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Title: Neto proteins regulate gating of the kainate-type glutamate receptor
GluK2 through two binding sites
Running Title: GluK2 gating by Netos
Authors: Yan-Jun Li1#, Gui-Fang Duan1#, Jia-Hui Sun1#, Dan Wu1, Chang Ye1,
Yan-Yu Zang1, Gui-Quan Chen12, Yong-Yun Shi3, Jun Wang4, Wei Zhang5*, and
Yun Stone Shi126*
1State Key Laboratory of Pharmaceutical Biotechnology, Department of
Neurology, Affiliated Drum Tower Hospital of Nanjing University Medical School,
and Minister of Education Key Laboratory of Model Animal for Disease Study,
Model Animal Research Center, Nanjing University, Nanjing 210032, China
2Institute for Brain Sciences, Nanjing University, Nanjing 210032, China
3Department of Orthopaedics, Luhe People's Hospital Affiliated to Yangzhou
University，Nanjing 211500, China
4Minister of Education Key Laboratory of Modern Toxicology, Department of
Toxicology, School of Public Health, Nanjing Medical University, Nanjing 211166,
China
5Institute of Chinese Integrative Medicine, Hebei Medical University,
Shijiazhuang 050017, China
6Chemistry and Biomedicine Innovation Center, Nanjing University, Nanjing
210032, China
#These authors contributed equally to this work.
*To whom correspondence may be addressed. Email: yunshi@nju.edu.cn and
weizhang@hebmu.edu.cn
Keywords: kainate receptor, glutamate receptor ionotropic kainate 2 (GluK2),
neuropilin and tolloid-like (Neto), channel gating, desensitization, deactivation,
recovery from desensitization, N-terminal domain (NTD), CUB domain,
glutamate ionotropic receptor kainate type subunit 2 (GriK2)
http://www.jbc.org/cgi/doi/10.1074/jbc.RA119.008631The latest version is at
JBC Papers in Press. Published on October 18, 2019 as Manuscript RA119.008631
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ABSTRACT
The neuropilin and tolloid-like (Neto)
proteins Neto1 and Neto2 are auxiliary
subunits of kainate-type glutamate
receptors (KARs) that regulate KAR
trafficking and gating. However, how
Netos bind and regulate the biophysical
functions of KARs remains unclear. Here,
we found that the N-terminal domain
(NTD) of glutamate receptor ionotropic
kainate 2 (GluK2) binds complement
C1r/C1s-Uegf-BMP (CUB1) domains of
Neto proteins (i.e. NTD–CUB1
interaction), and that the core of GluK2
(GluK2NTD) binds Netos through
domains other than CUB1s (core–Neto
interaction). Using electrophysiological
analysis in HEK293T cells, we examined
the effects of these interactions on GluK2
gating, including deactivation,
desensitization, and recovery from
desensitization. We found that NTD
deletion does not affect GluK2 fast gating
kinetics, the desensitization and the
deactivation. We also observed that
Neto1 and Neto2 differentially regulate
GluK2 fast gating kinetics which largely
rely on the NTD–CUB1 interactions. NTD
removal facilitated GluK2 recovery from
desensitization, indicating that the NTD
stabilizes the GluK2 desensitization state.
Co-expression with Neto1 or Neto2 also
accelerated GluK2 recovery from
desensitization, which fully relied on the
NTD–CUB1 interactions. Moreover, we
demonstrate that the NTD–CUB1
interaction involves electric attraction
between positively charged residues in
the GluK2_NTD and negatively charged
ones in the CUB1 domains.
Neutralization of these charges
eliminated the regulatory effects of the
NTD–CUB1 interaction on GluK2 gating.
We conclude that KARs bind Netos
through at least two sites and that the
NTD–CUB1 interaction critically
regulates Neto-mediated GluK2 gating.
INTRODUCTION
In the central nerve system,
excitatory synaptic transmission is
primarily mediated by glutamate.
Glutamate released from presynaptic
terminals excites three types of
ionotropic glutamate receptors, which
are pharmacologically classified as
AMPA (amino-3-hydroxy-5-
methylisoxazole-4-propionic acid)
receptors (AMPARs), NMDA (N-methyl-
D-aspartic acid) receptors（NMDARs）,
and kainate receptors (KARs) (1).
AMPARs mediate the majority of fast
transmission while NMDARs are
responsible for synaptic plasticity. KARs
are expressed in subsets of neuronal
types in the brain. They not only
contribute to EPSCs on postsynaptic cell,
but also regulate neurotransmitter
release on the presynaptic terminal (2).
Additionally, KAR activity is involved in
synaptic plasticity (3). Dysfunction of
KARs causes neurologic diseases such
as epilepsy, schizophrenia, and autism
(2,4).
Ionotropic glutamate receptors are
tetramers. Each subunit contains a large
N-terminal domain (NTD), accounting for
about 40% of the full length, followed by
a ligand binding domain (LBD), and a
transmembrane domain (TMD) forming
the ion channel pore, then an
intracellular C-terminal tail associating
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with scaffold proteins. Among these
domains, the NTD is the largest, but its
function, until recently, has been poorly
understood. The NTDs of NMDARs are
important for their gating and regulation
by allosteric modulators including Zn2+,
H+, ifenprodil etc (1,5,6). The NTDs of
AMPARs play little role in receptor fast
gating but instead contribute to the
stabilization of desensitization (7).
Additionally, NTD truncated AMPAR
subunits display increased mobility on
synapse and lose their ability to sustain
long term potentiation (8,9). Similar to
AMPARs, removal of GluK2_NTD does
not change the rates of deactivation and
desensitization in heterologous systems
(10), whereas NTDs of KARs are crucial
for synaptic localization (11,12).
Beside the pore-forming subunits,
native KARs and AMPARs associate
with auxiliary proteins (13). Neto proteins
bind KARs and regulate KAR
deactivation, desensitization,
rectification, synaptic trafficking similar to
the effects of TARPs on AMPARs (14-16).
Furthermore, Netos play critical roles in
determining the axonal distribution of
KARs in neurons (17,18). Netos are also
proposed to play a role in neural circuit
development (19-21) and be required for
normal fear expression (22,23). Netos
are single-pass transmembrane proteins
with a long extracellular N-terminal
sequence containing two Cir/C1s-Uegf-
BMP (CUB) domains and a low density
lipoprotein class A (LDLa) domain, and a
short intracellular C-terminal tail (24).
Neto2 mutations in LDLa eliminate its
effects on desensitization (25), and
mutations in the intracellular C-terminal
tail prevent the effects of Neto1/2 on
rectification (26). Biochemical studies
indicate that the CUB2 domain is critical
for Neto protein binding to GluK2 (27).
Nevertheless, the exact interaction
between Netos and KARs remains
elusive.
We previously reported that synaptic
targeting of GluK1 in hippocampal CA1
neurons relies on Neto proteins while
that of GluK2 does not(11,28). The
differential trafficking properties and
dependence on Netos between GluK1
and GluK2 rely on their NTDs(11,29). We
thus suspect that the NTDs of KARs
might directly bind Neto proteins. Here
we find that GluK2NTD specifically binds
the CUB1 domain of Neto proteins. In
addition, GluK2 without NTD
(GluK2_core) can still bind to Neto
proteins with or without CUB1 domain.
The effects of NTD-CUB1 and core-Neto
interactions on GluK2 gating, including
desensitization, deactivation and
recovery from desensitization, are
systemically studied. Our data suggest a
two-step model for Neto regulation of
KAR gating and emphasize an important
regulatory role for the NTD-CUB1
interaction.
RESULTS
Netos interact with GluK2 through
multiple sites
Previously we found the synaptic
targeting property of GluK1 and GluK2 is
differentially regulated by Netos in an
NTD-dependent manner (11,28,29),
indicating that NTDs of KARs might
directly interact with Netos. To test this
hypothesis, we expressed the NTD of
GluK2 by introducing a stop codon at
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position 401, thus the NTD (residues 31-
400) will be synthesized through the
secretory ER pathway under the
guidance of the signal peptide. To
facilitate its detection, an HA tag was
inserted after the signal peptide and a
FLAG epitope was inserted at the C-
termini of Netos. Consistent with our
prediction, GluK2NTD was co-
immunoprecipitated with Neto proteins
(Fig. 1, A and B). We also observed that
GluK2NTD, in which the NTD was
deleted, co-immunoprecipitated with
Netos (Fig. 1, A and B), indicating that
GluK2 interacts with Netos through
multiple sites. In Netos, two CUB
domains and an LDLa domain are
located extracellularly. Which of these
domains might interact with GluK2NTD?
Since the NTD is the distal extracellular
domain of KARs and is about 80 Å above
the membrane plane (30), it is
reasonable to suspect the very distal
CUB1 domain of Netos might interact
with GluK2NTD. Indeed, GluK2NTD was
efficiently co-immunoprecipitated with
Neto1CUB1 or Neto2CUB1 (Fig. 1C).
Meanwhile, GluK2NTD was pulled-
down by Neto1/2CUB1 (Fig. 1D),
indicating a second interaction which we
named as core-Neto interaction.
Furthermore, deletion of CUB1 domains
largely diminished Neto interaction with
GluK2NTD (Fig. 1, E-G), suggesting that
GluK2NTD specifically interacts with the
CUB1 domains of Neto proteins. Thus,
our observation suggested that GluK2
bind Netos through at least two
interaction sites, the NTD-CUB1
interaction and core-Neto interaction (Fig.
1H).
NTD truncation does not affect GluK2
desensitization and deactivation
Previous work demonstrated that
NTD truncated GluK2 receptors are
functional (10). We thus used
GluK2NTD to study Neto related gating
(Fig. S1A). Western blot analysis from
cell lysate showed similar expression
level of GluK2NTD and intact GluK2
receptors (Fig. S1B). Surface
biotinylation revealed that the surface
expression of GluK2 receptors was
unaffected by NTD truncation (Fig. S1B).
Furthermore, immunofluorescence
experiments examining an HA tag
inserted at N-termini of full-length and
NTD-deleted GluK2 showed that
GluK2NTD could express and traffic to
plasma membrane like full-length GluK2
receptors (Fig. S1C). Together, these
observations suggest that the NTD is not
required for GluK2 expression and
membrane trafficking.
We then tested the effects of NTD
truncation on gating kinetics by applying
saturating concentration of glutamate (10
mM) to outside-out patches excised from
the transfected HEK cells using a fast
piezoelectric system (25). The
desensitization kinetics recorded by 500
ms application of glutamate displayed no
difference between full-length and NTD-
deleted GluK2 receptors (Fig. 2A and Fig.
S2A). Similarly, the deactivation kinetics
recorded by brief application (1 ms) of 10
mM glutamate was unchanged by NTD
truncation (Fig. 3A and Fig. S2B). These
observations are consistent with
previous reports (10), suggesting that the
NTD itself has little effect on the
deactivation and desensitization kinetics
of KARs.
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NTD-CUB1 interactions distinguish
Neto1 and Neto2 on desensitization
Previous works show that Neto1
speeds and Neto2 slows GluK1
desensitization in recombinant system or
in neurons (28,31). Similarly Neto1
speeds and Neto2 slows GluK2
desensitization when overexpressed in
hippocampal CA1 neurons (11). Here we
found in HEK cells that Neto2
dramatically slows the desensitization of
GluK2 by ~4 times while Neto1 has little
effects (Fig. 2B and Fig. 2A), indicating
that Neto1 and Neto2 differentially
regulate GluK2 desensitization.
Interestingly, in the absence of NTD,
GluK2NTD was slightly slowed by both
Neto1 and Neto2 (Fig. 2C) to a similar
extent. When CUB1 domains of Netos
were removed, GluK2 desensitization
was not changed by Neto1CUB1 or
Neto2CUB1 (Fig. 2D). Furthermore,
GluK2NTD desensitization was
similarly slowed by Neto1CUB1 and
Neto2CUB1 (Fig. 2E). These results
thus demonstrated that the differential
regulation of GluK2 desensitization by
Neto1 and Neto2 relies on the NTD-
CUB1 interaction. In addition, in the
cases of NTD (Fig. 2C), CUB1(Fig. 2D)
and NTD+CUB1 (Fig. 2E), conditions
in which NTD-CUB1 interactions were
disrupted, KAR desensitization was
generally slowed by Netos, indicating
that the core-Neto interactions slow
desensitization. Comparing the Neto
effects on desensitization in Figure 2B
and those in Figure 2C-E, leads to the
conclusion that NTD-Neto1CUB1
speeds KAR desensitization and NTD-
Neto2CUB1 slows it.
NTD-CUB1 interactions distinguish
Neto1 and Neto2 on deactivation
We further studied the Neto
regulatory effects on GluK2 deactivation.
Neto2 but not Neto1 slowed GluK2
deactivation (Fig. 3A and Fig. S2B).
While the NTD was removed,
GluK2NTD was moderately slowed by
Neto1 and Neto2 to similar extent (Fig.
3C). When CUB1 domains were deleted,
GluK2 deactivation was slightly slowed
by Neto1CUB1 and not by
Neto2CUB1 (Fig. 3D), but the effects of
Neto1CUB1 and Neto2CUB1 were
not significantly different. Furthermore,
GluK2NTD deactivation was slowed by
Neto1CUB1 and Neto2CUB1 to
similar extent (Fig. 3E). The results thus
revealed that the different effects of
Neto1 and Neto2 on GluK2 deactivation
rely on the NTD-CUB1 interaction. Also
like the desensitization data, it can be
concluded that the core-Neto
interactions slow deactivation in general.
Taken together, these data revealed
that the NTD-CUB1 interaction is critical
for Neto1/2 modulation of GluK2 fast
gating kinetics.
NTD deletion facilitates GluK2
recovery from desensitization
Deletion of NTDs of AMPA receptors
facilitates their recovery from
desensitization (7). We wondered
whether this is the case for KARs. The
recovery rate of full-length and NTD-
deleted GluK2 receptors was monitored
through a pair of glutamate (10 mM for
50 ms) applications with variable
intervals. GluK2 receptors completely
recovered from desensitization in
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seconds with rec value of 2.62 ± 0.54 s,
similar to previous reports (25,32). NTD
truncation dramatically sped up this
process by about 3 times (rec = 0.91 ±
0.23 s) (Fig. 4A, 4B and Fig. S3). These
data thus suggest that the NTDs of KARs
strongly inhibit the recovery from
desensitization.
Netos speeds the recovery rate of
GluK2 but not GluK2NTD
When GluK2 was coexpressed with
Neto1 or Neto2, the recovery from
desensitization were facilitated (Fig. 4C),
consistent with previous reports (25,31-
33) that Netos speed KAR recovery.
Thus, removal of NTD and coexpression
with Netos had similar effects on GluK2
recovery from desensitization. Could
these two manipulations have synergistic
effects on the recovery rate? Very
surprisingly, we found the recovery rate
of GluK2NTD was notably slowed by
Neto2 with rec doubled but not by Neto1
(Fig. 4D). Neto2CUB1 also slowed the
recovery of GluK2 (Fig. 4E) and
GluK2NTD (Fig. 4F) by doubling the rec
while Neto1CUB1 had no effects (Fig. 4,
E and F). Thus, data in Figure 4D-F
indicated that core-Neto1 and core-
Neto2 differentially regulate GluK2
recovery; core-Neto2 slows recovery
while core-Neto1 has no effects.
Furthermore, the left shifting of the
recovery curve by Netos in Figure 4C
compared to the generally right shifting in
the absence of NTD-CUB1 interaction
(Fig. 4, D-F), indicated NTD-CUB1
interaction speeds KAR recovery from
desensitization.
The residues in Netos responsible for
NTD-CUB1 interactions
Thus far we have found that Neto
proteins interact with GluK2 through at
least two sites, the NTD and the core.
NTD-CUB1 interaction plays important
role in regulating GluK2 gating. We thus
wondered what exact sites are
responsible for this interaction. We first
made a model of Neto2CUB1 domain by
Homology Modeling using Deepview
software. The spindle-shaped CUB1
domain was polarized according to its
charge distribution (Fig. 5A). On one end
of the molecule, positively charged
arginine residues including Arg50, Arg81,
Arg83, Arg131 and Arg135 are clustered
to make the positive charge pole. On the
other end, negatively charged Asp144,
Glu145, Glu146 and Glu148 composed
of the negative charge pole. We then
examined whether these charges play a
role in the interaction with GluK2NTD by
mutating them to alanine residues. When
the 4 negatively charged residues were
mutated (DE4A), the Neto2CUB1
domain failed to pull down GluK2NTD
(Fig. 5B, arrow), while mutation of the 5
positively charged residues (R5A) did not
affect the pull-down efficiency. There are
3 negatively charged residues in the
corresponding region in the Neto1CUB1
domain (Fig. 5A, right panel). Mutation
on these negatively charged residues in
the Neto1CUB1 domain (DE3A) also
largely diminished the interaction with
GluK2NTD (Fig. 5C, arrow). We further
examined whether these negatively
charged residues were responsible for
the function of the NTD-CUB1 interaction.
When the 4 negatively charged residues
in Neto2 were mutated to alanines, the
slowing of GluK2 desensitization by
Neto2 was largely impaired (Fig. 5D and
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Fig. 2B). The Neto1(DE3A), in which the
3 negatively charged residues were
mutated, slightly slowed GluK2
desensitization (Fig. 5D). Interestingly,
when the negative charges neutralized,
the regulatory effects on GluK2
desensitization were now the same
between Neto1 and Neto2 (Fig. 5D),
resembling the CUB1 deleted Netos (Fig.
2D). The mutated Netos failed to
facilitate GluK2 recovery from
desensitization indicating the NTD-
CUB1 interaction was impaired (Fig. 5E),
resembling CUB1 deletions (Fig. 4E).
The residues in GluK2NTD
responsible for NTD-CUB1
interactions
Since the negatively charged pole
on the CUB1 domains are responsible
for the interaction with GluK2NTD, we
suspect that a positively charged patch
on the GluK2NTD surface might interact
with CUB1s. We then searched the
surface of GluK2NTD for highly positively
charged regions. The GluK2NTD dimer
was adapted from the full length GluK2
Cryo-EM structure (PDB ID: 5kuf). We
identified 6 positively charged clusters
which contain at least 2 positively
charged residues (in blue, Fig. 6A) from
one subunit of the NTD dimer. The
positively charged regions were
neutralized by mutating lysine and
arginine residues to alanine residues
(Fig. 6A). Other positively charged
residues scattered on the NTD surface
were not touched (in pink, Fig. 6A). Pull-
down experiments showed that when
group 1 positively charged residues in
GluK2NTD were mutated, the interaction
with Neto2CUB1 was largely diminished,
while mutations on groups 2-6 did not
affect NTD interaction with Neto2CUB1
(Fig. 6B). The group 1 residues contain
Arg50, Lys82, Lys93 and Lys94. Neither
single mutation on these residues nor
double mutations on Arg50 and Lys82
affected the pull-down efficiency (Fig.
6C). These data suggest the positively
charged residues in group 1 are
redundant. We then examined the
functional effects of mutating these
positively charged residues.
GluK2(RK4A), in which 4 positively
charged residues (Arg58, Lys82, Lys93
and Lys94) were mutated to alanines,
was modestly slowed by Neto1 and
Neto2 (Fig. 6D), resembling that of NTD
deletion (Fig. 2D). Netos failed to
facilitate the recovery of GluK2(RK4A)
from desensitization (Fig. 6E), indicating
the NTD-CUB1 interaction was impaired
with these mutations, resembling that of
GluK2NTD (Fig. 4D).
Differential regulation of
desensitization does not simply rely
on CUB1s
Neto1 and Neto2 have significant
differential effects on GluK2 fast gating
especially on the desensitization kinetics.
Our deletion and mutation experiments
indicate that the CUB1 domains might
account for the difference. To test this
hypothesis, we made chimeric
constructs of Netos by swapping the
CUB1 domains (Fig. S4A). Indeed,
Neto1(Neto2CUB1), Neto1 harboring
Neto2CUB1 slowed GluK2
desensitization in comparison to Neto1,
but the slowing effect was much less
than Neto2 (Fig. S4B and C). On the
other hand, Neto2(Neto1CUB1) slowed
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GluK2 desensitization just like Neto2 (Fig.
S4B and C). These data thus indicate
that the difference in CUB1 sequences,
at most, only partially accounts for the
differential regulatory effects on GluK2
fast gating by Netos.
NTD-CUB1 interactions do not affect
rectification
Another biophysical property of
KARs affected by Netos is voltage-
dependent blockage by polyamines
(26,34). We examined the rectification
property of GluK2 receptors after NTD
truncation in the presence of ConA to
prevent desensitization (35). NTD
deletion enhanced rectification
(Supporting Fig. S5, A and B), reducing
the rectification index (Fig. S5C). Both
Neto1 and Neto2 markedly reduced the
inward rectification of full-length GluK2
receptor (Fig. S5, A and C) and
GluK2NTD (Fig. S5, B and C). Neto1
effects were relatively stronger than
Neto2. These data thus suggested the
GluK2 NTD is not involved in Neto
modulation of receptor rectification.
DISCUSSION
In the present study, we have
experimentally defined two interactions
between GluK2 receptor and auxiliary
Neto proteins. The GluK2NTD directly
interacts with Neto proteins through
binding to CUB1 domains, defining the
NTD-CUB1 interactions. The core of
GluK2 interacts with Neto domains other
than CUB1, defining a second core-Neto
interaction. The NTD-CUB1 interaction
involves the static electric attraction
between a negatively charged cluster in
CUB1 domains and a positively charged
patch on the surface of GluK2NTD.
By coexpression of NTD truncated
GluK2 and CUB1 truncated Netos, we
have systemically examined 1. the NTD
alone, 2. the core-Neto interaction, 3. the
NTD-CUB1 interaction on KAR gating,
including desensitization, deactivation
and recovery from desensitization. The
three factors have different effects on
GluK2 fast gating (deactivation and
desensitization) and slow gating
(recovery from desensitization). To
facilitate the understanding of our data,
we made schematic models (Fig. 7) and
started from the smallest functional
receptor GluK2NTD. For fast gating
such as desensitization, comparison 1 in
Figure 7A suggests that NTD alone has
no effects on GluK2 desensitization.
Comparison 2 in Figure 7A suggests
core-Neto interactions slow KAR
desensitization. Comparison 3 in Figure
7A suggests NTD-Neto1CUB1
interaction speeds desensitization while
NTD-Neto2CUB1 slows desensitization.
For recovery from desensitization,
comparison 1 in Figure 7B suggests NTD
has strong inhibitory effects on GluK2
recovery. Comparison 2 and 2’ in Figure
7B suggest core-Neto1 has no effects on
KAR recovery from desensitization while
core-Neto2 slows the recovery.
Comparison 3 in Figure 7B suggest
NTD-CUB1 interactions facilitate GluK2
recovery.
Following the discovery of Netos as
KAR auxiliary subunits, numerous
studies have explored Neto regulation on
KAR kinetics, mostly through
recombinant systems or through
overexpression in neurons. A general
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conclusion is that the Neto regulation of
KARs is receptor subunit specific and
Neto-isoform specific. Neto1 speeds
GluK1 desensitization in recombinant
systems as well as overexpression in
hippocampal CA1 neurons (28,31).
Neto1 speeds GluK2 desensitization in
hippocampal CA1 neurons (11) but not in
HEK cells (this study). Neto1 has
relatively small or no effects on GluK1 or
GluK2 deactivation in neurons (11,28). In
contrast, Neto2 significantly slows the
desensitization and deactivation of
GluK1 and GluK2 in either recombinant
systems or neurons (11,25,28,36). We
find that deleting the NTD has no effect
on GluK2 decay kinetics. Interestingly,
the core-Neto interactions by Neto1 and
Neto2 similarly slow the desensitization
and deactivation of GluK2 under 5
conditions when NTD-CUB1 interactions
are disrupted: deletion of the NTD,
deletion of the CUB1,deletion of both,
mutation on the CUB1 negative charges,
or mutation on GluK2NTD positive
charges (Fig. 2C-E, 3C-E, 5D and 6D).
Only when full-length wt Neto1/2 are co-
expressed with GluK2, are the
desensitization and deactivation
dramatically different between Neto1 and
Neto2 (Fig. 2B and 3B). Therefore, the
differential modulatory effects of Neto1
and Neto2 on GluK2 desensitization and
deactivation rely on the CUB1 interaction
with the NTD of KARs. However, the
sequence difference in CUB1 domains
can’t explain the differential regulatory
effects of Neto1 and Neto2 on GluK2 fast
gating kinetics. By switching the CUB1
domains, the desensitization kinetics are
not switched (Fig. S4). Fisher (32) found
switching both CUB1 and CUB2 domain
between Neto1/2 can largely (yet
incompletely) switch the differential
gating properties on GluK1. Therefore,
the Neto isoform specific modulation on
KAR fast gating might require a more
sophisticated stereoscopic interaction
between NTD and CUB1. In addition, a
more complicated allosteric modulation
between NTD-CUB1 and core-Neto
interactions might exist. To fully
understand the differential modulatory
effects by Neto1 and Neto2 require the
structural picture of GluK/Neto complex
in future.
One interesting property of the
NTDs of glutamate receptors is their
effect on the recovery from
desensitization. NMDARs have little
desensitization, while non-NMDARs,
AMPARs and KARs, desensitize soon
after activation and recover in variable
time intervals (37). GluK2 fully recovers
from desensitization in seconds, while
AMPARs recover in hundreds of
milliseconds (25,38,39). During
desensitization, the association of LBD
dimers ruptures, and rearranges into
quasi-fourfold architecture (6,37,40).
Additionally, NTD dimers of GluA2
receptors appear to separate, whereas
the NTD dimers of GluK2 receptors
remain undisrupted. This may explain
why GluK2 is much more stable in the
desensitized state compared to GluA2
receptors (40-42). NTD truncated GluK2
receptors desensitize at the same rate as
intact receptors (Fig. 2A), but recover
three times faster (Fig. 4B). These
results are consistent with the structural
observations, suggesting that NTDs
stabilize the desensitized state of
AMPARs (7) or KARs.
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Netos were reported to facilitate the
recovery from desensitization of variable
homomeric and heteromeric KARs
(14,25,31-33,36). We also observed the
facilitation of GluK2 recovery by Netos
which completely rely on the NTD-CUB1
interactions. Under all conditions that
NTD-CUB1 interactions are disrupted,
including deletion of the NTD and/or
CUB1, and mutations on the interface,
the core-Neto interactions generally
stabilizes the desensitized states and
slows the recovery. For this effect, Neto2
is much stronger than Neto1.
A large amount of studies suggest
that KARs interact with Netos at multiple
sites. From the Neto side, CUB domains,
LDLa, TMD and CTD are involved
(25,26,32). However, which domains of
KARs that are involved in the interaction
remain largely unclear. A previous study
suggested that the linker between M3
and S2 is important for Neto-related
gating (43). Our biochemical results
demonstrate that both the isolated NTD
and the GluK2NTD are able to bind
Netos, consistent with the notion that
KARs bind Netos through multiple sites.
Functionally, the NTD-CUB1 interactions
and core-Neto interactions have
differential effects on GluK2 deactivation,
desensitization and recovery from
desensitization, suggesting a two-step
model for Neto regulation of KAR gating
(Fig. 7).
MATERIALS AND METHODS
Molecular Biology
GluK2 (Q form) and Neto2 from rat and
Neto1 from mouse were used in this
study. To ensure the co-expression of
Neto proteins and GluK2 receptor in the
same HEK293T cells, the cDNA of GluK2
was subcloned into vector pCAGGS-
IRES-EGFP, while Neto1 and Neto2
were subcloned into vector pCAGGS-
IRES-mCherry(28). Mutations in GluK2
and Netos were made by overlapping
PCR. Specifically, GluK2NTD was
made by deletion of Thr32-His400 of
GluK2 without disruption of the signal
peptide (residues 1-31). Additionally, we
subcloned the NTD together with the
signal peptide into vector pCAGGS-
IRES-EGFP to construct isolated NTD.
For pull-down experiments, an HA tag
was inserted into GluK2 constructs after
the sequence of signal peptide, and a
FLAG tag was inserted into C-termini of
Neto1 and Neto2 constructs. All the
constructs were confirmed using
sequencing over the entire length of the
coding region.
Western Blots
HEK293T cells were cultured using
DMEM with 10% FBS, and passaged
every two days. In western blot
experiment, HEK293T cell were
transiently transfected using
Lipofectamine 2000 Reagent (Invitrogen)
following manufacturer’s instructions.
The vectors used in transfection was
GluK2(mutation) : Neto(mutation) = 1:1
for biochemical experiments. 4 h later,
medium was changed and 100 M
DNQX (Abcam) was added to block
GluK2 receptor currents. Cells were
lysed in RIPA buffer 48 h later. The cell
lysates were kept on the ice for 30 min,
and then centrifuged at 13,800 x g, 4°C
for 30 min. After centrifugation, the
supernatant was transferred to a new
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tube and completely mixed with 4 X
loading buffer. Then the mix was
immediately loaded into 8% SDS-PAGE
gels in the presence of DTT. The protein
bands were transferred to PVDF
membranes (Millipore) at 100 V for 2 h,
and then blocked in 5% non-fat milk
dissolved in TBST at room temperature
for 1 h. Finally, the level of GluK2
receptor and Neto proteins were probed
with anti-HA antibody (Sigma, H3663),
anti-FLAG antibody (Sigma, F3165) or
anti-C-terminal GluR6/7 (Merk Millipore,
04-921) respectively, and detected using
the ECL substrate (Thermo) before
exposure.
Cell-Surface Biotinylation
Cells were washed three times with ice-
cold PBS before 1 mM solution of Sulfo-
NHS-LC-Biotin (Thermo Fisher Scientific
Life Sciences, catalog no. 21335) in PBS
for biotinylating cell surface proteins.
After incubating at 4°C for 30 min,
reactions were quenched with 50 mM
glycine, followed by rinsing three times
with ice-cold TBS. Cells were then
scraped in RIPA buffer (50 mM Tris HCl,
150 mM NaCl, 1% Triton X-100, 1%
sodium deoxycholate, 0.1% SDS, 10 mM
sodium phosphate, 2 mM EDTA, and 0.2%
sodium vanadate) supplemented with a
mixture of protease inhibitors (Roche
Applied Science) and solubilized for 1 h
at 4°C. Nonsolubilized particles was
removed by centrifugation at 13,800 x g
for 10 min at 4°C. The solubilized protein
concentration was determined by BCA
assay and mixed with monomeric avidin
agarose beads (Thermo Fisher Scientific
Life Sciences, catalog no. 20228). The
mixture was incubated for 1 h with
rotation at room temperature. Beads
were subsequently washed three times
with PBS. Finally, proteins were eluted by
boiling in Laemmli buffer and then
separated by electrophoresis on 8%
SDS-PAGE gels.
Immunocytochemistry and Confocal
Microscopy
Cell surface receptors were detected by
non-permeabilized
immunocytochemistry. HEK293T cells
were washed in PBS and fixed in 4% PFA
in PBS. After blocking in normal goat
serum, cell-surface GluK2 or
GluK2NTD staining was examined
using mouse anti-HA antibody (Sigma,
H3663), followed by goat anti-mouse
Alexa 549 secondary antibody. Samples
were then permeabilized with 0.1%
Triton X-100, and total GluK2 content
was determined by staining with rabbit
anti-C-terminal GluR6/7 (Merk Millipore,
04-921) and goat anti-rabbit Alexa 488.
After the secondary antibody was
washed by PBS for three times, the cells
were additionally incubated with Hoechst
33258 for nuclear staining. Samples
were examined and analyzed through a
63 X oil immersion lens on a Zeiss
LSM880 microscope.
Immunoprecipitation
Transfected cells were washed three
times with PBS, and harvested and
solubilized in Lysis buffer (50 mM Tris-Cl,
PH 7.2, 150 mM NaCl, 2 mM EDTA, and
0.1% Triton X-100), supplemented with a
mixture of protease inhibitors (Roche
Applied Science) and solubilized for 1h
at 4°C. After centrifuged at 13,800 x g for
10 min, the pellet was discarded. Lysates
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were then incubated with antibodies at
4°C overnight. Then lysates were
incubated with Protein G beads (GE
Healthcare) for 2 h at 4°C on a rotating
platform. After incubation, beads were
washed 4 times with Lysis buffer and
boiled in 40 ul 2 X Laemmli buffer. The
mixtures were then centrifuged at 13,800
x g and the supernatant was used for
detection by western blot. For all
samples, 1% of that used for IPs was
used for input in gel analysis.
Electrophysiology
Whole Cell Electrophysiology
Recording Whole cell recording was
performed on transfected HEK293T cells
as described previously (44). The
vectors used in transfection for
electrophysiology recording was
GluK2(mutation): Neto(mutation) = 1:2 to
ensure that Netos were expressed with
sufficient amount compared to GluK2.
The cells were bathed in the extracellular
solution (in mM): 145 NaCl, 2.5 KCl, 1
CaCl2, 1 MgCl2, 10 glucose, and 10
HEPES (pH 7.4). The positively
transfected cells were identified by
fluorescence via epifluorescence
microscopy. Whole cell patches were
performed with glass pipettes (3 to 5 MΩ)
filled with intracellular solution (in mM):
140 CsCl, 4 MgCl2, 1 EGTA, and 10
HEPES, 4 Na2ATP, 0.1 spermine (pH
7.4). Before recording, the transfected
cells were incubated with 1 mg/ml ConA
for at least 5 mins to prevent GluK2
receptor desensitization. The current-
voltage curves were recorded with a
ramp voltage protocol from -100 mV to
+100 mV in a period of 700 ms for 10
times in the presence of 1 mM glutamate
diluted in extracellular solution. All the
currents were collected with an
Axoclamp 700B amplifier and Digidata
1440A (MolecularDevices, Sunnyvale,
CA, USA), filtered at 3 kHz, and digitized
at 10 kHz. The current data were
analyzed using Clampfit software.
Outside-Out Patch Recording The
outside-out patches was pulled from
transfected HEK293T cells and recorded
as previously reported (25). The external
solution was (in mM): 140 NaCl, 2.5 KCl,
2 CaCl2, 1 MgCl2, 5 glucose, and 10
HEPES (pH 7.4). Patch pipettes
(resistance 3 to 5 MΩ) were filled with a
solution containing (in mM): 130 KF, 33
KOH, 2 MgCl2, 1 CaCl2, 11 EGTA, and 10
HEPES (pH 7.4). 10 mM glutamate
diluted into the external solution was
applied with theta glass pipettes
mounted on a piezoelectric bimorph. The
deactivation and desensitization were
recorded by 1 ms and 500 ms glutamate
application respectively, and analyzed by
fitting with a single exponential function
A= A0*exp(-t/
) + C or a double
exponential function A= A0*(f1*exp(-t/
f) +
f2*exp(-t/
s) ) + C. In these functions t is
the time. The currents amplitude (A)
starts at A0 and decays down to C. In our
recording, the steady state currents C
was generally undetectable. f1 and f2 are
the fractions of respective components
as percent (f1 + f2 = 1), and f and s are
decay kinetics of fast and slow
components. The weighted was
calculated using the formula: weighted
= f1*
f + f2*
s. The recovery from
desensitization was examined by pairs of
50 ms applications of 10 mM glutamate,
with intervals ranged from 5 ms to 8000
ms. The recovery ratio was calculated
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via dividing the second peak amplitude
by the first peak and analyzed by fitting
with a single exponential function: f =(fmax
- C)*exp(-t/
rec) + C, where t is the time;
fmax is the maximal recovery; C is non-
desensitized steady state fraction at the
end of 50 ms glutamate application;
rec
is the recovery constant.
Homology Modeling
A 3D model of Neto2CUB1 was made by
Homology Modeling using Deepview
software. The amino acid sequence of
Neto2CUB1 was loaded into the
workspace and BLAST against ExPDB
database for searching appropriate
templates. Cubilin (PDB ID: 3kq4) was
chosen as an optimal template because
of the high coverage rate (98%) and
sequence identity (39%). Homologous
sequence in Cubilin was residues 234-
346. The model was computed and built
by the SWISS-MODEL server. This
model and GluK2 NTD dimer structure
(adapted from PDB ID: 5kuf) were
viewed and depicted using Pymol
software.
Statistical Analysis
Data were presented as mean ± SD from
three or more independent experiments.
Statistical analyses were carried out
using GraphPad Prism 7 software and
analyzed using one-way ANOVA test,
two-way ANOVA or unpaired t-test if not
otherwise stated. All p < 0.05 was
considered significant and labeled as *, p
< 0.01 was labeled as **, and p < 0.001
was labeled as ***.
ACKNOWLEDGMENTS
We thank prof. Roger Nicoll in UCSF for his critical comments on the manuscript. This
work is supported by grants from the National Natural Science Foundation of China
(91849112, 31571060, 81573416, 31371061), the Natural Science Foundation of
Jiangsu Province (BE2019707) and the Fundamental Research Funds for the Central
Universities (0903-14380029).
CONFLICTS OF INTEREST
The authors declare no conflicts of interest.
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Figure 1. Neto1/2 have two separate interaction sites with GluK2.
A and B, Immunoblot of immunoprecipitates from transfected HEK293T cells. The
identities of the transfected constructs were indicated above each lane. Full-length
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GluK2, GluK2NTD and GluK2NTD were co-immunoprecipitated with Neto1 and
Neto2.
C, The GluK2NTD were co-immunoprecipitated with CUB1 domains of Neto1/2.
D, Co-immunoprecipitation of GluK2NTD and Neto proteins without CUB1 domains.
E and F, Co-immunoprecipitation of GluK2NTD and Neto proteins with or without
CUB1 domains. Deletion of CUB1 domains significantly suppressed the interaction
between GluK2 and Neto proteins (arrows).
G, Quantification of immunoprecipitation in (E) and (F). The GluK2NTD pulled-down
was normalized by the FLAG signal pulled-down. Compared to full-length Neto1 (1.00
± 0.32), the GluK2NTD precipitated by Neto1CUB1 was significantly reduced to 0.38
± 0.08 (n = 4 pairs, **p < 0.01, paired t-test). Compared to full-length Neto2 (1.00 ±
0.22), the GluK2NTD pulled down by Neto2CUB1 was significantly reduced to 0.24
± 0.03 (n = 4 pairs, ***p < 0.001, paired t-test).
H, A schematic model shows the two interaction sites between GluK2 and Neto
proteins.
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Figure 2. Neto regulation on GluK2 desensitization
A, Deletion of NTD has no effects on GluK2 desensitization. Up panel, superimposed
average desensitization traces of GluK2 and GluK2NTD. Left low panel, statistical
comparison between the des of GluK2 and GluK2NTD. Right low panel, a schematic
model depicts deletion of NTD.
B, The modulatory effects of Neto1 and Neto2 on GluK2 desensitization. Up panel
shows superimposed average desensitization traces. Left low panel, statistical
comparison among the des of GluK2 with and without Netos. While Neto1 has no
effects on GluK2 desensitization, Neto2 slows GluK2 desensitization by about 4 times
(***p < 0.001). Neto1 and Neto2 exhibited deferential modulation on GluK2
desensitization (GluK2+Neto1 vs. GluK2+Neto2, ***p < 0.001). Right low panel, a
schematic model depicts the NTD-CUB1 and core-Neto interactions.
C, The modulatory effects of Neto1 and Neto2 on GluK2NTD desensitization. Up
panel shows superimposed average desensitization traces. Left low panel, statistical
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comparison among the des of GluK2NTD with and without Netos. Both Neto1 and
Neto2 has modest slowing effects on GluK2NTD (*p < 0.05), no difference was found
between GluK2NTD+Neto1 and GluK2NTD+Neto2. Right low panel, a schematic
model depicts the core-Neto interaction under these conditions.
D, CUB1-deleted Netos have no apparent effects on GluK2 desensitization.
E, The modulatory effects of CUB1-deleted Netos on GluK2NTD. Neto1CUB1
slowed the desensitization of GluK2NTD (**p < 0.01). Neot2CUB1 slowed the
desensitization of GluK2NTD (*p < 0.05). No significant difference was found
between GluK2NTD+Neto1CUB1 and GluK2NTD+Neto2CUB1.
The raw desensitization traces were depicted and the average desensitization traces
calculated in supporting Figure S2A. The data were analyzed using Two-way ANOVA
with post hoc Tukey's multiple comparisons tests (Netos or mutants, F (4,151) = 22.80,
p < 0.001; NTD, F (1, 151) = 0.16, p = 0.69; interaction, F (4, 151) = 25.69, p < 0.001).
Statistical significance was denoted as: *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not
significant.
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Figure 3. Neto regulation on GluK2 deactivation.
A, Deletion of NTD has no effects on GluK2 deactivation.
B, The modulatory effects of Neto1 and Neto2 on GluK2 deactivation. Up panel shows
superimposed average deactivation traces. Low panel, while Neto1 has no effects on
GluK2 desensitization, Neto2 slows GluK2 deactivation (*p < 0.05). Neto1 and Neto2
exhibited deferential modulation on GluK2 deactivation (GluK2+Neto1 vs.
GluK2+Neto2, ***p < 0.001).
C, The modulatory effects of Neto1 and Neto2 on GluK2NTD deactivation. Up panel
shows superimposed average desensitization traces. Low panel, both Neto1 and
Neto2 has modest slowing effects on GluK2NTD (*p < 0.05). No difference was found
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between GluK2NTD+Neto1 and GluK2NTD+Neto2.
D, Neto1CUB1 slightly slowed (*p < 0.05) while Neto2CUB1 has no effects on GluK2
deactivation. However, the effects were not significantly different between
Neto1CUB1 and Neto2CUB1.
E, The modulatory effects of CUB1-deleted Netos on GluK2NTD deactivation. Both
Neto1CUB1 and Neto2CUB1 slowed the deactivation of GluK2NTD (***p < 0.001).
No significant difference was found between GluK2NTD+Neto1CUB1 and
GluK2NTD+Neto2CUB1.
The raw deactivation traces were depicted and the average deactivation traces
calculated in supporting Figure S2B. The data were analyzed using Two-way ANOVA
with post hoc Tukey's multiple comparisons tests (Netos or mutants, F (4, 129) = 14.68,
p < 0.001; NTD, F (1, 129) = 47.39, p < 0.001; interaction, F (4, 129) = 10.00, p <
0.001). Statistical significance was denoted as: *p < 0.05, **p < 0.01, ***p < 0.001, n.s.,
not significant.
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Figure 4. Neto regulation on GluK2 recovery from desensitization
A, Representative recording traces of GluK2 and GluK2NTD evoked by pairs of 50
ms applications of 10 mM glutamate. The interval time between two applications
ranged from 5 ms to 8000 ms. The amplitude was normalized to the first peak.
B, Analysis of the recovery rates of GluK2 and GluK2NTD. Left, the recovery was
calculated by the peak amplitude of the 2nd response divided by that of the 1st
response. Data points represented mean ± SD. The average data was fitted with a
single exponential equation (GluK2, black; GluK2NTD, red). Right, the time constants
(rec) were compared between GluK2 and GluK2NTD. Deletion of NTD significantly
speeded the receptor recovery from desensitization (***p < 0.001).
C, Netos regulate GluK2 recovery from desensitization. Both Neto1 and Neto2
speeded GluK2 recovery from desensitization (*p < 0.05 and **p < 0.01 respectively).
D, Netos differentially regulate GluK2NTD recovery from desensitization. Neto1 has
no effects on GluK2NTD recovery from desensitization. Neto2 slowed GluK2NTD
recovery from desensitization (*p < 0.05).
E, CUB1 deleted Netos differentially regulate GluK2 recovery from desensitization.
Neto1CUB1 has no effects on GluK2 recovery from desensitization. Neto2CUB1
slowed GluK2 recovery from desensitization (***p < 0.001).
F, CUB1 deleted Netos differentially regulate GluK2NTD recovery from
desensitization. Neto1CUB1 has no effects on GluK2NTD recovery from
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desensitization. Neto2CUB1 slowed GluK2NTD recovery from desensitization (*p <
0.05)
The representative traces for recovery from desensitization were depicted in
supporting Figure S3. The data were analyzed using Two-way ANOVA with post hoc
Tukey's multiple comparisons tests (Netos or mutants, F (4, 93) = 29.92, p < 0.001;
NTD, F (1, 93) = 109.80, p < 0.001; interaction, F (4, 93) = 20.22, p < 0.001). Statistical
significance was denoted as: *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant.
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Figure 5. Critical residues on CUB1 domains for NTD-CUB1 interactions.
A, A homology model for Neto2CUB1 domain. Left panel, distribution of the charges
on Neto2CUB1 surface. Negatively charged residues are shown in red, and positively
charged residues are shown in blue. The molecule is polarized according to its charge
distribution. Middle panel, the model is rotated for better view of the negatively charged
pole. Right panel, sequence alignment around the negatively charged residues
between Neto1 and Neto2.
B, Co-immunoprecipitation of Neto2CUB1 mutants with GluK2NTD. The interaction
between GluK2NTD and Neto2CUB1 was significantly diminished when 4 negatively
charged residues were mutated to alanine residues (arrow). The mutation on 5
residues on the positively charged pole did not affect CUB1 interaction with GluK2NTD.
Right panel, bar graph shows the relative pull-down efficiency from 3 experiments. The
GluK2NTD pulled-down were normalized by the FLAG signal pulled-down.
C, Mutation on the 3 negatively charged residues in Neto1CUB1 domain diminished
its binding to GluK2NTD (arrow). Right panel, bar graph shows the relative pull-down
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efficiency from 3 experiments. The GluK2NTD pulled-down were normalized by the
FLAG signal pulled-down.
D, Neutralization on the negative charges affect Neto regulation on GluK2
desensitization. Left, superimposed average desensitization traces of GluK2 with or
without mutated Netos recorded in a parallel experiment. Right panel, bar graph shows
the weighteddes. GluK2, 2.69 ± 0.35 ms, n = 8; GluK2 + Neto1(DE3A), 4.10 ± 0.91
ms, n = 10; GluK2 + Neto2(DE4A), 3.75 ± 0.83 ms, n = 8. One-way ANOVA with post
hoc Tukey's multiple comparisons tests, F (2, 23) = 7.24, p < 0.01. *p < 0.05; **p <
0.01; n.s., not significant.
E, Neutralization on the negative charges affect Neto regulation on GluK2 recovery
from desensitization. Left, the recovery of GluK2 with or without mutant Netos recorded
in a parallel experiment. Right, analysis of the recovery rates. GluK2+Neto1(DE3A)
(2.36 ± 0.77 s, n = 13) was not different from GluK2 (2.45 ± 0.92 s, n = 12). Neto2(DE4A)
slowed GluK2 recovery (4.24 ± 1.40 s, n = 8, **p < 0.01, one-way ANOVA with post
hoc Tukey's multiple comparisons, F (2, 30) = 9.17, p < 0.001).
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Figure 6. Critical residues in GluK2NTD for NTD-CUB1 interaction.
A, The NTD dimer of GluK2 is adapted from Cryo-EM structure of GluK2 (PDB ID:
5kuf). Highly positive patches on the surface of subunit A containing at least two
positively charged residues were identified (in blue). Positively charged residues
scattered on NTD surface were shown in pink. Right, the residues composed of the 6
highly positive patches on NTD surface.
B, Co-immunoprecipitation of Neto2CUB1 domain coexpressed with GluK2NTD with
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or without mutations. Lanes 1-6 are GluK2NTD mutations carry alanine replacement
of positively charged residue identified in (A). The interaction between GluK2NTD and
CUB1 domain was significantly disrupted when the 4 positively charged residues in
group 1 were mutated to alanine residues (arrow). Low panel, bar graph shows the
pull-down efficiency from 3 experiments.
C, Co-immunoprecipitation of Neto2CUB1 domain with GluK2NTD mutations. 4A, the
same mutant as lane 1 in (B). 2A, R58A_K82A. The interaction between GluK2NTD
and CUB1 domain was significantly disrupted in 4A mutation of the GluK2NTD but not
in the series of single mutation or double mutation. Low panel, bar graph quantified the
pull-down efficiency from 3 experiments.
D, Neto1 and Neto2 regulation on the desensitization of GluK2(RK4A). Both Neto1
(5.89 ± 3.01 ms, n = 19) and Neto2 (5.78 ± 1.72, n = 12) slowed the desensitization of
GluK2(RK4A) (2.47 ± 0.23 ms, n = 10). **p < 0.01, one-way ANOVA with post hoc
Tukey's multiple comparisons, F (2, 30) = 9.17, p < 0.001.
E, GluK2(RK4A) recovery from desensitization with or without Netos. Neto1 (1.27 ±
0.22 s, n = 13) had no effects on GluK2(RK4A) recovery (1.19 ± 0.38 s, n = 18). Neto2
(1.95 ± 0.69 s, n = 15) slowed GluK2(RK4A) recovery. ***p < 0.001, one-way ANOVA
with post hoc Tukey's multiple comparisons, F (2, 43) = 11.08, p < 0.001.
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Figure 7. Schematic models summarizing NTD and Neto modulation of GluK2
gating.
A, The effects of the three factors, NTD, core-Neto interaction and NTD-CUB1
interaction on GluK2 desensitization. The model starts from the smallest functional
receptor GluK2NTD. NTD has no effects on desensitization (comparison 1). Core-
Neto interaction with either Neto1 or Neto2 slows desensitization (comparison 2). NTD-
Neto1 speeds up desensitization while NTD-Neto2 slows desensitization (comparison
3). These models can be applied for GluK2 deactivation.
B, The three factors on the recovery from desensitization. Among the three factors,
NTD appear to have most dramatic effects, which stabilizes the receptor in
desensitization state (comparison 1). Core-Neto1 has little effects on while core-Neto2
slows the recovery speed either on NTD truncated (comparison 2) or full-length GluK2
(comparison 2’). The NTD-CUB1 interaction speeds GluK2 recovery (comparison 3).
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Chen, Yong-Yun Shi, Jun Wang, Wei Zhang and Yun Stone Shi
Yan-Jun Li, Gui-Fang Duan, Jia-Hui Sun, Dan Wu, Chang Ye, Yan-Yu Zang, Gui-Quan
through two binding sites
Neto proteins regulate gating of the kainate-type glutamate receptor GluK2
published online October 18, 2019J. Biol. Chem.
10.1074/jbc.RA119.008631Access the most updated version of this article at doi:
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