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AMPA receptors: mechanisms of auxiliary protein action
Accepted Manuscript Title: AMPA receptors: mechanisms of auxiliary protein action Author: Clarissa Eibl Andrew J.R. Plested PII: DOI: Reference:
S2468-8673(17)30032-9 https://doi.org/doi:10.1016/j.cophys.2017.12.009 COPHYS 32
To appear in: Received date: Revised date: Accepted date:
14-10-2017 13-12-2017 21-12-2017
Please cite this article as: Eibl, C., Plested, A.J.R., AMPA receptors: mechanisms of auxiliary protein action, Current Opinion in Physiology (2017), https://doi.org/10.1016/j.cophys.2017.12.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
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AMPA receptors: mechanisms of auxiliary protein action
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Clarissa Eibl and Andrew J.R. Plested
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4 Institute of Biology, Cellular Biophysics, Humboldt Universität zu Berlin, 10115
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Berlin;
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Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), 13125 Berlin;
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Cluster of Excellence NeuroCure, Charité Universitätsmedizin, 10117 Berlin,
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Germany.
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cr
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10
an
11 Abstract
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AMPA receptors are the prime mediators of fast excitatory transmission in the
14
brain. Auxiliary subunits directly interact with AMPA receptors, hustling them to
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synapses and fine-tuning their responses to glutamate in multiple ways. Recent
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structural and functional studies have clarified the scope of molecular
17
interactions underlying AMPAR co-assembly with and modulation by auxiliary
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proteins. Complementary physiological and pharmacological work has expanded
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our view of what auxiliary proteins are capable of. Here we review how these
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studies advance a more refined outline of mechanisms of auxiliary protein
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action, but still leave some tantalizing parts of the puzzle missing.
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22 Introduction
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AMPA-type glutamate receptors (AMPARs) are ligand-gated ion-channels that
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are abundantly expressed in the neurons and glia of vertebrates, where they
26
mediate the majority of fast excitatory transmission underlying basal and higher
27
brain functions. AMPARs respond to glutamate released from synaptic terminals
28
within hundreds of microseconds, allowing rapid signal propagation in the CNS.
29
The AMPA receptor family comprises four principal subunits (GluA1-A4) and
30
most receptors in the brain are hetero-tetramers that include GluA2. Extensive
31
modifications to receptor subunits occur both during biogenesis with post-
32
transcriptional RNA editing (including Q/R editing in GluA2) and splicing
33
(flip/flop variants). Further, a myriad of post-translational modifications
34
including glycosylation, ubiquitination and phosphorylation are reported (for a
35
thorough review see ref. 1). Although these events alter the amplitude, duration
36
and ionic character of postsynaptic currents, they cannot explain some aspects of
37
glutamate receptor biology in neurons, including slower kinetics of native
38
receptors. This gap was filled by the discovery of a menagerie of membrane-
39
embedded “auxiliary proteins” that associate early in receptor biogenesis.
40
Numerous proteins modify synaptic transmission and co-precipitate with AMPA
41
receptors, so consensus characteristics for auxiliary proteins were formulated to
42
focus the discussion. First, auxiliary proteins should bind selectively to mature
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49
and 2 Cornichon-homologs pass muster. With the arrival of the first structures of
50
complexes between AMPA receptors and auxiliary proteins (Fig. 1), the time is
51
ripe to highlight the scope and subtlety of action, and consider how decades of
52
structural and biophysical studies on GluA subunits must now be weighed in a
53
new context.
43 44 45 46 47
AMPARs. Second, auxiliary proteins modulate the functional properties of AMPARs and/or mediate surface trafficking and third, the necessity of auxiliary
proteins in vivo is expected2,3. Exciting candidates including CPT1c, FRRS1, SAC1 and SynDig 4–7 fall short of these somewhat arbitrary criteria. But a baker’s dozen of auxiliary proteins comprising 6 transmembrane AMPA receptor regulatory
proteins (TARPs), the germ cell-specific gene 1-like (GSG1L), 4 CKAMPs/Shisas
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2
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AMPA Receptor assemblies
56
The first clue to auxiliary protein structure came from the Claudin family of tight-
57
junction proteins 8,9,10 that are related to TARPs and GSG1L. Sequence alignments
58
suggested a common architecture of four transmembrane helices (TM1-4),
59
connected by one short intracellular loop and two extracellular segments (ECS1
60
and ECS2). Initially, the ECS1 of TARPs was considered to be unstructured,
61
perhaps reaching up to the receptor’s amino terminal domain (ATD)
62
TARP-γ2 structures showed that, as in Claudins, the ECS1 is folded into four
63
antiparallel β-sheets. A snorkel-like flexible loop (L1), absent in Claudins, is too
64
short to reach above the LBD layer (Figure 2). ECS2 consists of a flexible loop
65
(L2) and β5, which forms an antiparallel sheet with ECS1-β1 in a “hat” domain.
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But
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Cryo-EM structures of AMPA receptors in complex with TARP-γ2 and GSG1L
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subunits showed auxiliary proteins decorating the perimeter of the complex
69
14.
70
independently confirmed by an elegant biochemical and functional study
71
Extensive hydrophobic interactions between TARP TM3 and TM4 and the
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receptor’s M1 and M4 helices (Fig. 2) suggest a tight assembly. Other regions
73
may be important – both TARP-γ2
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immediately intracellular regions (not resolved in structures of complexes to
75
date) for complex formation (Fig. 3).
12–
15,16
and CKAMP44
17
15.
rely on interactions at
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Each auxiliary protein protomer interacts across two GluA subunits,
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different stoichiometries, either 4:4 or (probably) 2:4 19,20. To combat this, recent
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structure-function work has relied on tandem constructs or other measures to
84
isolate complexes. Further complexity is offered by the symmetry switch in the
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AMPAR complex, between the two-fold extracellular domains and the four-fold
86
membrane domain (and thus principal TARP association sites). This geometry
76 77 78 79 80
The tandem constructs used for the structural studies might have been expected to produce a 4:4 stoichiometry, but instead complexes with 1, 2 and 4 TARPs illustrate preferential assembly sites 12,13,18, with the lack of membrane allowing
spare TARPs to “hang” outside the complex. The apparent independence of
association chimes with previous work showing that TARPs could assemble with
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produces two distinct TARP association sites and therefore likely asymmetric
88
roles for TARPs to control receptor function (Fig. 2).
89 The TARPs (γ2, γ3, γ4, γ5, γ7 and γ8) have sequence identity of around 30-70%.
91
The remarkably similar structure and mode of co-assembly of GSG1L, despite
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being as dissimilar to TARPs as Claudins and the Calcium channel γ1 subunit,
93
suggests, for practical purposes, that GSG1L is a TARP. The biggest differences
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are found within the flexible loops 1 and 2, the β4-TM2 loop (containing a patch
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of negatively charged residues) and the C-terminal tail. These sites deviate most
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from the Claudins and have received the most diligent attention in mutagenesis
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studies. All TARPs probably have a similar mode of association through the
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membrane domains, but subtype-specific co-assembly remains a possibility. The
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main differences in functional modulation likely arise from differential
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stoichiometry, and the modulatory strength of the variable regions (Fig. 3)
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23,23,24 25.
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Synaptic receptor complexes
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Experiments on the stargazer mouse, which lacks the γ2 subunit, showed
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impaired AMPA receptor function in cerebellar granule neurons (CGNs)
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identified γ2 as being essential to deliver functional AMPA receptors to synapses
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108 109 110 111 112 113
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in these cells. With TARP-γ2 being a relatively weak modulator of AMPAR
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21–
gating, the essence of TARP activity appeared to be receptor trafficking. But this simple story does not play out in the wider brain, or with other TARPs. The
deletion or overexpression of an auxiliary protein can alter synaptic currents, but can do so either by changing cell surface expression, synaptic trapping or receptor modulation. Thus TARPs allow selective expression of AMPA receptors with atypical properties – the postsynaptic response of a given glutamatergic
synapse cannot be known from first principles.
114 115
An important observation in this context is that the absence of synaptic AMPARs
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in CGNs of the stargazer mouse is due to γ7 mediating intracellular retention of
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complexes in the absence of γ2 28. The absence of γ2 has limited effects in other
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neurons in the stargazer mouse. In less clear-cut cases, effects of deletion and
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overexpression on synaptic current kinetics appear asymmetric. For example, 4
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overexpression of γ4 elongates decay of miniature currents (mEPSCs) 22,23 but its
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deletion has a minor effect on synaptic currents. In the hippocampus, the effect
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of γ8 deletion is substantial, but its overexpression does little29. In both cases,
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the synaptic complexes are not simply those that either lack or gain γ4 or γ8, but
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which have gained or lost other auxiliary proteins as a consequence, altering
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their trafficking and gating. In the hippocampus, γ2 and γ8 assemble
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competitively into complexes, with γ2 producing “High density” synapses
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Functional tests of the incorporation of multiple auxiliary subunits are not yet
128
mature, but CKAMP44 and CKAMP52 bind complementarily to the same AMPA
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complexes as γ8 or γ2
130
subunits into complexes is likely widespread and brain region specific
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Importantly, some interacting proteins form transient complexes in the ER to
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regulate trafficking, and dysregulation of these mechanisms has grave
133
consequences 4. Until methods to examine the heterogeneity and complexity of
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AMPAR-complexes at the cellular level are devised (but see ref. 24), the mosaic
135
of auxiliary proteins at synapses will remain opaque.
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Overall, promiscuous co-assembly of multiple 32.
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30.
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To date, little subunit specificity (within GluAs) of auxiliary protein action could
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be identified. Some TARPs seem to preferentially modulate GluA2
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example arises in the cornichon family
140
homologs (CNIH1-4) have three transmembrane helices, but CNIH2 and CNIH3
33.
21.
One clear
The four mammalian cornichon
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Mutation of Ala793 on M1 to Phe resulted in a loss of function of γ2 and a gain of
148
function for CNIH3
149
5KK2), Ala793 of GluA2 is sandwiched by Ile153 and Ile157 of γ2, leaving no
150
space for Phe at position 793 (Fig 3C).
141 142 143 144 145
have longer extracellular loops, probably accounting for their modulation of
gating (Fig. 3) 34. TARP-γ8 blocks association of CNIH with non-GluA1 subunits faster synaptic currents upon CNIH deletion come from preferential trafficking of GluA2/A3 receptors that intrinsically have faster kinetics
35.
Whether CNIH2/3
and TARPs share a common mode of association is currently unclear. The same residues in M1 and M4 of the AMPAR are important for γ2 and CNIH complexes. 36.
Within AMPAR-γ2 complexes (pdb codes: 5WEO and
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Altering the number and properties of AMPA receptors at the postsynaptic
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density (PSD) is presumed to be central to synaptic plasticity from a postsynaptic
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locus (for one of many excellent reviews see ref. 37). TARPs are ideally suited to
155
participate, being both synaptic anchors and functional modulators. Critically,
156
simulations indicate that progressive clustering is more important than receptor
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number 38. The activity dependent phosphorylation of TARPs 39,40 is implicated in
158
long-term potentiation (LTP). Evidence that PSD95 is arranged perpendicular to
159
the membrane supports the idea that TARPs with longer C-tails can anchor at
160
higher affinity, deep-lying PDZ domains following phosphorylation
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deletion reduces LTP 29, whereas phosphorylation of TARP-γ2 appears essential
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for LTP 40. But at least some types of LTP rely on a reserve pool of receptors 42:
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surface expression promoted by TARPs might be more important than
164
differential modulation of trapping, pharmacology and channel conductance at
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synaptic sites 43.
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TARP-γ8
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Recent reports have uncovered a dynamic role for TARP modulation of gating in
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synaptic receptors. TARP-γ2 is responsible for a slow rebound current in the
169
cerebellar brush cells
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These observations are striking because canonical AMPA receptors open rapidly
171
and exclusively in response to brief, high concentrations of glutamate, and
172
otherwise desensitize at concentrations of transmitter that do not open the
and also for slow currents in the Purkinje neuron
45.
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seem certain to be related 49. The humped equilibrium current curve was initially
180
presumed to come from a glutamate-driven “autoinactivation” and dissociation
181
of the complex
182
concentrations of glutamate seen during incomplete transmitter uptake or
183
"spillover". In line with this finding, earlier work identified that spillover
184
currents must be mediated by a desensitization-resistant class of AMPA
173 174 175 176 177
channel 46,47. However, careful biophysical work shows that TARP-γ2 can flip this
dogma,
with
intermediate
concentrations
of
glutamate
producing
disproportionately large standing currents, and a “hump” in the steady state concentration-response curve. This phenomenon arises because γ2 increases the potency of glutamate to activate the channel, whilst preventing desensitization 48.
The boost of kainate efficacy and the conversion of CNQX to a partial agonist
50.
Instead, γ2, and likely other TARPs, boost activity at
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receptors51.
186 187
At high concentrations of glutamate, AMPAR-TARP complexes can enter a slow
188
gating mode, with high-open-probability and high conductance single channel
189
bursts more reminiscent of NMDA receptors
190
mode is itself promoted by the activation of AMPARs, in a positive-feedback loop
191
53.
192
“baseline” of the postsynaptic current can appear to get “leaky”. Disturbingly,
193
receptors in this mode are not blocked by NBQX, although they are inhibited by
194
pentobarbital 45– which receptors without TARPs are not. If, indeed, we imagine
195
that the fastest receptor in the brain also moonlights as a slow follower of
196
repetitive stimulation, these slow-gating currents last hundreds of milliseconds,
197
therefore spanning instantaneous spike frequencies of around 10 Hz.
The switch to this “superactive”
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Such observations might explain why, during repetitive stimulation, the
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Progressive slowing of decays increases charge transfer, whilst rebound currents
200
amplify it further, with important implications for experimental designs
201
involving repetitive stimulation that assume a monolithic post-synaptic response
202
(such as mEPSC deconvolution in the presence of the desensitization blocker CTZ
203
54).
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obtain (and indeed to avoid) slow and rebound currents are an exciting future
205
research area. In terms of complexes, one clue might be that CNIH, which
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Unraveling what synaptic geometries, and what complexes, are required to
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conducting pathway (pdb code: 5WEO) (pdb code: 5VOT), and offer a tentative
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picture of the gating mechanism 18,56. Upon quisqualate or glutamate binding, the
214
LBD layer expands laterally. This movement is translated to the pore principally
215
via the M3-S2 linker of subunits B & D, although the S1-M1 linker and the S2-M4
216
linker also undergo movement
217
either a subconductance or the full conductance - is unclear. According to
206 207 208 209 210
produces very slow EPSCs in the mossy cells of the hippocampus
55,
has the
opposite effect in CA1 pyramidal cells, where it blocks superactivating currents 24.
Gating modulation
The latest cryo-EM GluA2-γ2 structures of active states revealed a widened ion
56.
The precise state of the cryo-EM structures –
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218
molecular dynamics studies, the pores in the derived models can conduct cations
219
18,57.
220
be wider than observed in these structures (7-8 Å)
221
substantial dilation in a maximally conducting pore. TARP effects on
222
conductance, calcium permeability and polyamine block are all reported, but a
223
mechanistic understanding of the interaction between these effects is lacking.
224
The selectivity filter was not well resolved in previous structures but appears
225
stabilized by TARP-γ2. Consistent with this observation, residues at the Q/R site
226
21,60
227
greatest effects on polyamine block are seen at the proximal intracellular region
228
59.
But the diameter of a conducting, non-selective AMPAR pore is expected to suggesting a more
59
could alter TARP association and modulation. The
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229
Extracellular regions of TARPs are only partly resolved in complexes to date, but
231
their close proximity to the LBD and LBD-TMD linkers suggests modulatory
232
action
233
detected in peptide mapping arrays 11,61, with the major caveat that these assays
234
lack steric constraints from the normal complex. Further analysis identified both
235
loops 1 and 2 being necessary for TARP modulation61. The transmembrane
236
segments of TARPs are nearly identical between closed and open states,
237
suggesting that extracellular interactions suffice. The S1-M1 and S2-M4 linkers of
Direct interactions of the flexible loop 1 and GluA2 LBDs were
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12–14,18.
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suggest the NP might be an inhibitory site for γ2 action, speaking for an
245
independent role of the KGK motif
246
structure (pdb code: 5WEO) the distal end of the negative patch, not studied in
247
functional experiments to date, is in close proximity to the KGK motif 56.
238 239 240 241 242
GluA2 were identified as a plausible area of action for loop 2 61. The first cryo-EM structures identified the conserved negatively-charged patch (NP) of γ2 as a potential interaction site with a positively charged area (KGK motif) on the LBD
for modulation (Figure 3)
12,13.
Replacing the KGK motif by an aspartate almost
completely abolishes the effects of γ2, but neutralization or reversing charges in
the proximal NP made γ2 an even stronger modulator. These experiments 62 36,61.
However, in the recent complex
248 249
Modulation of desensitization 8
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AMPA receptor desensitization, through rupture of the active dimer interface
251
between LBDs, allows tension to ebb from the LBD-TMD linkers and the channel
252
to close. Structures from several labs pictured the extracellular domains
253
spreading widely63,64, but complexes with auxiliary proteins have produced
254
much more compact arrangements. The longer loop 1 segments of TARPs and
255
GSG1L are prime candidates for stabilizing desensitized states - TARPs with
256
shorter or absent loop 1 do not alter desensitized states much 21. Several reports
257
of slowed kinetics of recombinant AMPARs in heterologous systems contrast
258
with faster recovery kinetics of AMPARs following overexpression of GSG1L in
259
pyramidal neurons 65.
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260
Accumulating evidence suggests a secondary desensitization mechanism
262
following from the linker regions
263
auxiliary subunit in proteomic studies
264
positioned next to the AMPA receptor reveals, that the cysteine-knot motif,
265
important for gating modulation
266
movement during gating. Mechanisms of modulation of desensitization that
267
depend on an interaction between the shaft of CKAMP and the LDB-TMD linker,
268
via its 40 residue flexible linker are also conceivable (see model in Fig. 1).
269
CKAMP family members exhibit opposing modulatory function. CKAMP44 and
270
CKAMP39 facilitate desensitization whilst, like TARPs, CKAMP52 and CKAMP59
CKAMP44 was identified as an AMPA 68.
A structural model of CKAMP44
M
66,67.
an
261
can reach to the LBD layer to restrict its
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277
linker region
278
PMP causes unfortunate neurological side effects, probably due to hindbrain
279
inhibition of AMPARs. A series of studies now reveal that subtype-specific drugs
280
can target particular AMPAR-TARP complexes. Targeting the forebrain-enriched
281
TARP-γ8 identified a promising inhibitor, LY3130481. Site directed mutagenesis
282
suggests LY3130481 binds within the membrane, between the third and fourth
271 272 273 274 275
slow desensitization and, reduce short-term synaptic depression 17,31,69.
Pharmaceutical perspective The regional expression patterns of the different auxiliary proteins in the brain, make them an attractive target for brain-region selective drugs
70.
Perampanel
(PMP; Eisai) is a non-competitive AMPAR inhibitor, binding at the LBD-TMD 71
and FDA approved as an adjunct antiepileptic drug. However,
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transmembrane segments of TARP-γ8 and the AMPAR M1 (Fig. 3C). Inhibition
284
may result from partially disrupting the complex to hinder TMD movements
285
during gating (Fig. 3C)72–74. A residue that discriminates between TARP and
286
CNIH modulation (Ala793) is immediately adjacent to the putative binding site
287
(Fig. 3C). In homology models of γ8, the residue corresponding to I153 in γ2 (γ8
288
Val176) is pointing away from Ala793 towards TM4 of γ8 (Gly210), potentially
289
forming an binding pocket for LY3130481
290
subtype selectivity. Recent high throughput screening identified positive and
291
negative allosteric modulators that selectively act on GluA2-TARP-γ2 and GluA2-
292
CNIH3 complexes, but not on GluA2 alone or in complex with GSG1L. These
293
initial hits need further optimization and validation but illustrate the power of
294
this approach 76.
and speaking to a mechanism of
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Conclusions
297
Structures of AMPA-receptor TARP complexes open a new era of investigation
298
into auxiliary protein action. This work should facilitate sophisticated studies of
299
their roles in neurons, where AMPA receptors have a wider repertoire of
300
functional traits than expected. Unpicking how distinct AMPA receptor
301
complexes might influence to synaptic dynamics, circuits and ultimately
302
cognition will remain important research topics for some time to come.
303
309 310
Figure Legends
304 305 306 307 308
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Acknowledgements
We thank Jelena Baranovic for comments on the manuscript, and we apologize to those colleagues whose work we could not cite due to space restrictions. A.J.R.P. is a Heisenberg Professor of the DFG and C.E. is a recipient of an Erwin-
Schrödinger Postdoctoral Fellowship (J3682-B21) of the Austrian Science fund (FWF).
311 312
Figure 1: Surface representation of AMPAR complexes with three different
313
auxiliary proteins (GluA2 subunits colored blue and grey). In the complex with
314
GSG1L (cyan, left), the antagonist MPQX bound to the ligand binding domain
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Page 10 of 23
(LBD) is shown as red spheres. The complex with TARP-γ2 (orange, centre) is
316
bound by glutamate (hidden within closed LBDs) in an active conformation. On
317
the right, a prospective model of a CKAMP44 complex is also shown (green) on
318
the same GluA2 scaffold. This complex model was built using the SwissModel
319
server 77,78 to generate the CKAMP44 extracellular Cys-knot structure based on
320
pdb code: 5M0W 79 and the transmembrane helix using pdb code: 2MET, 80 as a
321
template. These structures were manually connected via a 21 residue long
322
flexible linker and positioned next to the AMPA receptor (using chains A-D from
323
pdb code: 5WEO 56 to illustrate the reach of CKAMP44. Due to the lack of any
324
structural model for around 40 and 60 residues at the N-terminus and C-
325
terminus, respectively, these segments were omitted from the model.
326
Receptors are depicted to scale with an opposed vesicle releasing glutamate,
327
with about 3% of the total number of glutamate molecules in such a vesicle
328
shown.
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329
Figure 2. Surface representation of glutamate -activated GluA2 receptor in
331
complex with TARP-γ2 (in cartoon representation, two pairs of γ2 protomers in
332
yellow and orange, respectively). Schematic views of the domain organization of
333
the distinct layers in the complex. At the TMD level, all four TARP-γ2 molecules
334
engage in same receptor interactions (indicated by orange and yellow circles). At
335
the LBD level, TARP interactions are expected to be asymmetric – being intra-
341
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342
domain (LBD, Pac-Man dotted outline, glutamate bound) are extracellular. The
343
LBD is composed from two segments (S1 and S2 colored light and dark grey
344
respectively) divided by the M1, M2 and M3 helices of the transmembrane
345
domain (TMD). The re-entrant loop M2 harbors the Q/R site (yellow diamond).
346
The flexible carboxyl-terminal domain (CTD) is in the cytoplasm. Sites involved in
347
complex formation with, or modulation of gating by, TARPs and GSG1L are
336 337 338 339 340
dimeric between subunits A-D and B-C, indicated by orange triangles, and interdimeric between subunits A-B and C-D, indicated by yellow rectangles.
Figure 3 A) AMPA receptor interaction sites with auxiliary proteins. The amino terminal domain (ATD, black) and the clamshell like ligand-binding
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indicated by orange and cyan patches respectively, and marked with numbered
349
citations (blue circles). B) Auxiliary protein interaction sites with AMPARs.
350
Differences between tetra-spanning TARP types I, II and GSG1L lie in the variable
351
lengths of the extracellular loops 1 and 2 (L1 and L2) and the intracellular carboxyl
352
terminal tail, with the PSD-binding motif (red pentagon) in type I TARPs but not in
353
GSG1L. Type Ia and type Ib TARPs are shown, type II TARPs are omitted. Sites
354
reported to modulate receptor gating and/or necessary for complex formation are
355
indicated in orange and cyan for TARPs and GSG1L, respectively and labeled with
356
numbered citations as in panel A. The tri-spanning CNIH2 and CNIH3 exhibit a
357
flexible extracellular loop (colored magenta) with a short predicted helix,
358
important in CNIH3 for modulation via the receptor LBD. CKAMPs possesses an
359
extracellular cysteine-knot motif (green) connected via a predicted flexible linker
360
to the single transmembrane helix, short interacting stretch (green) followed by a
361
long C-terminal tail containing the PDZ type II motif (red pentagon). C) Auxiliary
362
subunit specific drug binding. A pink circle encloses Val176 and Gly210 (light
363
blue rectangles), residues in TM3 and TM4 (blue) of TARP-γ8 implicated in
364
binding the selective compound LY3130481. In GluA2, Ala793 in M4 (grey),
365
implicated in TARP-γ2 and CNIH modulation is adjacent. The corresponding
366
residues in TARP- γ2 (Ile157 and Ala184, model from pdb code 5KK2) are
367
pointing in opposite directions, presumably disfavoring LY3130881 binding. Of
368
note, in the TARP-γ2 complex, Ile157 and Ile153 surround Ala793 from the M4
370
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segment of GluA2.
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370 371
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* The first structures of AMPA receptors with auxiliary subunits were published * Structural studies allow a molecular understanding of auxiliary protein action * Complexes with auxiliary proteins in brain can exhibit non-canonical slow gating * Auxiliary protein selective drugs target specific AMPA receptor complexes in the brain
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S2468-8673(17)30032-9 https://doi.org/doi:10.1016/j.cophys.2017.12.009 COPHYS 32
To appear in: Received date: Revised date: Accepted date:
14-10-2017 13-12-2017 21-12-2017
Please cite this article as: Eibl, C., Plested, A.J.R., AMPA receptors: mechanisms of auxiliary protein action, Current Opinion in Physiology (2017), https://doi.org/10.1016/j.cophys.2017.12.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. 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.
1
AMPA receptors: mechanisms of auxiliary protein action
2
Clarissa Eibl and Andrew J.R. Plested
3
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4 Institute of Biology, Cellular Biophysics, Humboldt Universität zu Berlin, 10115
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Berlin;
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Leibniz Forschungsinstitut für Molekulare Pharmakologie (FMP), 13125 Berlin;
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Cluster of Excellence NeuroCure, Charité Universitätsmedizin, 10117 Berlin,
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Germany.
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10
an
11 Abstract
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AMPA receptors are the prime mediators of fast excitatory transmission in the
14
brain. Auxiliary subunits directly interact with AMPA receptors, hustling them to
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synapses and fine-tuning their responses to glutamate in multiple ways. Recent
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structural and functional studies have clarified the scope of molecular
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interactions underlying AMPAR co-assembly with and modulation by auxiliary
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proteins. Complementary physiological and pharmacological work has expanded
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our view of what auxiliary proteins are capable of. Here we review how these
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studies advance a more refined outline of mechanisms of auxiliary protein
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action, but still leave some tantalizing parts of the puzzle missing.
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22 Introduction
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AMPA-type glutamate receptors (AMPARs) are ligand-gated ion-channels that
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are abundantly expressed in the neurons and glia of vertebrates, where they
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mediate the majority of fast excitatory transmission underlying basal and higher
27
brain functions. AMPARs respond to glutamate released from synaptic terminals
28
within hundreds of microseconds, allowing rapid signal propagation in the CNS.
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The AMPA receptor family comprises four principal subunits (GluA1-A4) and
30
most receptors in the brain are hetero-tetramers that include GluA2. Extensive
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modifications to receptor subunits occur both during biogenesis with post-
32
transcriptional RNA editing (including Q/R editing in GluA2) and splicing
33
(flip/flop variants). Further, a myriad of post-translational modifications
34
including glycosylation, ubiquitination and phosphorylation are reported (for a
35
thorough review see ref. 1). Although these events alter the amplitude, duration
36
and ionic character of postsynaptic currents, they cannot explain some aspects of
37
glutamate receptor biology in neurons, including slower kinetics of native
38
receptors. This gap was filled by the discovery of a menagerie of membrane-
39
embedded “auxiliary proteins” that associate early in receptor biogenesis.
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Numerous proteins modify synaptic transmission and co-precipitate with AMPA
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receptors, so consensus characteristics for auxiliary proteins were formulated to
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focus the discussion. First, auxiliary proteins should bind selectively to mature
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49
and 2 Cornichon-homologs pass muster. With the arrival of the first structures of
50
complexes between AMPA receptors and auxiliary proteins (Fig. 1), the time is
51
ripe to highlight the scope and subtlety of action, and consider how decades of
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structural and biophysical studies on GluA subunits must now be weighed in a
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new context.
43 44 45 46 47
AMPARs. Second, auxiliary proteins modulate the functional properties of AMPARs and/or mediate surface trafficking and third, the necessity of auxiliary
proteins in vivo is expected2,3. Exciting candidates including CPT1c, FRRS1, SAC1 and SynDig 4–7 fall short of these somewhat arbitrary criteria. But a baker’s dozen of auxiliary proteins comprising 6 transmembrane AMPA receptor regulatory
proteins (TARPs), the germ cell-specific gene 1-like (GSG1L), 4 CKAMPs/Shisas
54
2
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AMPA Receptor assemblies
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The first clue to auxiliary protein structure came from the Claudin family of tight-
57
junction proteins 8,9,10 that are related to TARPs and GSG1L. Sequence alignments
58
suggested a common architecture of four transmembrane helices (TM1-4),
59
connected by one short intracellular loop and two extracellular segments (ECS1
60
and ECS2). Initially, the ECS1 of TARPs was considered to be unstructured,
61
perhaps reaching up to the receptor’s amino terminal domain (ATD)
62
TARP-γ2 structures showed that, as in Claudins, the ECS1 is folded into four
63
antiparallel β-sheets. A snorkel-like flexible loop (L1), absent in Claudins, is too
64
short to reach above the LBD layer (Figure 2). ECS2 consists of a flexible loop
65
(L2) and β5, which forms an antiparallel sheet with ECS1-β1 in a “hat” domain.
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But
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Cryo-EM structures of AMPA receptors in complex with TARP-γ2 and GSG1L
68
subunits showed auxiliary proteins decorating the perimeter of the complex
69
14.
70
independently confirmed by an elegant biochemical and functional study
71
Extensive hydrophobic interactions between TARP TM3 and TM4 and the
72
receptor’s M1 and M4 helices (Fig. 2) suggest a tight assembly. Other regions
73
may be important – both TARP-γ2
74
immediately intracellular regions (not resolved in structures of complexes to
75
date) for complex formation (Fig. 3).
12–
15,16
and CKAMP44
17
15.
rely on interactions at
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Each auxiliary protein protomer interacts across two GluA subunits,
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different stoichiometries, either 4:4 or (probably) 2:4 19,20. To combat this, recent
83
structure-function work has relied on tandem constructs or other measures to
84
isolate complexes. Further complexity is offered by the symmetry switch in the
85
AMPAR complex, between the two-fold extracellular domains and the four-fold
86
membrane domain (and thus principal TARP association sites). This geometry
76 77 78 79 80
The tandem constructs used for the structural studies might have been expected to produce a 4:4 stoichiometry, but instead complexes with 1, 2 and 4 TARPs illustrate preferential assembly sites 12,13,18, with the lack of membrane allowing
spare TARPs to “hang” outside the complex. The apparent independence of
association chimes with previous work showing that TARPs could assemble with
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87
produces two distinct TARP association sites and therefore likely asymmetric
88
roles for TARPs to control receptor function (Fig. 2).
89 The TARPs (γ2, γ3, γ4, γ5, γ7 and γ8) have sequence identity of around 30-70%.
91
The remarkably similar structure and mode of co-assembly of GSG1L, despite
92
being as dissimilar to TARPs as Claudins and the Calcium channel γ1 subunit,
93
suggests, for practical purposes, that GSG1L is a TARP. The biggest differences
94
are found within the flexible loops 1 and 2, the β4-TM2 loop (containing a patch
95
of negatively charged residues) and the C-terminal tail. These sites deviate most
96
from the Claudins and have received the most diligent attention in mutagenesis
97
studies. All TARPs probably have a similar mode of association through the
98
membrane domains, but subtype-specific co-assembly remains a possibility. The
99
main differences in functional modulation likely arise from differential
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stoichiometry, and the modulatory strength of the variable regions (Fig. 3)
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23,23,24 25.
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Synaptic receptor complexes
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Experiments on the stargazer mouse, which lacks the γ2 subunit, showed
104
impaired AMPA receptor function in cerebellar granule neurons (CGNs)
105
identified γ2 as being essential to deliver functional AMPA receptors to synapses
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108 109 110 111 112 113
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in these cells. With TARP-γ2 being a relatively weak modulator of AMPAR
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21–
gating, the essence of TARP activity appeared to be receptor trafficking. But this simple story does not play out in the wider brain, or with other TARPs. The
deletion or overexpression of an auxiliary protein can alter synaptic currents, but can do so either by changing cell surface expression, synaptic trapping or receptor modulation. Thus TARPs allow selective expression of AMPA receptors with atypical properties – the postsynaptic response of a given glutamatergic
synapse cannot be known from first principles.
114 115
An important observation in this context is that the absence of synaptic AMPARs
116
in CGNs of the stargazer mouse is due to γ7 mediating intracellular retention of
117
complexes in the absence of γ2 28. The absence of γ2 has limited effects in other
118
neurons in the stargazer mouse. In less clear-cut cases, effects of deletion and
119
overexpression on synaptic current kinetics appear asymmetric. For example, 4
Page 4 of 23
overexpression of γ4 elongates decay of miniature currents (mEPSCs) 22,23 but its
121
deletion has a minor effect on synaptic currents. In the hippocampus, the effect
122
of γ8 deletion is substantial, but its overexpression does little29. In both cases,
123
the synaptic complexes are not simply those that either lack or gain γ4 or γ8, but
124
which have gained or lost other auxiliary proteins as a consequence, altering
125
their trafficking and gating. In the hippocampus, γ2 and γ8 assemble
126
competitively into complexes, with γ2 producing “High density” synapses
127
Functional tests of the incorporation of multiple auxiliary subunits are not yet
128
mature, but CKAMP44 and CKAMP52 bind complementarily to the same AMPA
129
complexes as γ8 or γ2
130
subunits into complexes is likely widespread and brain region specific
131
Importantly, some interacting proteins form transient complexes in the ER to
132
regulate trafficking, and dysregulation of these mechanisms has grave
133
consequences 4. Until methods to examine the heterogeneity and complexity of
134
AMPAR-complexes at the cellular level are devised (but see ref. 24), the mosaic
135
of auxiliary proteins at synapses will remain opaque.
ip t
120
cr
us
Overall, promiscuous co-assembly of multiple 32.
M
an
17,31.
30.
d
136
To date, little subunit specificity (within GluAs) of auxiliary protein action could
138
be identified. Some TARPs seem to preferentially modulate GluA2
139
example arises in the cornichon family
140
homologs (CNIH1-4) have three transmembrane helices, but CNIH2 and CNIH3
33.
21.
One clear
The four mammalian cornichon
146
Ac ce p
te
137
147
Mutation of Ala793 on M1 to Phe resulted in a loss of function of γ2 and a gain of
148
function for CNIH3
149
5KK2), Ala793 of GluA2 is sandwiched by Ile153 and Ile157 of γ2, leaving no
150
space for Phe at position 793 (Fig 3C).
141 142 143 144 145
have longer extracellular loops, probably accounting for their modulation of
gating (Fig. 3) 34. TARP-γ8 blocks association of CNIH with non-GluA1 subunits faster synaptic currents upon CNIH deletion come from preferential trafficking of GluA2/A3 receptors that intrinsically have faster kinetics
35.
Whether CNIH2/3
and TARPs share a common mode of association is currently unclear. The same residues in M1 and M4 of the AMPAR are important for γ2 and CNIH complexes. 36.
Within AMPAR-γ2 complexes (pdb codes: 5WEO and
151
5
Page 5 of 23
Altering the number and properties of AMPA receptors at the postsynaptic
153
density (PSD) is presumed to be central to synaptic plasticity from a postsynaptic
154
locus (for one of many excellent reviews see ref. 37). TARPs are ideally suited to
155
participate, being both synaptic anchors and functional modulators. Critically,
156
simulations indicate that progressive clustering is more important than receptor
157
number 38. The activity dependent phosphorylation of TARPs 39,40 is implicated in
158
long-term potentiation (LTP). Evidence that PSD95 is arranged perpendicular to
159
the membrane supports the idea that TARPs with longer C-tails can anchor at
160
higher affinity, deep-lying PDZ domains following phosphorylation
161
deletion reduces LTP 29, whereas phosphorylation of TARP-γ2 appears essential
162
for LTP 40. But at least some types of LTP rely on a reserve pool of receptors 42:
163
surface expression promoted by TARPs might be more important than
164
differential modulation of trapping, pharmacology and channel conductance at
165
synaptic sites 43.
cr
ip t
152
TARP-γ8
an
us
41.
M
166
Recent reports have uncovered a dynamic role for TARP modulation of gating in
168
synaptic receptors. TARP-γ2 is responsible for a slow rebound current in the
169
cerebellar brush cells
170
These observations are striking because canonical AMPA receptors open rapidly
171
and exclusively in response to brief, high concentrations of glutamate, and
172
otherwise desensitize at concentrations of transmitter that do not open the
and also for slow currents in the Purkinje neuron
45.
178
Ac ce p
te
44
d
167
179
seem certain to be related 49. The humped equilibrium current curve was initially
180
presumed to come from a glutamate-driven “autoinactivation” and dissociation
181
of the complex
182
concentrations of glutamate seen during incomplete transmitter uptake or
183
"spillover". In line with this finding, earlier work identified that spillover
184
currents must be mediated by a desensitization-resistant class of AMPA
173 174 175 176 177
channel 46,47. However, careful biophysical work shows that TARP-γ2 can flip this
dogma,
with
intermediate
concentrations
of
glutamate
producing
disproportionately large standing currents, and a “hump” in the steady state concentration-response curve. This phenomenon arises because γ2 increases the potency of glutamate to activate the channel, whilst preventing desensitization 48.
The boost of kainate efficacy and the conversion of CNQX to a partial agonist
50.
Instead, γ2, and likely other TARPs, boost activity at
6
Page 6 of 23
185
receptors51.
186 187
At high concentrations of glutamate, AMPAR-TARP complexes can enter a slow
188
gating mode, with high-open-probability and high conductance single channel
189
bursts more reminiscent of NMDA receptors
190
mode is itself promoted by the activation of AMPARs, in a positive-feedback loop
191
53.
192
“baseline” of the postsynaptic current can appear to get “leaky”. Disturbingly,
193
receptors in this mode are not blocked by NBQX, although they are inhibited by
194
pentobarbital 45– which receptors without TARPs are not. If, indeed, we imagine
195
that the fastest receptor in the brain also moonlights as a slow follower of
196
repetitive stimulation, these slow-gating currents last hundreds of milliseconds,
197
therefore spanning instantaneous spike frequencies of around 10 Hz.
The switch to this “superactive”
ip t
52.
an
us
cr
Such observations might explain why, during repetitive stimulation, the
198
Progressive slowing of decays increases charge transfer, whilst rebound currents
200
amplify it further, with important implications for experimental designs
201
involving repetitive stimulation that assume a monolithic post-synaptic response
202
(such as mEPSC deconvolution in the presence of the desensitization blocker CTZ
203
54).
204
obtain (and indeed to avoid) slow and rebound currents are an exciting future
205
research area. In terms of complexes, one clue might be that CNIH, which
d
M
199
211
Ac ce p
te
Unraveling what synaptic geometries, and what complexes, are required to
212
conducting pathway (pdb code: 5WEO) (pdb code: 5VOT), and offer a tentative
213
picture of the gating mechanism 18,56. Upon quisqualate or glutamate binding, the
214
LBD layer expands laterally. This movement is translated to the pore principally
215
via the M3-S2 linker of subunits B & D, although the S1-M1 linker and the S2-M4
216
linker also undergo movement
217
either a subconductance or the full conductance - is unclear. According to
206 207 208 209 210
produces very slow EPSCs in the mossy cells of the hippocampus
55,
has the
opposite effect in CA1 pyramidal cells, where it blocks superactivating currents 24.
Gating modulation
The latest cryo-EM GluA2-γ2 structures of active states revealed a widened ion
56.
The precise state of the cryo-EM structures –
7
Page 7 of 23
218
molecular dynamics studies, the pores in the derived models can conduct cations
219
18,57.
220
be wider than observed in these structures (7-8 Å)
221
substantial dilation in a maximally conducting pore. TARP effects on
222
conductance, calcium permeability and polyamine block are all reported, but a
223
mechanistic understanding of the interaction between these effects is lacking.
224
The selectivity filter was not well resolved in previous structures but appears
225
stabilized by TARP-γ2. Consistent with this observation, residues at the Q/R site
226
21,60
227
greatest effects on polyamine block are seen at the proximal intracellular region
228
59.
But the diameter of a conducting, non-selective AMPAR pore is expected to suggesting a more
59
could alter TARP association and modulation. The
us
or adjacent to it
cr
ip t
58,59,
an
229
Extracellular regions of TARPs are only partly resolved in complexes to date, but
231
their close proximity to the LBD and LBD-TMD linkers suggests modulatory
232
action
233
detected in peptide mapping arrays 11,61, with the major caveat that these assays
234
lack steric constraints from the normal complex. Further analysis identified both
235
loops 1 and 2 being necessary for TARP modulation61. The transmembrane
236
segments of TARPs are nearly identical between closed and open states,
237
suggesting that extracellular interactions suffice. The S1-M1 and S2-M4 linkers of
Direct interactions of the flexible loop 1 and GluA2 LBDs were
243
Ac ce p
te
d
12–14,18.
M
230
244
suggest the NP might be an inhibitory site for γ2 action, speaking for an
245
independent role of the KGK motif
246
structure (pdb code: 5WEO) the distal end of the negative patch, not studied in
247
functional experiments to date, is in close proximity to the KGK motif 56.
238 239 240 241 242
GluA2 were identified as a plausible area of action for loop 2 61. The first cryo-EM structures identified the conserved negatively-charged patch (NP) of γ2 as a potential interaction site with a positively charged area (KGK motif) on the LBD
for modulation (Figure 3)
12,13.
Replacing the KGK motif by an aspartate almost
completely abolishes the effects of γ2, but neutralization or reversing charges in
the proximal NP made γ2 an even stronger modulator. These experiments 62 36,61.
However, in the recent complex
248 249
Modulation of desensitization 8
Page 8 of 23
AMPA receptor desensitization, through rupture of the active dimer interface
251
between LBDs, allows tension to ebb from the LBD-TMD linkers and the channel
252
to close. Structures from several labs pictured the extracellular domains
253
spreading widely63,64, but complexes with auxiliary proteins have produced
254
much more compact arrangements. The longer loop 1 segments of TARPs and
255
GSG1L are prime candidates for stabilizing desensitized states - TARPs with
256
shorter or absent loop 1 do not alter desensitized states much 21. Several reports
257
of slowed kinetics of recombinant AMPARs in heterologous systems contrast
258
with faster recovery kinetics of AMPARs following overexpression of GSG1L in
259
pyramidal neurons 65.
us
cr
ip t
250
260
Accumulating evidence suggests a secondary desensitization mechanism
262
following from the linker regions
263
auxiliary subunit in proteomic studies
264
positioned next to the AMPA receptor reveals, that the cysteine-knot motif,
265
important for gating modulation
266
movement during gating. Mechanisms of modulation of desensitization that
267
depend on an interaction between the shaft of CKAMP and the LDB-TMD linker,
268
via its 40 residue flexible linker are also conceivable (see model in Fig. 1).
269
CKAMP family members exhibit opposing modulatory function. CKAMP44 and
270
CKAMP39 facilitate desensitization whilst, like TARPs, CKAMP52 and CKAMP59
CKAMP44 was identified as an AMPA 68.
A structural model of CKAMP44
M
66,67.
an
261
can reach to the LBD layer to restrict its
276
Ac ce p
te
d
17,
277
linker region
278
PMP causes unfortunate neurological side effects, probably due to hindbrain
279
inhibition of AMPARs. A series of studies now reveal that subtype-specific drugs
280
can target particular AMPAR-TARP complexes. Targeting the forebrain-enriched
281
TARP-γ8 identified a promising inhibitor, LY3130481. Site directed mutagenesis
282
suggests LY3130481 binds within the membrane, between the third and fourth
271 272 273 274 275
slow desensitization and, reduce short-term synaptic depression 17,31,69.
Pharmaceutical perspective The regional expression patterns of the different auxiliary proteins in the brain, make them an attractive target for brain-region selective drugs
70.
Perampanel
(PMP; Eisai) is a non-competitive AMPAR inhibitor, binding at the LBD-TMD 71
and FDA approved as an adjunct antiepileptic drug. However,
9
Page 9 of 23
transmembrane segments of TARP-γ8 and the AMPAR M1 (Fig. 3C). Inhibition
284
may result from partially disrupting the complex to hinder TMD movements
285
during gating (Fig. 3C)72–74. A residue that discriminates between TARP and
286
CNIH modulation (Ala793) is immediately adjacent to the putative binding site
287
(Fig. 3C). In homology models of γ8, the residue corresponding to I153 in γ2 (γ8
288
Val176) is pointing away from Ala793 towards TM4 of γ8 (Gly210), potentially
289
forming an binding pocket for LY3130481
290
subtype selectivity. Recent high throughput screening identified positive and
291
negative allosteric modulators that selectively act on GluA2-TARP-γ2 and GluA2-
292
CNIH3 complexes, but not on GluA2 alone or in complex with GSG1L. These
293
initial hits need further optimization and validation but illustrate the power of
294
this approach 76.
and speaking to a mechanism of
an
us
cr
74,75
ip t
283
Conclusions
297
Structures of AMPA-receptor TARP complexes open a new era of investigation
298
into auxiliary protein action. This work should facilitate sophisticated studies of
299
their roles in neurons, where AMPA receptors have a wider repertoire of
300
functional traits than expected. Unpicking how distinct AMPA receptor
301
complexes might influence to synaptic dynamics, circuits and ultimately
302
cognition will remain important research topics for some time to come.
303
309 310
Figure Legends
304 305 306 307 308
te
d
M
296
Ac ce p
295
Acknowledgements
We thank Jelena Baranovic for comments on the manuscript, and we apologize to those colleagues whose work we could not cite due to space restrictions. A.J.R.P. is a Heisenberg Professor of the DFG and C.E. is a recipient of an Erwin-
Schrödinger Postdoctoral Fellowship (J3682-B21) of the Austrian Science fund (FWF).
311 312
Figure 1: Surface representation of AMPAR complexes with three different
313
auxiliary proteins (GluA2 subunits colored blue and grey). In the complex with
314
GSG1L (cyan, left), the antagonist MPQX bound to the ligand binding domain
10
Page 10 of 23
(LBD) is shown as red spheres. The complex with TARP-γ2 (orange, centre) is
316
bound by glutamate (hidden within closed LBDs) in an active conformation. On
317
the right, a prospective model of a CKAMP44 complex is also shown (green) on
318
the same GluA2 scaffold. This complex model was built using the SwissModel
319
server 77,78 to generate the CKAMP44 extracellular Cys-knot structure based on
320
pdb code: 5M0W 79 and the transmembrane helix using pdb code: 2MET, 80 as a
321
template. These structures were manually connected via a 21 residue long
322
flexible linker and positioned next to the AMPA receptor (using chains A-D from
323
pdb code: 5WEO 56 to illustrate the reach of CKAMP44. Due to the lack of any
324
structural model for around 40 and 60 residues at the N-terminus and C-
325
terminus, respectively, these segments were omitted from the model.
326
Receptors are depicted to scale with an opposed vesicle releasing glutamate,
327
with about 3% of the total number of glutamate molecules in such a vesicle
328
shown.
an
us
cr
ip t
315
M
329
Figure 2. Surface representation of glutamate -activated GluA2 receptor in
331
complex with TARP-γ2 (in cartoon representation, two pairs of γ2 protomers in
332
yellow and orange, respectively). Schematic views of the domain organization of
333
the distinct layers in the complex. At the TMD level, all four TARP-γ2 molecules
334
engage in same receptor interactions (indicated by orange and yellow circles). At
335
the LBD level, TARP interactions are expected to be asymmetric – being intra-
341
Ac ce p
te
d
330
342
domain (LBD, Pac-Man dotted outline, glutamate bound) are extracellular. The
343
LBD is composed from two segments (S1 and S2 colored light and dark grey
344
respectively) divided by the M1, M2 and M3 helices of the transmembrane
345
domain (TMD). The re-entrant loop M2 harbors the Q/R site (yellow diamond).
346
The flexible carboxyl-terminal domain (CTD) is in the cytoplasm. Sites involved in
347
complex formation with, or modulation of gating by, TARPs and GSG1L are
336 337 338 339 340
dimeric between subunits A-D and B-C, indicated by orange triangles, and interdimeric between subunits A-B and C-D, indicated by yellow rectangles.
Figure 3 A) AMPA receptor interaction sites with auxiliary proteins. The amino terminal domain (ATD, black) and the clamshell like ligand-binding
11
Page 11 of 23
indicated by orange and cyan patches respectively, and marked with numbered
349
citations (blue circles). B) Auxiliary protein interaction sites with AMPARs.
350
Differences between tetra-spanning TARP types I, II and GSG1L lie in the variable
351
lengths of the extracellular loops 1 and 2 (L1 and L2) and the intracellular carboxyl
352
terminal tail, with the PSD-binding motif (red pentagon) in type I TARPs but not in
353
GSG1L. Type Ia and type Ib TARPs are shown, type II TARPs are omitted. Sites
354
reported to modulate receptor gating and/or necessary for complex formation are
355
indicated in orange and cyan for TARPs and GSG1L, respectively and labeled with
356
numbered citations as in panel A. The tri-spanning CNIH2 and CNIH3 exhibit a
357
flexible extracellular loop (colored magenta) with a short predicted helix,
358
important in CNIH3 for modulation via the receptor LBD. CKAMPs possesses an
359
extracellular cysteine-knot motif (green) connected via a predicted flexible linker
360
to the single transmembrane helix, short interacting stretch (green) followed by a
361
long C-terminal tail containing the PDZ type II motif (red pentagon). C) Auxiliary
362
subunit specific drug binding. A pink circle encloses Val176 and Gly210 (light
363
blue rectangles), residues in TM3 and TM4 (blue) of TARP-γ8 implicated in
364
binding the selective compound LY3130481. In GluA2, Ala793 in M4 (grey),
365
implicated in TARP-γ2 and CNIH modulation is adjacent. The corresponding
366
residues in TARP- γ2 (Ile157 and Ala184, model from pdb code 5KK2) are
367
pointing in opposite directions, presumably disfavoring LY3130881 binding. Of
368
note, in the TARP-γ2 complex, Ile157 and Ile153 surround Ala793 from the M4
370
cr
us
an
M
d
te
Ac ce p
369
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348
segment of GluA2.
12
Page 12 of 23
370 371
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