Upload
others
View
2
Download
0
Embed Size (px)
Citation preview
Article
Interaction between the L
inker, Pre-S1, and TRPDomains Determines Folding, Assembly, andTrafficking of TRPV ChannelsHighlights
d TRPV4 folding and trafficking require intrasubunit interaction
d It involves an alternating hydrogen network between pre-S1,
TRP, and pre-S1 linker
d This interaction seems to be common among TRPV channels
Garcia-Elias et al., 2015, Structure 23, 1404–1413August 4, 2015 ª2015 Elsevier Ltd All rights reservedhttp://dx.doi.org/10.1016/j.str.2015.05.018
Authors
Anna Garcia-Elias,
Alejandro Berna-Erro,
Fanny Rubio-Moscardo, ...,
Ruben Vicente,
Fernando Gonzalez-Nilo,
Miguel A. Valverde
In Brief
Transient receptor potential (TRP)
cationic channels are important cellular
sensors of the environment. Garcia-Elias
et al. show that the interactions among
different domains of the same subunit are
structural determinants of TRP channel
folding and assembly.
Structure
Article
Interaction between the Linker, Pre-S1, and TRPDomains Determines Folding, Assembly, andTrafficking of TRPV ChannelsAnna Garcia-Elias,1,4 Alejandro Berna-Erro,1,4 Fanny Rubio-Moscardo,1 Carlos Pardo-Pastor,1 Sanela Mrkonji�c,1
Romina V. Sepulveda,2,3 Ruben Vicente,1 Fernando Gonzalez-Nilo,2,3 and Miguel A. Valverde1,*1Laboratory of Molecular Physiology and Channelopathies, Department of Experimental and Health Sciences, Universitat Pompeu Fabra,
C/ Dr. Aiguader 88, Barcelona 08003, Spain2Universidad Andres Bello, Center for Bioinformatics and Integrative Biology, Facultad de Ciencias Biologicas, Av. Republica 239,
Santiago 8320000, Chile3Centro Interdisciplinario de Neurociencia de Valparaıso, Facultad de Ciencias, Universidad de Valparaıso, Valparaıso 2366103, Chile4Co-first author*Correspondence: [email protected]
http://dx.doi.org/10.1016/j.str.2015.05.018
SUMMARY
Functional transient receptor potential (TRP) chan-nels result from the assembly of four subunits.Here, we show an interaction between the pre-S1,TRP, and the ankyrin repeat domain (ARD)-S1linker domains of TRPV1 and TRPV4 that is essen-tial for proper channel assembly. Neutralization ofTRPV4 pre-S1 K462 resulted in protein retentionin the ER, defective glycosylation and trafficking,and unresponsiveness to TRPV4-activating stimuli.Similar results were obtained with the equivalentmutation in TRPV1 pre-S1. Molecular dynamicssimulations revealed that TRPV4-K462 generatedan alternating hydrogen network with E745 (TRPbox) and D425 (pre-S1 linker), and that K462Qmutation affected subunit folding. Consistently,single TRPV4-E745A or TRPV4-D425A mutationsmoderately affected TRPV4 biogenesis while dou-ble TRPV4-D425A/E745A mutation resumed theTRPV4-K462Q phenotype. Thus, the interaction be-tween pre-S1, TRP, and linker domains is manda-tory to generate a structural conformation thatallows the contacts between adjacent subunits topromote correct assembly and trafficking to theplasma membrane.
INTRODUCTION
Transient receptor potential (TRP) cationic channels are impor-
tant cellular sensors of the environment due to their role in the
transduction of physical and chemical stimuli (Voets et al.,
2005). Each subunit of the TRP family contains six transmem-
brane segments (S1–S6) with a pore region between S5 and
S6, and intracellular N- and C-terminal tails (Montell, 2005).
The classical (TRPC) and vanilloid (TRPV) subfamilies as well
as the TRPA1 channel present several N-terminal ankyrin
1404 Structure 23, 1404–1413, August 4, 2015 ª2015 Elsevier Ltd Al
repeat domains (ARDs) (Montell, 2005). A C-terminal TRP box
domain immediately after S6 is reported for members of the
TRPV, TRPC, and melastatin (TRPM) subfamilies. Similar to
voltage-gated K+ channels, TRP channels assemble as tetra-
mers with 4-fold symmetry and a central ion permeation pore
(Huynh et al., 2014; Liao et al., 2013; Maruyama et al., 2007;
Mio et al., 2007; Moiseenkova-Bell et al., 2008; Shigematsu
et al., 2010). Heteromeric TRP channels formed within the
same or different subfamilies are possible (Cheng et al., 2010;
Hoenderop et al., 2003; Schaefer, 2005), adding functional
diversity to a family of channels already characterized by their
polymodal nature.
The proper folding and assembly of different ion-channel sub-
units is likely mediated bymultiple domains generating intra- and
intersubunit interactions (Green andMillar, 1995). In this context,
TRP tetramerization involves transmembrane segments (Hellwig
et al., 2005), ARD (Arniges et al., 2006; Erler et al., 2004; Hellwig
et al., 2005), and different regions of the N tails (Chang et al.,
2004; Myeong et al., 2014; Pertusa et al., 2014) and C tails
(Becker et al., 2008; Erler et al., 2006; Hellwig et al., 2005; Lei
et al., 2013; Zhang et al., 2011), including the TRP box (Garcia-
Sanz et al., 2004).
Within the vanilloid subfamily of TRP channels, the heat-
activated TRPV1 and TRPV4 channels present a high degree
of similarity in their sequence and biophysical properties
(Owsianik et al., 2006). The TRPV4 cationic channel is widely
distributed and participates in the transduction of osmotic
(Arniges et al., 2004; Liedtke et al., 2000; Tian et al., 2009), me-
chanical (Andrade et al., 2005; Liedtke et al., 2003; Suzuki
et al., 2003), heat (Garcia-Elias et al., 2013; Guler et al., 2002;
Watanabe et al., 2002a), and UVB stimuli (Moore et al., 2013).
TRPV1 is expressed primarily on nociceptive neurons and
can be activated by capsaicin, noxious heat, and protons (Ca-
terina et al., 1997).
In the present study we have addressed structural determi-
nants governing TRPV subunit folding and assembly, focusing
mainly on TRPV4 but also on TRPV1. We have shown how
the electrostatic interactions of a triad of residues within the
ARD-S1 linker, pre-S1, and TRP box govern the overall channel
architecture.
l rights reserved
Figure 1. Sequence Alignment of Human
TRPVs ARD-S1 Linker, Pre-S1, and TRP
Domains
RESULTS
Neutralization of Positive Charges in the Pre-S1 DomainThe alignment of the pre-S1 region of TRPV channels (Figure 1)
showed the presence of positive residues (arginine, lysine,
histidine) close to conserved aromatic residues common to all
TRPVs. This feature, attributed to an evolutionary pressure on
all TRPVs (Donate-Macian and Peralvarez-Marın, 2014), points
to an important role of this region in channel regulation. The
recently resolved structure of rat TRPV1 (rTRPV1) suggests the
interaction of pre-S1 with the TRP box located immediately after
S6, and its role in channel gating (Liao et al., 2013). In an exper-
imental evaluation of the importance of the human TRPV4 pre-S1
region, we neutralized the three positive charges within the
sequence TRPV4-462KWRK465. Wild-type TRPV4 (TRPV4-WT)
(Figure 2A) protein displayed a robust cell surface expression
while TRPV4-462AWAA (Figure 2B) protein showed a reticular
intracellular immunostaining, indicative of retention in the ER.
Mock transfected cells incubated with anti-TRPV4 antibody are
shown in Figure S1A. Functional evaluation of the TRPV4 mutant
consisted of the transient transfection of HeLa cells and the
measurement of intracellular Ca2+ concentration ([Ca2+]i) in
response to three different well-known TRPV4-activating stimuli
(Figure 2C). As a negative control, HeLa cells were transfected
with GFP. Heat (38�C), hypotonicity (30%), and the synthetic
agonist GSK1016790A (10 nM) (Thorneloe et al., 2008) induced
increases in [Ca2+]i in TRPV4-WT while similar responses were
obtained in TRPV4-462AWAA and GFP transfected cells.
Patch-clamp whole-cell electrophysiological analysis of HeLa
cells (Figures 2D and 2E) showed that hypotonicity and the syn-
thetic agonist 4a-phorbol 12,13-didecanoate (4a-PDD, 10 mM)
(Watanabe et al., 2002b) only activated cationic currents in
TRPV4-WT but not in TRPV4-462AWAA-expressing cells. Human
TRPV1 also presents positive as well as negative residues
around the equivalent tryptophan (hTRPV1-426KWDR429). Similar
to TRPV4-462AWAA, neutralization of all three charges in hTRPV1
(hTRPV1-426AWAA) resulted in a protein that was retained
intracellularly (Figure 2F) and was unable to elicit responses to
heat (45�C) and capsaicin (1 mM) when expressed in HeLa cells
(Figure 2G).
Subcellular Localization and Homomerization of TRPV4Pre-S1 MutantsThe localization and lack of channel activity of the TRPV4-462AWAA mutant resembles that reported for TRPV4 spliced
variants unable to oligomerize and leave the ER due to partial
deletions of ARD (Arniges et al., 2006). Thus, we next studied
the assembly of the TRPV4 mutant. Cross-complementation
Structure 23, 1404–1413, August 4, 2015
assays were run in which HeLa cells
were co-transfected with TRPV4-WT-
Flag and TRPV4-WT-YFP or TRPV4-462AWAA-YFP (all fused to the channel C
tail). We first checked that the presence
of the tag does not affect the localization of the protein
(Figure S1B). Flag-TRPV4-WT and TRPV4-WT-YFP double-
transfected HeLa cells showed an almost identical localization
(Figure 3A), with a clear staining of the plasma membrane and
overlapping of the signals plot profiles. On the other hand, co-
transfection of TRPV4-WT-Flag with TRPV4-462AWAA-YFP (Fig-
ure 3B) generated two well-differentiated patterns of localization
without overlapping at the plasma membrane (merge image
and plot profile analysis). TRPV4-462AWAA-YFP was retained
at the ER and TRPV4-WT-Flag stained the plasma membrane,
although the latter also showed a little intracellular retention
(Figure 3B). Co-immunoprecipitation studies confirmed a
weak interaction between TRPV4-WT and TRPV4-462AWAA,
compared with TRPV4-WT only subunits (Figure 3C). The prox-
imity of CFP- and YFP-tagged TRPV4 proteins, as an indication
of subunit assembly, was checked by the fluorescent resonance
energy transfer (FRET) technique (Arniges et al., 2006; Hellwig
et al., 2005). Figure 3D shows the maximal CFP increase
(FRET efficiency) after bleaching of the YFP signal in HeLa
cells transiently co-transfected with expression plasmids en-
coding CFP or YFP fused to the C tails of TRPV4-WT and
TRPV4-462AWAA. The location of the fluorescent tags (N or C
tails) did not affect the FRETmeasurements (Figure S2). Maximal
FRET efficiency of the TRPV4-462AWAA was one-third of that
obtained with TRPV4-WT, and comparable with the control con-
dition using soluble YFP.
Together, the results obtained with TRPV4-462AWAA and
hTRPV1-426AWAA mutants suggested that this charged region
is important for proper channel folding, assembly, and trafficking
to the plasma membrane. The positive charge conserved in this
region among the TRPV channels is a lysine located at position
462 in hTRPV4. Moreover, the published structure of rTRPV1
(Liao et al., 2013) already pointed to the presence of a hydrogen
bond between the equivalent lysine, rTRPV1-K425, in the pre-S1
and rTRPV-E709 in the TRP box. The predicted homology model
of TRPV4 based on the rTRPV1 coordinates revealed similar
hydrogen bonding between TRPV4-K462 and TRPV4-E745
(Figures 8A and 8D). Neutralization of TRPV4-K462 resulted
in intracellular retention of the protein, thereby losing their co-
localization with the plasma membrane marker concanavalin A
(Figures 4A and 4C) and showing a marked co-localization with
the ER marker calreticulin (Figure 4B). Analysis of the TRPV4-
K462Q glycosylation profile, unlike the WT, demonstrated no
Golgi-mediated glycosylation, further confirming the retention
of this mutant in the ER (Figure S3).
Calcium imaging experiments showed that the response of
TRPV4-K462Q transfected cells to heat (Figure 4D), hypotonicity
(Figure 4E), and GSK1016790A (Figure 4F) was indistinguishable
ª2015 Elsevier Ltd All rights reserved 1405
Figure 2. Expression and Activity of TRPV1
and TRPV4 Pre-S1 Mutants
(A and B) Representative immunofluorescence
localization of TRPV4-WT (A) and TRPV4-462AWAA
(B) proteins in transiently transfectedHEK293cells.
Corresponding phase-contrast images are shown
at the right. Scale bar, 20 mm.
(C) Fura-2 ratios obtained in HeLa cells transfected
with GFP, TRPV4-WT, and TRPV4-462AWAA, and
perfused with warm solutions (38�C), 30% hypo-
tonic solutions (HTS), and 10 nM GSK1016790A.
Traces are means ± SEM of 186–373 cells
measured in 5–13 independent experiments.
(D) Ramp current-voltage relations of cationic
currents recorded from HeLa cells transfected
with TRPV4-WT or TRPV4-462AWAA and exposed
to 30% hypotonic shocks and 10 mM 4a-PDD.
(E) Mean current density measured at +100 mV in
response to a 30% hypotonic shock and 10 mM
4a-PDD in TRPV4-WT or TRPV4-462AWAA. Num-
ber of cells recorded is shown for each condition.
**P < 0.01.
(F) Immunofluorescence localization of TRPV1-WT
and TRPV1-426AWAA proteins in transiently
transfected HeLa cells. Scale bar, 20 mm.
(G) Fura-2 ratios obtained in HeLa cells transfected
with GFP, TRPV1-WT, and TRPV1-426AWAA, and
perfused with solutions heated at 45�C and 1 mM
capsaicin. Traces aremeans±SEMof 76–146cells
measured in 4–7 independent experiments.
See also Figure S1.
from the response of GFP transfected cells. Similar results
were obtained using a different cell line (HEK293 cells) to overex-
press WT and mutant TRPV4 channels (Figure S4). FRET exper-
iments confirmed defective assembly of channels containing
the mutant TRPV4-K462Q subunit alone or in combination with
WT subunits (Figure 4G). The analysis of TRPV4-426QQWQ and
TRPV4-K462A provided results identical to those obtained with
TRPV4-K462Q (Figures S5A–S5C), suggesting that the charge,
rather than the size of the residue, is the relevant factor in correct
functioning of the channel. Neutralization of a set of positive
charges in the distal end of the TRPV4 C tail (862RKWR865) did
not affect the response of the channel (Figure S5D). Changes
1406 Structure 23, 1404–1413, August 4, 2015 ª2015 Elsevier Ltd All rights reserved
in the total expression levels between
WT and mutant TRPV4 proteins were
also discarded as the reason for the func-
tional differences observed (Figure S6).
Similar to TRPV4, single mutation of the
equivalent hTRPV1 residue (TRPV1-
K426Q) resulted in a non-functional
hTRPV1 channel (Figure 5).
Interaction between Pre-S1, ARD-S1 Linker, and TRP DomainsTo further analyze the relevance of the
pre-S1 lysine in the architectural organi-
zation of the channel complex, the inter-
action of this lysine with other negative
residues was studied using molecular
dynamics simulations. Similar to previous
dynamics studies (Lindy et al., 2014; Poblete et al., 2015; Teng
et al., 2015), we used the structural data available for rTRPV1 to
simulate 100 ns of both rTRPV1 and hTRPV4 (Movies S1 and
S2). First, rTRPV1 presented amean of 4.3 ± 2.1 hydrogen bonds
within 3 A from K425 and hTRPV4 presented 5.6 ± 1.9 bonds
within 3 A fromK462 (Figure S7). Second, we confirmed the inter-
action between K425 and E709 in rTRPV1 (Figure S7A) and
between K462 and E745 in hTRPV4 (Figures S7B and 8A). Third,
we identified a negative residue, D388, in the linker domain be-
tweenARDandpre-S1 of rTRPV1 (Figure S7A) and the equivalent
D425 in TRPV4 (Figure 8A) with the ability to generate hydrogen
bonds with rTRPV1-K425 and hTRPV4-K462, respectively. To
Figure 3. Assembly of TRPV4-WT and
TRPV4-462AWAA Proteins
(A and B) HeLa cells were co-transfected with
TRPV4-WT-Flag and TRPV4-WT-YFP (A) or
TRPV4-WT-Flag and TRPV4-462AWAA-YFP (B).
The merge panels display the co-localization be-
tween the Flag and YFP signals (yellow). The white
line on the merged figures indicates the plot profile
analysis (right) performed on each image using
ImageJ software. Arrows show the location of the
plasma membrane at both ends of the line. Scale
bar, 20 mm.
(C) Co-immunoprecipitation of TRPV4-WT and
TRPV4-462AWAA proteins. Left panels show
expression of TRPV4-WT-YFP, TRPV4-WT-Flag,
and TRPV4-462AWAA-YFP proteins. TRPV4 was
immunoprecipitated with anti-Flag antibody and
analyzed by western blots with either anti-Flag
(bottom right) or anti-GFP (top right). The combi-
nation of plasmids transfected into HeLa cells is
also shown.
(D) Maximal high FRET efficiencies corresponding
to homomultimer formation could only be demon-
strated for TRPV4-WT. Number of cells recorded is
shown for each condition. Mean ± SEM. **P < 0.01
versus all other conditions.
See also Figure S2.
establish the relative contribution of these negatively charged
residues to the biogenesis of the TRPV4 channel, we neutralized
these residues either individually or simultaneously.
Both TRPV4-D425A and TRPV4-E745A, unlike the TRPV4-WT,
showed preferential co-localization with the ER marker (Figures
6A and 6B). The double mutant TRPV4-D425A/E745A also co-
localized with the ERmarker (Figures 7A and 7B) and completely
abolished channel response to heat and hypotonicity (Figure 7C).
TRPV4-E745A and TRPV4-D425A response to heat and hypoto-
nicity was greatly reduced (Figure 7C), though not abolished
(more evident in the case of TRPV4-D425A). The fact that no sta-
tistically significant calcium increases betweenGFP, TRPV4-WT,
and mutant transfected cells were obtained in the absence of
extracellular Ca2+ (Figure S8) suggested that the Ca2+ signal re-
corded in response to the TRPV4 stimuli is generated by Ca2+
influx via TRPV4 channels. Therefore, it is plausible that a few
TRPV4-D425A and TRPV4-E745A channel subunits may have
escaped theERquality controlmechanismand formed functional
channels that reached the plasma membrane and responded to
the TRPV4 stimuli. Consistent with the defective channel activity
of mutants in the linker and TRP domains, all these mutants
showed markedly reduced FRET values (Figure 7D).
Impact of Mutations in the Pre-S1 on the OverallChannel ArchitectureOur in vitro experiments are consistent with a defect in the
assembly of the mutant channels, and we used molecular dy-
Structure 23, 1404–1413, August 4, 2015
namics simulations to further interrogate
how pre-S1 mutations affect the overall
architecture of a preformed channel. The
calculated distance between rTRPV1-
K425(Cd) and rTRPV1-E709(Cd) was
5.48 ± 0.4 A (n = 4), and that between
TRPV1-K425(Cd) and TRPV1-D388(Cg) was 8.17 ± 0.7 A (n =
4). In hTRPV4 the distance between K462(Cd) and E745(Cd)
was 4.6 ± 0.4 A (n = 4), and that between K462(Cd) and
D425(Cg) was 8.5 ± 0.9 A (n = 4). All these distances were
increased in the rTRPV1-K425Q (Figure S7A) and hTRPV4-
K462Q (Figures 8A and S7B): K425(Cd)-E709(Cd) 8.2 ± 0.8 A
(n = 4), K425(Cd)-D388(Cg)10.6 ± 0.7 A (n = 4), K462(Cd)-
E745(Cd) 7 ± 0.6 A (n = 4) and K462(Cd)-D425(Cg) 12.3 ± 0.8 A
(n = 4). Moreover, hydrogen bonding in close proximity to the
key lysine of pre-S1 was disrupted in the case of simulations
with rTRPV1-K425Q and hTRPV4-K462Q mutants, resulting in
the reduction of hydrogen bonds (1.7 ± 1.3 and 1.4 ± 1.1, respec-
tively). The total number of hydrogen bonds in the TRPV1
tetramer fell from 24 in the WT to 11 in the TRPV1-K425Q, with
an approximate loss of 39 kcal/mol. Similarly, the TRPV4-
K462Q tetramer implied a loss of ten hydrogen bonds (from 26
to 16) and 30 kcal/mol.
We also analyzed the impact of K462Q mutation in the curva-
ture of the surrounding region. Figure 8B shows the predicted
tetrameric structure highlighting the four pre-S1 and S1 do-
mains. A noticeable change in the curvature is observed in
the TRPV4-K462Q model (Figure 8B), which resulted in the
generation of de novo angles between residues 466 and 470
(Figure 8C). In addition, this mutation affected the positioning
of the TRP domain (yellow in Figure 8D). Although more difficult
to quantify due to its unstructured nature, the ARD-pre-S1
linker that projects into the neighboring subunit (green in
ª2015 Elsevier Ltd All rights reserved 1407
Figure 4. Subcellular Localization and Function of TRPV4-K462Q
(A and B) Co-localization of TRPV4-WT and TRPV4-K462Q proteins (green) expressed in HeLa cells with the plasmamembranemarker concanavalin A (A, red) or
with the ER marker calreticulin (B, red). Co-localization is shown in white (merge panels). Scale bar, 20 mm.
(C) Determination of the Pearson correlation coefficient between the TRPV4 and concanavalin A signals using the ‘‘Intensity Correlation Analysis’’ plugin of
ImageJ. Number of cells recorded is shown for each condition. Mean ± SEM. *P < 0.05 versus all other conditions.
(D–F) Fura-2 ratios obtained in HeLa cells transfected with GFP, TRPV4-WT, and TRPV4-K462Q, and perfused with warm solutions (38�C), 30% hypotonic
solutions (HTS), and 10 nM GSK1016790A. Traces are means ± SEM of 215–362 cells measured in 8–13 independent experiments.
(G) Maximal high FRET efficiencies corresponding to homomultimer formation could only be demonstrated for TRPV4-WT. Number of cells recorded is shown for
each condition. Mean ± SEM. **P < 0.01 versus all other conditions.
See also Figures S3–S5.
Figure 8D) was also affected, moving away from the neigh-
boring subunit (Figure 8D).
Together, the molecular dynamics simulations of a preassem-
bled channel suggested a considerable change in the folding of
the individual subunits and in the overall interactions in the
mutant tetramers. These changes are consistent with the exper-
imental data showing defective assembly and no function of
mutants, in which the hydrogen bonds established between
the pre-S1 and the linker/TRP domains were lost.
DISCUSSION
Misfolded channel proteins and/or oligomerization-deficient
subunits can be identified and intracellularly retained by quality
control mechanisms in the ER (Green and Millar, 1995). There-
fore, it is particularly important to identify the structural domains
involved in the folding and oligomerization process. Biological
organization and functioning of ion channels, similar to other
transmembrane proteins, is assured by their interaction with
lipids of the plasma membrane (Laganowsky et al., 2014) as
well as interactions between different protein subunits (Schwap-
pach, 2008). In the folding and oligomerization process, electro-
static interactions between positively and negatively charged
1408 Structure 23, 1404–1413, August 4, 2015 ª2015 Elsevier Ltd Al
amino acids in cytoplasmic regions and membrane lipids are
of paramount relevance (Raja, 2011). In the present study, we
interrogated the role of such electrostatic interactions between
the pre-S1, ARD-S1 linker (from the ARD6 to the pre-S1) and
TRP box domains in the biogenesis and function of the TRPV
channels, particularly TRPV4.
Previous studies have reported that TRPV4 splice variants
lacking ARD3 and/or part of the outer helix or ARD6 do not oligo-
merize correctly and are retained in the ER (Arniges et al., 2006).
Similarly, TRPV5 and TRPV6 oligomerization involves ARDs in a
process that has been described as a molecular zippering (Erler
et al., 2004). The resolved structure of rTRPV1 provides further
insight into the role of ARD in channel assembly (Liao et al.,
2013). A b sheet from the linker region and a b strand from the
C terminus contact two ARD from an adjacent subunit, corre-
sponding to the ARDs missing in the TRPV4 splice variants
that do not oligomerize (Arniges et al., 2006). This intersubunit
interaction may be affected if the architectural organization of
the ARD-containing N terminus is altered. In this sense, the
rTRPV1 structure proposed the interaction between the pre-S1
(K425) and the TRP box (E709) that was interpreted as a
structural determinant of channel gating and allosteric modula-
tion (Liao et al., 2013). We now report that the evolutionarily
l rights reserved
Figure 5. Activity of hTRPV1-WT and hTRPV1-K426Q Channels
Fura-2 ratios obtained in HeLa cells transfected with GFP (lozenges), TRPV1-
WT (squares), and TRPV1-K426Q (circles) and perfused with solutions heated
at 45�C and 1 mM capsaicin. Traces are means ± SEM of 100–200 cells
measured in 3–4 independent experiments.
conserved lysine in TRPVs pre-S1 region (hTRPV1-K426 and
hTRPV4-K462) is critical for folding and subunit assembly.
In fact, rather than a hydrogen bond pair, our molecular dy-
namics simulation and in vitro analysis identified a salt bridge/
hydrogen bond triad formed between the pre-S1 lysine, the
aspartate in the linker region (rTRPV1-D388 and hTRPV4-
D425), and the glutamate in the TRP box (rTRPV1-E709 and
hTRPV4-E745) as the actual determinant of functional folding.
Mutation of pre-S1 lysine reduced the electrostatic interactions
(loss of 13 and 10 salt bridges in TRPV1 and TRPV4, respec-
tively), and particularly, the hydrogen bonds in close proximity
to the lysine, which fell from four to two in TRPV1 and from six
to two in TRPV4. The molecular dynamics simulations showed
Figure 6. Subcellular Localization of ARD-S1 Linker and TRP Box Mut
(A) Co-localization (white) of TRPV4-WT, TRPV4-D425A, and TRPV4-E745A (gree
(B) Co-localization of channel proteins with the ER marker calreticulin (red).
Scale bars, 20 mm. See also Figure S6.
Structure 23, 1404
that mutation of the pre-S1 lysine exerted a large effect on
the structure of the pre-S1 region, introducing novel twists
that, in turn, affect the ARD-S1 linker. Most likely, such changes
affected the positioning of the intracellular N terminus and, there-
fore, the interaction between adjacent subunits and their assem-
bly at the early stages of channel tetramerization.
In the case of hTRPV4, alanine substitution of D425 or E745
partially affected channel function while double mutation
D425A-E745A fully abolished channel activity. The reason why
single (hTRPV4-D425A and hTRPV4-E745A) and double
(TRPV4-D425A-E745A) mutants showed slight differences in
channel activity despite similar FRET efficiencies and ER reten-
tion patterns is still unresolved. TRPV4 subunits oligomerize in
the ER (Arniges et al., 2006), thereby discarding posterior as-
sembly at the plasma membrane. It is possible that the FRET
technique is not sensitive enough to detect tetramerization of a
limited number of mutant subunits that may escape ER quality
control mechanisms and are capable of responding to activating
stimuli.
In summary, our data show that the intrasubunit interaction
between the pre-S1 and ARD-S1 linker in the N terminus and
the TRP domain in the C terminus of TRPV1 and TRPV4 channels
determines the correct folding to promote channel assembly,
most likely by setting the interfaces connecting adjacent sub-
units in the early stages of tetramerization.
EXPERIMENTAL PROCEDURES
Cells, Transfection, and Solutions
For electrophysiological or calcium imaging experiments, HeLa and
HEK293 cells were transiently transfected with hTRPV4 and hTRPV1 WT
and/or mutants as previously described (Fernandes et al., 2008). Isotonic
bath solutions used for imaging experiments contained 140 mM NaCl,
2.5 mM KCl, 1.2 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, and 10 mM
ants
n) expressed in HeLa cells with plasmamembranemarker concanavalin A (red).
–1413, August 4, 2015 ª2015 Elsevier Ltd All rights reserved 1409
Figure 7. Subcellular Localization and Function of ARD-S1 Linker
and TRP Box Mutants
(A and B) Co-localization (white) of TRPV4-D425A/E745A (green) expressed in
HeLa cells with plasma membrane marker concanavalin A (A, red) and the ER
marker calreticulin (B, red). Scale bar, 20 mm.
(C) Fura-2 ratios obtained in HeLa cells transfected with GFP, TRPV4-
WT, TRPV4-D425A, TRPV4-E745A, and TRPV4-D425A/E745A. Traces are
means ± SEM of 206–345 cells measured in 10–15 independent experiments.
(D) Maximal high FRET efficiencies corresponding to homomultimer formation
could only be demonstrated for TRPV4-WT. Number of cells recorded is
shown for each condition. Mean ± SEM. **P < 0.01 versus all other conditions.
See also Figure S8.
Figure 8. Effect of TRPV4-K462Q Mutation on Atomic-Level Interac-
tions between Pre-S1, Linker, and TRP Box Domains
(A) Atomic-level interactions between pre-S1 (blue), S3-S4 linker (red), ARD-S1
linker (green), and TRP domains (yellow) in TRPV4-WT and TRPV4-K462Q 3D
models. Note the increased distance between K462, E745, and D425 residues
in the mutant channel structure.
(B) Curvature analysis of the pre-S1 to proximal S1 fragment (residues 456–
476). Side view of the four subunits of the TRPV4 model after 100 ns of
molecular dynamics simulation. Spatial location of the pre-S1 and domains, in
which curvatures over 30� are shown in green and those over 50� are shown in
red.
(C) Measurements of the bending angle in the pre-S1-S1 domains of TRPV4-
WT (left) and TRPV4-K462Q (right) models.
(D) Side view of the four subunits of the TRPV4-WT (left) and TRPV4-K462Q
(right) models in which one subunit is highlighted in cyan. Pre-S1 (dark blue),
ARD-S1 linker (green), and TRP domains (yellow) are also highlighted.
See also Figure S7.
HEPES (pH 7.3) with Tris. Bath solutions for whole-cell recordings
contained 100 mM NaCl, 1 mM MgCl2, 6 mM CsCl, 10 mM HEPES,
1 mM EGTA, and 5 mM glucose (pH 7.3) with Tris. Osmolarity was adjusted
to 310 mOsm using mannitol and 30% hypotonic solutions (220 mOsm)
1410 Structure 23, 1404–1413, August 4, 2015 ª2015 Elsevier Ltd Al
were obtained by removing mannitol. Whole-cell pipette solution contained
20 mM CsCl2, 100 mM CsAcetate, 1 mM MgCl2, 0.1 mM EGTA, 10 mM
HEPES, 4 mM Na2ATP, and 0.1 mM NaGTP; 300 mOsm (pH 7.25). All
l rights reserved
chemicals were obtained from Sigma-Aldrich except HC-067047 (Tocris
Biosciences) and Fura-2 (Invitrogen).
Electrophysiological and Ratiometric Ca2+ Recordings
Patch-clamp whole-cell currents were recorded at room temperature (�24�C,unless otherwise indicated) as previously described (Fernandes et al., 2008).
Cells were perfused at 0.8 ml/min. Cytosolic Ca2+ signals, relative to the ratio
(340/380) measured prior to cell stimulation, were obtained from cells loaded
with 4.5 mM fura-2 AM as previously described (Fernandes et al., 2008).
Western Blot, Co-immunoprecipitation, and Deglycosylation with
PNGaseF and EndoH
To compare the expression levels of different TRPV4 proteins, 40 mg of total
protein was obtained from HeLa cells harvested 24–48 hr after transfection
with TRPV4-WT and different mutants. The protein was separated on a pre-
cast polyacrylamide gel NuPAGE (4%–12%, Invitrogen). Co-immunoprecipi-
tation experiments were run as previously described (Fernandes et al.,
2008). Analysis of TRPV4 glycosylations was carried out as previously
described (Arniges et al., 2006). Total protein was extracted from HeLa cells
24 hr after transfection. Cells were lysed in a buffer containing 150 mM
NaCl, 5 mM EDTA, 1% NP-40, 1 mM sodium orthovanadate, 1 mM PMSF,
1 mM DTT; 0.05% aprotinin (1 hr at 4�C). The lysis buffer was supplemented
with a Complete Mini protease inhibitor cocktail (1:7 v/v; Roche). The nuclear
fraction was pelleted by centrifugation at 12,000 rpm for 15 min. Following the
manufacturer’s instructions, 20 mg of total protein were digested with 2 ml of
EndoH or PNGaseF (New England Biolabs) for 1.5 hr at 37�C. Proteins were
subjected to SDS-PAGE (8%) and subsequently electroblotted onto nitrocel-
lulose membranes. Incubation with anti-human TRPV4 antibody (1:500) for
1 hr at room temperature was followed by incubation for 1 hr at room temper-
ature with the horseradish peroxidase-conjugated donkey anti-rabbit immu-
noglobulin G (Amersham Biosciences) at a dilution of 1:2,000. Detection was
done with a SuperSignal West chemiluminescent substrate (Pierce).
Confocal Microscopy
Cells transiently transfected with hTRPV1 and hTRPV4 WT and/or mutants
(pcDNA3.1) were probed with a polyclonal affinity-purified anti-human
TRPV4 (1:1,000) (Arniges et al., 2004, 2006; Fernandes et al., 2008) and/or
mouse anti-calreticulin (1:500, BD Biosciences). Anti-TRPV1 antibody was a
free gift from Alomone. In cross-complementation assays, cells were co-trans-
fected with pcDNA3.1 TRPV4-WT-Flag and pcDNA3.1-YFP constructs and
probed with a rabbit anti-Flag (1:500, Sigma-Aldrich). Alexa Fluor 488 goat
anti-rabbit or Alexa 555 goat anti-mouse (Molecular Probes, 1:2,000) were
used as secondary antibodies. For membrane detection, cells were stained
for 20 min, then fixed in ice with 100 mg/ml concanavalin A tetramethylrhod-
amine (Invitrogen). Digital images were taken and analyzed using a Leica
TCS SP2 and the NIH ImageJ software (http://rsb.info.nih.gov/ij/).
FRET Measurements
Acceptor photobleaching FRET measurements of HeLa cells transfected with
TRPV4-WT andmutants with a YFP or CFP tag were carried out in a Leica TCS
SP2 confocal microscope (Leica) attached to an inverted microscope. FRET
efficiencies were expressed as the increase of the FRET donor CFP after
bleaching the FRET acceptor YFP (Arniges et al., 2006; Garcia-Elias et al.,
2013).
TRPV4 Molecular Structure and Molecular Dynamics Simulations
The sequence of hTRPV4 from V148 to A755 (UniProt: Q9HBA0) was aligned
along the cryo-electron microscopic structure of the rTRPV1 (PDB: 3J5P,
closed conformation) of 3.2 A resolution (Liao et al., 2013). Given a high simi-
larity in the transmembranal zone, 69% of sequence identity was achieved
from the whole sequence alignment. Afterward, five molecular models were
obtained by using Modeller v9.10 (Eswar et al., 2006) and the model with the
lowest DOPE energy was selected for the next stage. Four molecular systems
were set up: rTRPV1 WT, rTRPV1 K425Q, hTRPV4 WT, and hTRPV4 K462Q.
The protein structures were embedded into a phosphatidyloleoyl phosphati-
dylcholine (POPC) bilayer. To mimic the experimental conditions, the systems
were solvated with the TIP3P water model (Boiteux and Berneche, 2011), then
neutralized and ionized with 0.11 M KCl. The CHARMM36 force fields for lipids
Structure 23, 1404
(Klauda et al., 2010) and proteins (Huang and MacKerell, 2013) were applied.
The final dimensions of the systems were �1703 �1703 �170 A3. The initial
systems were subjected to a standard energy minimization and then equili-
brated for 3.6 ns. Position restraints of 1 (kcal/mol A2) were assigned to the
alpha carbons, which were diminished by 0.2 (kcal/mol A2) for 0.5 ns to reach
0.0 (kcal/mol A2). The molecular dynamics simulations were performed
with periodic boundary conditions. The systems were run using an isobaric-
isothermal ensemble, and the temperature and pressure applied were 300 K
and 1 atm, respectively. The temperature was controlled using Langevin
dynamics with a damping coefficient of 1 ps�1. The computation of long-range
electrostatic interactions was calculated with the particle-mesh Ewaldmethod
(Darden et al., 1993). The motion equations were integrated with a time step of
2, 2, and 4 fs for bonded, short-range, and long-range non-bonded interac-
tions, respectively. An 8-A spherical cut-off was used for short-range non-
bonded interactions, including a switching function from 7 A for the van der
Waals term and shifted electrostatics (Wells et al., 2012). Once the systems
were totally unrestricted, 100 ns of production data were collected for each
system. Measurements of the bending angle in the hinge formed by preS1-
S1 of the last state of TRPV4-WT and TRPV4-K462Q of molecular dynamics
simulations were calculated using the VMDBendix package (Dahl et al., 2012).
Statistical Analysis
Data are expressed as mean ± SEM (or mean ± SD in Figure S7) of n experi-
ments. Statistical analysis was assessed with Student unpaired test or one-
way ANOVA and Bonferroni post hoc using Sigma-Plot software.
SUPPLEMENTAL INFORMATION
Supplemental Information includes eight figures and two movies and can be
found with this article online at http://dx.doi.org/10.1016/j.str.2015.05.018.
AUTHOR CONTRIBUTIONS
A.G.-E., A.B.-E., F.G.-N., and M.A.V. designed research; A.G.-E., A.B.-E.,
F.R.-M., S.M., C.P.-P., RV.S., and R.V. performed research; A.G.-E., A.B.-E.,
R.V.S., F.G.-N., and M.A.V. analyzed data; and M.A.V. wrote the paper. All
authors collaborated in editing the manuscript. The authors declare no conflict
of interest.
ACKNOWLEDGMENTS
We thank Jaume Bonet and Baldo Oliva (GRIB, Universitat Pompeu Fabra,
Barcelona) for initial structural modeling, and Cristina Plata for technical
support. This work was supported by the Spanish Ministry of Science and
Innovation (SAF2010-16725 and SAF2012-38140); Fondo de Investigacion
Sanitaria (Red HERACLES RD12/0042/0014); and FEDER Funds. F.G.-N. ac-
knowledges the support of Millennium Scientific Initiative of the Ministerio de
Economıa, Fomento y Turismo (P029-022-F), FONDECYT grant 1131003
and Anillo grant ACT-1107, CONICYT-PIA. R.V.S. is funded by CONICYT-
PCHA/Doctorado Nacional 2013-21130631 fellowship.
Received: February 16, 2015
Revised: May 14, 2015
Accepted: May 25, 2015
Published: July 2, 2015
REFERENCES
Andrade, Y.N., Fernandes, J., Vazquez, E., Fernandez-Fernandez, J.M.,
Arniges, M., Sanchez, T.M., Villalon, M., Valverde, M.A., Vazquez, E.,
Fernandez-Fernandez, J.M., et al. (2005). TRPV4 channel is involved in the
coupling of fluid viscosity changes to epithelial ciliary activity. J. Cell Biol.
168, 869–874.
Arniges, M., Vazquez, E., Fernandez-Fernandez, J.M., Valverde, M.A.,
Vazquez, E., and Fernandez-Fernandez, J.M. (2004). Swelling-activated
Ca2+ entry via TRPV4 channel is defective in cystic fibrosis airway epithelia.
J. Biol. Chem. 279, 54062–54068.
–1413, August 4, 2015 ª2015 Elsevier Ltd All rights reserved 1411
Arniges, M., Fernandez-Fernandez, J.M., Albrecht, N., Schaefer, M., and
Valverde, M.A. (2006). Human TRPV4 channel splice variants revealed a key
role of ankyrin domains in multimerization and trafficking. J. Biol. Chem.
281, 1580–1586.
Becker, D., Muller, M., Leuner, K., Jendrach, M., and Muller, M. (2008). The
C-terminal domain of TRPV4 is essential for plasma membrane localization.
Mol. Membr. Biol. 25, 139–151.
Boiteux, C., and Berneche, S. (2011). Absence of ion-binding affinity in the
putatively inactivated low-[K+] structure of the KcsA potassium channel.
Structure 19, 70–79.
Caterina, M.J., Schumacher, M.A., Tominaga, M., Rosen, T.A., Levine, J.D.,
and Julius, D. (1997). The capsaicin receptor: a heat-activated ion channel in
the pain pathway. Nature 389, 816–824.
Chang, Q., Gyftogianni, E., van de Graaf, S.F., Hoefs, S., Weidema, F.A.,
Bindels, R.J., and Hoenderop, J.G. (2004). Molecular determinants in TRPV5
channel assembly. J. Biol. Chem. 279, 54304–54311.
Cheng, W., Sun, C., and Zheng, J. (2010). Heteromerization of TRP channel
subunits: extending functional diversity. Protein Cell 1, 802–810.
Dahl, A.C.E., Chavent, M., and Sansom, M.S.P. (2012). Bendix: intuitive helix
geometry analysis and abstraction. Bioinformatics 28, 2193–2194.
Darden, T., York, D., and Pedersen, L. (1993). Particle mesh Ewald: An
N,log(N) method for Ewald sums in large systems. J. Chem. Phys. 98, 10089.
Donate-Macian, P., and Peralvarez-Marın, A. (2014). Dissecting domain-
specific evolutionary pressure profiles of transient receptor potential vanilloid
subfamily members 1 to 4. PLoS One 9, e110715.
Erler, I., Hirnet, D., Wissenbach, U., Flockerzi, V., and Niemeyer, B.A. (2004).
Ca2+-selective transient receptor potential V channel architecture and function
require a specific ankyrin repeat. J. Biol. Chem. 279, 34456–34463.
Erler, I., Al-Ansary, D.M.M., Wissenbach, U., Wagner, T.F.J., Flockerzi, V., and
Niemeyer, B.A. (2006). Trafficking and assembly of the cold-sensitive TRPM8
channel. J. Biol. Chem. 281, 38396–38404.
Eswar, N., Webb, B., Marti-Renom, M.A., Madhusudhan, M.S., Eramian, D.,
Shen, M.-Y., Pieper, U., and Sali, A. (2006). Comparative protein structure
modeling using Modeller. Curr. Protoc. Bioinformatics, Chapter 5, Unit 5.6.
Fernandes, J., Lorenzo, I.M., Andrade, Y.N., Garcia-Elias, A., Serra, S.A.,
Fernandez-Fernandez, J.M., and Valverde, M.A. (2008). IP3 sensitizes
TRPV4 channel to the mechano- and osmotransducing messenger 50-60-epoxyeicosatrienoic acid. J. Cell Biol. 181, 143–155.
Garcia-Elias, A., Mrkonjic, S., Pardo-Pastor, C., Inada, H., Hellmich, U.A.,
Rubio-Moscardo, F., Plata, C., Gaudet, R., Vicente, R., and Valverde, M.A.
(2013). Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of
TRPV4 cytosolic tails enables channel activation by physiological stimuli.
Proc. Natl. Acad. Sci. USA 110, 9553–9558.
Garcia-Sanz, N., Fernandez-Carvajal, A., Morenilla-Palao, C., Planells-Cases,
R., Fajardo-Sanchez, E., Fernandez-Ballester, G., and Ferrer-Montiel, A.
(2004). Identification of a tetramerization domain in the C terminus of the vanil-
loid receptor. J. Neurosci. 24, 5307–5314.
Green, W.N., and Millar, N.S. (1995). Ion-channel assembly. Trends Neurosci.
18, 280–287.
Guler, A.D., Lee, H., Iida, T., Shimizu, I., Tominaga, M., Caterina, M., and Guler,
A.D. (2002). Heat-evoked activation of the ion channel, TRPV4. J. Neurosci. 22,
6408–6414.
Hellwig, N., Albrecht, N., Harteneck, C., Schultz, G., and Schaefer, M. (2005).
Homo- and heteromeric assembly of TRPV channel subunits. J. Cell Sci. 118,
917–928.
Hoenderop, J.G., Voets, T., Hoefs, S., Weidema, F., Prenen, J., Nilius, B., and
Bindels, R.J. (2003). Homo- and heterotetrameric architecture of the epithelial
Ca2+ channels TRPV5 and TRPV6. EMBO J. 22, 776–785.
Huang, J., and MacKerell, A.D. (2013). CHARMM36 all-atom additive protein
force field: validation based on comparison to NMR data. J. Comput. Chem.
34, 2135–2145.
Huynh, K.W., Cohen, M.R., Chakrapani, S., Holdaway, H.A., Stewart, P.L., and
Moiseenkova-Bell, V.Y. (2014). Structural insight into the assembly of TRPV
channels. Structure 22, 260–268.
1412 Structure 23, 1404–1413, August 4, 2015 ª2015 Elsevier Ltd Al
Klauda, J.B., Venable, R.M., Freites, J.A., O’Connor, J.W., Tobias, D.J.,
Mondragon-Ramirez, C., Vorobyov, I., MacKerell, A.D., and Pastor, R.W.
(2010). Update of the CHARMM all-atom additive force field for lipids: valida-
tion on six lipid types. J. Phys. Chem. B 114, 7830–7843.
Laganowsky, A., Reading, E., Allison, T.M., Ulmschneider, M.B., Degiacomi,
M.T., Baldwin, A.J., and Robinson, C.V. (2014). Membrane proteins bind lipids
selectively to modulate their structure and function. Nature 510, 172–175.
Lei, L., Cao, X., Yang, F., Shi, D.-J., Tang, Y.-Q., Zheng, J., and Wang, K.
(2013). A TRPV4 channel C-terminal folding recognition domain critical for
trafficking and function. J. Biol. Chem. 288, 10427–10439.
Liao, M., Cao, E., Julius, D., and Cheng, Y. (2013). Structure of the TRPV1 ion
channel determined by electron cryo-microscopy. Nature 504, 107–112.
Liedtke, W., Choe, Y., Marti-Renom, M.A., Bell, A.M., Denis, C.S., Sali, A.,
Hudspeth, A.J., Friedman, J.M., and Heller, S. (2000). Vanilloid receptor-
related osmotically activated channel (VR-OAC), a candidate vertebrate
osmoreceptor. Cell 103, 525–535.
Liedtke, W., Tobin, D.M., Bargmann, C.I., and Friedman, J.M. (2003).
Mammalian TRPV4 (VR-OAC) directs behavioral responses to osmotic and
mechanical stimuli in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA
100 (Suppl 2 ), 14531–14536.
Lindy, A.S., Parekh, P.K., Zhu, R., Kanju, P., Chintapalli, S.V., Tsvilovskyy, V.,
Patterson, R.L., Anishkin, A., van Rossum, D.B., and Liedtke, W.B. (2014).
TRPV channel-mediated calcium transients in nociceptor neurons are
dispensable for avoidance behaviour. Nat. Commun. 5, 4734.
Maruyama, Y., Ogura, T., Mio, K., Kiyonaka, S., Kato, K., Mori, Y., and Sato, C.
(2007). Three-dimensional reconstruction using transmission electron micro-
scopy reveals a swollen, bell-shaped structure of transient receptor potential
melastatin type 2 cation channel. J. Biol. Chem. 282, 36961–36970.
Mio, K., Ogura, T., Kiyonaka, S., Hiroaki, Y., Tanimura, Y., Fujiyoshi, Y., Mori,
Y., and Sato, C. (2007). The TRPC3 channel has a large internal chamber
surrounded by signal sensing antennas. J. Mol. Biol. 367, 373–383.
Moiseenkova-Bell, V.Y., Stanciu, L.A., Serysheva, I.I., Tobe, B.J., and Wensel,
T.G. (2008). Structure of TRPV1 channel revealed by electron cryomicroscopy.
Proc. Natl. Acad. Sci. USA 105, 7451–7455.
Montell, C. (2005). The TRP superfamily of cation channels. Sci. STKE 2005,
re3.
Moore, C., Cevikbas, F., Pasolli, H.A., Chen, Y., Kong, W., Kempkes, C.,
Parekh, P., Lee, S.H., Kontchou, N., Ye, I., et al. (2013). Correction for
Moore, et al., UVB radiation generates sunburn pain and affects skin by acti-
vating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.
Proc. Natl. Acad. Sci. USA 110, 15502.
Myeong, J., Kwak, M., Hong, C., Jeon, J.-H., and So, I. (2014). Identification of
a membrane targeting domain of the transient receptor potential canonical
(TRPC)4 channel unrelated to its formation of a tetrameric structure. J. Biol.
Chem. 289, 34990–35002.
Owsianik, G., Talavera, K., Voets, T., and Nilius, B. (2006). Permeation and
selectivity of TRP channels. Annu. Rev. Physiol. 68, 685–717.
Pertusa, M., Gonzalez, A., Hardy, P., Madrid, R., and Viana, F. (2014).
Bidirectional modulation of thermal and chemical sensitivity of TRPM8
channels by the initial region of the N-terminal domain. J. Biol. Chem. 289,
21828–21843.
Poblete, H., Oyarzun, I., Olivero, P., Comer, J., Zuniga, M., Sepulveda, R.V.,
Baez-Nieto, D., Gonzalez Leon, C., Gonzalez-Nilo, F., and Latorre, R. (2015).
Molecular determinants of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2)
binding to transient receptor potential V1 (TRPV1). Channels J. Biol. Chem.
290, 2086–2098.
Raja, M. (2011). The potassium channel KcsA: a model protein in studying
membrane protein oligomerization and stability of oligomeric assembly?
Arch. Biochem. Biophys. 510, 1–10.
Schaefer, M. (2005). Homo- and heteromeric assembly of TRP channel
subunits. Pflugers Arch. 451, 35–42.
Schwappach, B. (2008). An overview of trafficking and assembly of
neurotransmitter receptors and ion channels (Review). Mol. Membr. Biol. 25,
270–278.
l rights reserved
Shigematsu, H., Sokabe, T., Danev, R., Tominaga, M., and Nagayama, K.
(2010). A 3.5-nm structure of rat TRPV4 cation channel revealed by
Zernike phase-contrast cryoelectron microscopy. J. Biol. Chem. 285,
11210–11218.
Suzuki, M., Mizuno, A., Kodaira, K., and Imai, M. (2003). Impaired pressure
sensation in mice lacking TRPV4. J. Biol. Chem. 278, 22664–22668.
Teng, J., Loukin, S.H., Anishkin, A., and Kung, C. (2015). L596-W733 bond
between the start of the S4-S5 linker and the TRP box stabilizes the closed
state of TRPV4 channel. Proc. Natl. Acad. Sci. USA 112, 3386–3391.
Thorneloe, K.S., Sulpizio, A.C., Lin, Z., Figueroa, D.J., Clouse, A.K.,
McCafferty, G.P., Chendrimada, T.P., Lashinger, E.S.R., Gordon, E., Evans,
L., et al. (2008). N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-
hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothio-
phene-2-carboxamide (GSK1016790A), a novel and potent transient receptor
potential vanilloid 4 channel agonist induces urinar. J. Pharmacol. Exp. Ther.
326, 432–442.
Tian, W., Fu, Y., Garcia-Elias, A., Fernandez-Fernandez, J.M., Vicente, R.,
Kramer, P.L., Klein, R.F., Hitzemann, R., Orwoll, E.S., Wilmot, B., et al.
(2009). A loss-of-function nonsynonymous polymorphism in the osmoregula-
Structure 23, 1404
tory TRPV4 gene is associated with human hyponatremia. Proc. Natl. Acad.
Sci. USA 106, 14034–14039.
Voets, T., Talavera, K., Owsianik, G., and Nilius, B. (2005). Sensing with TRP
channels. Nat. Chem. Biol. 1, 85–92.
Watanabe, H., Vriens, J., Suh, S.H., Benham, C.D., Droogmans, G., and Nilius,
B. (2002a). Heat-evoked activation of TRPV4 channels in a HEK293 cell
expression system and in native mouse aorta endothelial cells. J. Biol.
Chem. 277, 47044–47051.
Watanabe, H., Davis, J.B., Smart, D., Jerman, J.C., Smith, G.D., Hayes, P.,
Vriens, J., Cairns, W., Wissenbach, U., Prenen, J., et al. (2002b). Activation
of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives. J. Biol. Chem.
277, 13569–13577.
Wells, D.B., Bhattacharya, S., Carr, R., Maffeo, C., Ho, A., Comer, J., and
Aksimentiev, A. (2012). Optimization of the molecular dynamics method for
simulations of DNA and ion transport through biological nanopores.
Methods Mol. Biol. 870, 165–186.
Zhang, F., Liu, S., Yang, F., Zheng, J., and Wang, K. (2011). Identification of a
tetrameric assembly domain in the C terminus of heat-activated TRPV1 chan-
nels. J. Biol. Chem. 286, 15308–15316.
–1413, August 4, 2015 ª2015 Elsevier Ltd All rights reserved 1413