluR2 is dictated by mRNA editing. These data establish gamma-5 and gamma-7 as a separate family of "type II TARPs" that impart distinct physiological features to specific AMPA receptors.

Evolutionary Conserved Role for TARPs in the Gating of Glutamate Receptors and Tuning of Synaptic Function

Neurotransmission in the brain is critically dependent on excitatory synaptic signaling mediated by AMPA-class ionotropic glutamate receptors (AMPARs). AMPARs are known to be associated with Transmembrane AMPA receptor Regulatory Proteins (TARPs). In vertebrates, at least four TARPs appear to have redundant roles as obligate chaperones for AMPARs, thus greatly complicating analysis of TARP participation in synaptic function. We have overcome this limitation by identifying and mutating the essential set of TARPs in C. elegans (STG-1 and STG-2). In TARP mutants, AMPAR-mediated currents and worm behaviors are selectively disrupted despite apparently normal surface expression and clustering of the receptors. Reconstitution experiments indicate that both STG-1 and STG-2 can functionally substitute for vertebrate TARPs to modify receptor function. Thus, we show that TARPs are obligate auxiliary subunits for AMPARs with a primary, evolutionarily conserved functional role in the modification of current kinetics.

The Interface between Extracellular and Transmembrane Domains of Homomeric Cys-Loop Receptors Governs Open-Channel Lifetime and Rate of Desensitization

The lifetimes of activated postsynaptic receptor channels contribute to the efficiency of synaptic transmission. Here we show that structural differences within the interface dividing extracellular and transmembrane domains of homomeric alpha7 and 5-HT(3A) receptors account for the large differences in open-channel lifetime and time of desensitization onset between these contrasting members of the Cys-loop receptor superfamily. For alpha7 receptors, agonist-evoked single-channel currents appear mainly as isolated brief openings (tau(o) = 0.35 ms), whereas macroscopic currents after a step pulse of agonist desensitize rapidly (tau(d) = 0.4 ms). In contrast for 5-HT(3A) receptors, agonist-evoked single-channel currents appear as clusters of many long openings in quick succession (tau(cluster) = 1.2 s), whereas macroscopic currents desensitize slowly (tau(d) = 1.1 s). A chimeric alpha7-5HT(3A) receptor exhibits functional properties intermediate between those of the parent receptors, but the functional signatures of each parent are reconstituted after substituting the major loops within the interface of the extracellular and transmembrane domains from the corresponding parent receptor. Furthermore, these structural loops contribute to open-channel lifetime and time of desensitization onset in a nonadditive manner. The results suggest that desensitization is the major determinant of the lifetimes of activated alpha7 and 5-HT(3A) receptors and that functional differences between the two receptors arise primarily through structural differences at the interface between extracellular and transmembrane domains.