Article

Interaction between the L

inker, Pre-S1, and TRPDomains Determines Folding, Assembly, andTrafficking of TRPV Channels

Highlights

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

Correspondencemiguel.valverde@upf.edu

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: miguel.valverde@upf.edu

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

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