Papers by Stuart Cull-candy
Desensitization is a canonical property of ligand-gated ion channels, causing progressive current... more Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors, which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we report the surprising finding that homomeric Q/R edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits ( -2, -8 or GSG1L), generates large steady-state currents and anomalous current-variance relationships. Using fluctuation analysis, single-channel recording, and kinetic modeling we demonstrate that the steady-state current is mediated predominantly by 'conducting desensitized' receptors. When combined with crystallography this unique functional readout of a hitherto silent state enabled us to examine cross-linked cysteine mutants to probe the conformation of the desensitized ligand binding domain of functioning AMPAR complexes within the plasma membrane.
Nature Neuroscience, Oct 9, 2011
Oligodendrocyte precursor cells (OPCs), a major glial cell type giving rise to myelinating oligod... more Oligodendrocyte precursor cells (OPCs), a major glial cell type giving rise to myelinating oligodendrocytes in the CNS, express calcium-permeable (CP-) AMPARs. Although CP-AMPARs are important in OPC proliferation and neuron-glia signalling, they render OPCs susceptible to ischemic damage in early development. Here we identify factors controlling dynamic regulation of AMPAR subtypes in OPCs from rat optic nerve and mouse cerebellar cortex. We find that activation of group 1 mGluRs drives an increase in the proportion of CP-AMPARs, reflected in increased single-channel conductance and inward rectification. This plasticity requires elevation of intracellular calcium, utilizes PI3 kinase, PICK-1 and the JNK pathway. In white matter, neurons and astrocytes release both ATP and glutamate. Surprisingly, activation of purinergic receptors in OPCs decreases CP-AMPAR expression, suggesting a capacity for homeostatic regulation. Finally, we show that stargazin-related transmembrane AMPAR regulatory proteins, which are key for AMPAR surface expression in neurons, regulate CP-AMPAR plasticity in OPCs.
eLife
Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic ... more Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent channel block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits – including transmembrane AMPAR regulatory proteins, cornichons, and GSG1L – which are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here, we show that the inclusion of the spider toxin analog 1-naphthylacetyl spermine (NASPM) in the intracellular solution results in a complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spe...
Molecular Pharmacology, 2022
AMPA-type gultamate receptors (AMPARs) mediate excitatory signaling in the brain and are therapeu... more AMPA-type gultamate receptors (AMPARs) mediate excitatory signaling in the brain and are therapeutic targets for the treatment of diverse neurological disorders. The receptors interact with a variety of auxiliary subunits, including the transmembrane AMPAR regulatory proteins (TARPs). The TARPs influence AMPAR biosynthesis and trafficking and enhance receptor responses by slowing desensitization and deactivation and increasing single-channel conductance. TARP c8 has an expression pattern that is distinct from that of other TARPs, being enriched in the hippocampus. Recently, several compounds have been identified that selectivity inhibit c8containing AMPARs. One such inhibitor, JNJ-55511118, has shown considerable promise for the treatment of epilepsy. However, key details of its mechanism of action are still lacking. Here, using patch-clamp electrophysiological recording from heterologously expressed AMPARs, we show that JNJ-55511118 inhibits peak currents of c8-containing AMPARs by decreasing their singlechannel conductance. The drug also modifies hallmark features of AMPAR pharmacology, including the TARP-dependent actions of intracellular polyamines and the partial agonist kainate. Moreover, we find that JNJ-55511118 reduces the influence of c8 on all biophysical measures, aside from its effect on the recovery from desensitization. The drug is also effective when applied intracellularly, suggesting it may access its binding site from within the membrane. Additionally, we find that AMPARs incorporating TARP c2 mutated to contain the JNJ-55511118 binding site, exhibit greater block than seen with AMPARs containing c8, potentially reflecting differences in TARP stoichiometry. Taken together, our data provide new insight into the mechanism by which c8-selective drugs inhibit AMPARs. Although modulation of AMPA-type glutamate receptors shows promise for the treatment various neurological conditions, the absence of subtype-selective drugs has hindered adoption of this therapeutic strategy. We made patch-clamp recordings to characterize the actions of the g8-selective AMPAR inhibitor JNJ-55511118 on GluA2(Q) receptors expressed in HEK cells. We report that JNJ-55511118 inhibits AMPAR-mediated currents by reducing single-channel conductance, providing clear insight into the mechanism of action of this important class of AMPAR modulators.
The Journal of Physiology, 2021
AMPA receptors are tetrameric glutamate‐gated ion channels that mediate a majority of fast excita... more AMPA receptors are tetrameric glutamate‐gated ion channels that mediate a majority of fast excitatory neurotransmission in the brain. They exist as calcium‐impermeable (CI‐) and calcium‐permeable (CP‐) subtypes, the latter of which lacks the GluA2 subunit. CP‐AMPARs display an array of distinctive biophysical and pharmacological properties that allow them to be functionally identified. This has revealed that they play crucial roles in diverse forms of central synaptic plasticity. Here we summarise the functional hallmarks of CP‐AMPARs and describe how these are modified by the presence of auxiliary subunits that have emerged as pivotal regulators of AMPARs. A lasting change in the prevalence of GluA2‐containing AMPARs, and hence in the fraction of CP‐AMPARs, is a feature in many maladaptive forms of synaptic plasticity and neurological disorders. These include modifications of glutamatergic transmission induced by inflammatory pain, fear conditioning, cocaine exposure, and anoxia‐in...
Neuropharmacology, 2021
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic ... more Calcium-permeable AMPA-type glutamate receptors (CP-AMPARs) contribute to many forms of synaptic plasticity and pathology. They can be distinguished from GluA2-containing calcium-impermeable AMPARs by the inward rectification of their currents, which reflects voltage-dependent block by intracellular spermine. However, the efficacy of this weakly permeant blocker is differentially altered by the presence of AMPAR auxiliary subunits – including transmembrane AMPAR regulatory proteins, cornichons and GSG1L – that are widely expressed in neurons and glia. This complicates the interpretation of rectification as a measure of CP-AMPAR expression. Here we show that inclusion of the spider toxin analogue 1-naphthylacetyl spermine (NASPM) in the intracellular recording solution results in complete block of GluA1-mediated outward currents irrespective of the type of associated auxiliary subunit. In neurons from GluA2-knockout mice expressing only CP-AMPARs, intracellular NASPM, unlike spermine...
Desensitization is a canonical property of ligand-gated ion channels, causing progressive current... more Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors, which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we report the surprising finding that homomeric Q/R edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits (γ-2, γ-8 or GSG1L), generates large steady-state currents and anomalous current-variance relationships. Using fluctuation analysis, single-channel recording, and kinetic modeling we demonstrate that the steady-state current is mediated predominantly by ‘conducting desensitized’ receptors. When combined with crystallography this unique functional readout of a hith-erto silent state enabled us to examine cross-linked cysteine muta...
Nature Communications, 2019
Desensitization is a canonical property of ligand-gated ion channels, causing progressive current... more Desensitization is a canonical property of ligand-gated ion channels, causing progressive current decline in the continued presence of agonist. AMPA-type glutamate receptors (AMPARs), which mediate fast excitatory signaling throughout the brain, exhibit profound desensitization. Recent cryo-EM studies of AMPAR assemblies show their ion channels to be closed in the desensitized state. Here we present evidence that homomeric Q/R-edited AMPARs still allow ions to flow when the receptors are desensitized. GluA2(R) expressed alone, or with auxiliary subunits (γ-2, γ-8 or GSG1L), generates large fractional steady-state currents and anomalous current-variance relationships. Our results from fluctuation analysis, single-channel recording, and kinetic modeling, suggest that the steady-state current is mediated predominantly by conducting desensitized receptors. When combined with crystallography this unique functional readout of a hitherto silent state enabled us to examine cross-linked cyst...
eneuro, 2018
Juvenile Batten disease is the most common progressive neurodegenerative disorder of childhood. I... more Juvenile Batten disease is the most common progressive neurodegenerative disorder of childhood. It is associated with mutations in the CLN3 gene, causing loss of function of CLN3 protein and degeneration of cerebellar and retinal neurons. It has been proposed that changes in granule cell AMPA-type glutamate receptors (AMPARs) contribute to the cerebellar dysfunction. In this study, we compared AMPAR properties and synaptic transmission in cerebellar granule cells from wild-type and Cln3 knock-out mice. In Cln3 ⌬ex1-6 cells, the amplitude of AMPA-evoked whole-cell currents was unchanged. Similarly, we found no change in the amplitude, kinetics, or rectification of synaptic currents evoked by individual quanta, or in their underlying single-channel conductance. We found no change in cerebellar expression of GluA2 or GluA4 protein. By contrast, we observed a reduced number of quantal events following mossy-fiber stimulation in Sr 2ϩ , altered short-term plasticity in conditions of reduced extracellular Ca 2ϩ , and reduced mossy fiber vesicle number. Thus, while our results suggest early presynaptic changes in the Cln3 ⌬ex1-6 mouse model of juvenile Batten disease, they reveal no evidence for altered postsynaptic AMPARs.
The Journal of Neuroscience, 2017
In the brain, transmembrane AMPAR regulatory proteins (TARPs) critically influence the distributi... more In the brain, transmembrane AMPAR regulatory proteins (TARPs) critically influence the distribution, gating, and pharmacology of AMPARs, but the contribution of these auxiliary subunits to AMPAR-mediated signaling in the spinal cord remains unclear. We found that the Type I TARP ␥-2 (stargazin) is present in lamina II of the superficial dorsal horn, an area involved in nociception. Consistent with the notion that ␥-2 is associated with surface AMPARs, CNQX, a partial agonist at AMPARs associated with Type I TARPs, evoked whole-cell currents in lamina II neurons, but such currents were severely attenuated in ␥-2-lacking stargazer (stg/stg) mice. Examination of EPSCs revealed the targeting of ␥-2 to be synapse-specific; the amplitude of spontaneously occurring miniature EPSCs (mEPSCs) was reduced in neurons from stg/stg mice, but the amplitude of capsaicin-induced mEPSCs from C-fiber synapses was unaltered. This suggests that ␥-2 is associated with AMPARs at synapses in lamina II but excluded from those at C-fiber inputs, a view supported by our immunohistochemical colabeling data. Following induction of peripheral inflammation, a model of hyperalgesia, there was a switch in the current-voltage relationships of capsaicin-induced mEPSCs, from linear to inwardly rectifying, indicating an increased prevalence of calcium-permeable (CP) AMPARs. This effect was abolished in stg/stg mice. Our results establish that, although ␥-2 is not typically associated with calcium-impermeable AMPARs at C-fiber synapses, it is required for the translocation of CP-AMPARs to these synapses following peripheral inflammation.
Neuron, 2017
AMPA receptors (AMPARs) are tetrameric ion channels that together with other ionotropic glutamate... more AMPA receptors (AMPARs) are tetrameric ion channels that together with other ionotropic glutamate receptors (iGluRs), the NMDA-and kainate receptors, mediate a majority of excitatory neurotransmission in the central nervous system. Whereas NMDA receptors gate channels with slow kinetics, responsible primarily for generating long-term synaptic potentiation and depression, AMPARs are the main fast transduction elements at synapses and are critical for the expression of plasticity. The kinetic and conductance properties of AMPARs are laid down during their biogenesis, and are regulated by post-transcriptional RNA editing, splice variation, post-translational modification and subunit composition. Furthermore, AMPAR assembly, trafficking and functional heterogeneity depends on a large repertoire of auxiliary subunits -a feature that is particularly striking for this type of iGluR. Here, we discuss how the subunit structure, stoichiometry and auxiliary subunits generate a heterogeneous plethora of receptors, each tailored to fulfill a vital role in fast synaptic signaling and plasticity.
Cell reports, 2017
Fast excitatory transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPA... more Fast excitatory transmission in the CNS is mediated mainly by AMPA-type glutamate receptors (AMPARs) associated with transmembrane AMPAR regulatory proteins (TARPs). At the high glutamate concentrations typically seen during synaptic transmission, TARPs slow receptor desensitization and enhance mean channel conductance. However, their influence on channels gated by low glutamate concentrations, as encountered during delayed transmitter clearance or synaptic spillover, is poorly understood. We report here that TARP γ-2 reduces the ability of low glutamate concentrations to cause AMPAR desensitization and enhances channel gating at low glutamate occupancy. Simulations show that, by shifting the balance between AMPAR activation and desensitization, TARPs can markedly facilitate the transduction of spillover-mediated synaptic signaling. Furthermore, the dual effects of TARPs can account for biphasic steady-state glutamate concentration-response curves-a phenomenon termed "autoinact...
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 9, 2015
AMPA-type glutamate receptors are ligand-gated cation channels responsible for a majority of the ... more AMPA-type glutamate receptors are ligand-gated cation channels responsible for a majority of the fast excitatory synaptic transmission in the brain. Their behavior and calcium permeability depends critically on their subunit composition and the identity of associated auxiliary proteins. Calcium-permeable AMPA receptors (CP-AMPARs) contribute to various forms of synaptic plasticity, and their dysfunction underlies a number of serious neurological conditions. For CP-AMPARs, the prototypical transmembrane AMPAR regulatory protein stargazin, which acts as an auxiliary subunit, enhances receptor function by increasing single-channel conductance, slowing channel gating, increasing calcium permeability, and relieving the voltage-dependent block by endogenous intracellular polyamines. We find that, in contrast, GSG1L, a transmembrane auxiliary protein identified recently as being part of the AMPAR proteome, acts to reduce the weighted mean single-channel conductance and calcium permeability...
The Journal of Physiology, 2015
The hippocampal CA1 region is highly vulnerable to ischaemic stroke. Two forms of AMPA receptor (... more The hippocampal CA1 region is highly vulnerable to ischaemic stroke. Two forms of AMPA receptor (AMPAR) plasticity -an anoxic form of long-term potentiation and a delayed increase in Ca 2+ -permeable (CP) AMPARs -contribute to this susceptibility by increasing excitotoxicity. r In CA1, the acid-sensing ion channel 1a (ASIC1a) is known to facilitate LTP and contribute to ischaemic acidotoxicity. r We have examined the role of ASIC1a in AMPAR ischaemic plasticity in organotypic hippocampal slice cultures exposed to oxygen glucose deprivation (a model of ischaemic stroke), and in hippocampal pyramidal neuron cultures exposed to acidosis. r We find that ASIC1a activation promotes both forms of AMPAR plasticity and that neuro- protection, by inhibiting ASIC1a, circumvents any further benefit of blocking CP-AMPARs. r Our observations establish a new interaction between acidotoxicity and excitotoxicity, and provide insight into the role of ASIC1a and CP-AMPARs in neurodegeneration. Specifically, we propose that ASIC1a activation drives certain post-ischaemic forms of CP-AMPAR plasticity.
F1000 - Post-publication peer review of the biomedical literature, 2011
Reduction in synaptic transmission and plasticity in mice lacking the hippocampus-enriched AMPAR ... more Reduction in synaptic transmission and plasticity in mice lacking the hippocampus-enriched AMPAR auxiliary subunit, TARP©-8, could be due to reduction in AMPAR expression or a direct role of ©-8. Here, we generated TARP©-8Δ4 knock-in mice lacking the C-terminal PDZ ligand. We found that synaptic transmission and AMPAR were reduced without changes in extrasynaptic AMPAR expression, but LTP was unaltered. Our findings indicate distinct TARP-dependent mechanisms for synaptic transmission and LTP. TARPs are auxiliary subunits of AMPARs that modulate expression, channel properties and localization of AMPARs in the brain 1 . Genetic disruption of TARPs causes reduction of AMPARs 2-4 , and in the ©-8 knockout (γ-8 -/-, also known as CACNG8 -/-) mouse, cellsurface AMPAR function and NMDA receptor-mediated LTP are severely impaired 3 . Interestingly, these phenotypes were also observed in mice lacking the AMPAR subunit, GluA1 5 . Indeed, expression of GluA1 and GluA2/3 in hippocampus of γ-8 -/-mice is reduced to 20-30% of wild type levels . Therefore, it remains unclear whether the impaired synaptic transmission and plasticity observed in γ-8 -/-mice are caused directly by loss of ©-8 or indirectly by reduction in AMPAR expression. Furthermore, although the TARP/ AMPAR complex is proposed to localize at synapses by interacting with PSD-95 through the C-terminal PDZ ligand 6,7 , overexpression of ©-8 lacking the PDZ ligand (⊗4) increases Users may view, print, copy, download and text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
The Journal of Physiology, 2010
Recent evidence suggests that lithium, which is used in the treatment of bipolar disorders, may a... more Recent evidence suggests that lithium, which is used in the treatment of bipolar disorders, may act by influencing AMPAR properties at central glutamatergic synapses. While it is clear that lithium potentiates recombinant AMPAR responses in a subunit specific way, the origin of this potentiation is not known. We examined the effects of lithium on native AMPAR channels in CA1 pyramidal cells in hippocampal slices where AMPARs are expected to be associated with auxiliary subunits. We found that lithium produced a selective increase in single-channel open probability (P open ), with little effect on single-channel conductance or burst length. From the present and previous finding it is likely that lithium causes a reduction in the time to recovery from desensitization, resulting in the observed increase in P open . This would be consistent with the view that lithium acts like certain other allosteric AMPAR modulators to reduce the time spent in the desensitized state, but differs from those that act by slowing dissociation of glutamate.
The Journal of Physiology, 2006
We have determined the functional properties of single AMPA receptor (AMPAR) and kainate receptor... more We have determined the functional properties of single AMPA receptor (AMPAR) and kainate receptor channels present in CA1 cells in hippocampal slices, to shed light on the relationship between single‐channel behaviour and synaptic currents in these cells. To derive basic properties of AMPA and kainate channels activated by their excitatory transmitter, we examined outside‐out patches exposed to glutamate. The kainate agonist SYM 2081, was used to confirm the presence of kainate receptors. Channels activated by glutamate or SYM 2081 exhibited conductance levels of 2–20 pS. Properties of single channels depended on the glutamate or AMPA concentration used. We observed a marked increase in mean channel conductance (γ) from γ= 6.9, to 11.2 pS, when glutamate was increased from 10 μm to 10 mm. The kinetic behaviour of AMPAR channels was also influenced by agonist concentration, with an increase in ‘bursty’ events at higher concentrations. In contrast, kainate channels were characterized ...
The Journal of Physiology, 1998
EPSCs were recorded under whole‐cell voltage clamp at room temperature from Purkinje cells in sli... more EPSCs were recorded under whole‐cell voltage clamp at room temperature from Purkinje cells in slices of cerebellum from 12‐ to 14‐day‐old rats. EPSCs from individual climbing fibre (CF) inputs were identified on the basis of their large size, paired‐pulse depression and all‐or‐none appearance in response to a graded stimulus. Synaptic transmission was investigated over a wide range of experimentally imposed release probabilities by analysing fluctuations in the peak of the EPSC. Release probability was manipulated by altering the extracellular [Ca2+] and [Mg2+]. Quantal parameters were estimated from plots of coefficient of variation (CV) or variance against mean conductance by fitting a multinomial model that incorporated both spatial variation in quantal size and non‐uniform release probability. This ‘multiple‐probability fluctuation’ (MPF) analysis gave an estimate of 510 ± 50 for the number of functional release sites (N) and a quantal size (q) of 0.5 ± 0.03 nS (n= 6). Control e...
The Journal of Physiology, 2012
• Signalling of information in the nervous system relies on the activation of specific neurotrans... more • Signalling of information in the nervous system relies on the activation of specific neurotransmitter receptors. • Here we characterise some of the properties of GluA1 AMPA receptors, whose ion-permeable channel is opened by the neurotransmitter glutamate. • We found that the individual single-channel openings exhibit several discrete conductance levels that persist in the presence of saturating glutamate concentrations, and that the presence of modulatory accessory subunits differentially influences the durations of these channel openings. • Our data also indicate that there are at least two kinetically distinguishable stable open states for each conductance level. • These observations place constraints on models of GluA1 function that can be used to relate receptor properties to synaptic function.
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Papers by Stuart Cull-candy