TRP ion channels: Proteins with conformational flexibility

Abstract

Ion channels display conformational changes in response to binding of their agonists and antagonists. The study of the relationships between the structure and the function of these proteins has witnessed considerable advances in the last two decades using a combination of techniques, which include electrophysiology, optical approaches (i.e. patch clamp fluorometry, incorporation of non-canonic amino acids, etc.), molecular biology (mutations in different regions of ion channels to determine their role in function) and those that have permitted the resolution of their structures in detail (X-ray crystallography and cryo-electron microscopy). The possibility of making correlations among structural components and functional traits in ion channels has allowed for more refined conclusions on how these proteins work at the molecular level. With the cloning and description of the family of Transient Receptor Potential (TRP) channels, our understanding of several sensory-related processes has also greatly moved forward. The response of these proteins to several agonists, their regulation by signaling pathways as well as by protein-protein and lipid-protein interactions and, in some cases, their biophysical characteristics have been studied thoroughly and, recently, with the resolution of their structures, the field has experienced a new boom. This review article focuses on the conformational changes in the pores, concentrating on some members of the TRP family of ion channels (TRPV and TRPA subfamilies) that result in changes in their single-channel conductances, a phenomenon that may lead to fine-tuning the electrical response to a given agonist in a cell.

Full text

REVIEW
TRP ion channels: Proteins with conformational flexibility
Ana Elena López-Romero
a
,&
, Ileana Hernández-Araiza
a
,&
, Francisco Torres-Quiroz
b
, Luis B. Tovar-Y-Romo
c
,
León D. Islas
d
, and Tamara Rosenbaum
a
a
Departamento de Neurociencia Cognitiva, División Neurociencias, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México,
Mexico, Mexico;
b
Departamento de Bioquímica y Biología Estructural, División Investigación Básica, Instituto de Fisiología Celular,
Universidad Nacional Autónoma de México, Mexico City, Mexico;
c
Departamento de Neuropatología Molecular, División Neurociencias,
Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Mexico City, Mexico.;
d
Departamento de Fisiología, Facultad de
Medicina, Universidad Nacional Autónoma de México, Mexico City, Mexico
ABSTRACT
Ion channels display conformational changes in response to binding of their agonists and antagonists.
The study of the relationships between the structure and the function of these proteins has witnessed
considerable advances in the last two decades using a combination of techniques, which include
electrophysiology, optical approaches (i.e. patch clamp fluorometry, incorporation of non-canonic
amino acids, etc.), molecular biology (mutations in different regions of ion channels to determine their
role in function) and those that have permitted the resolution of their structures in detail (X-ray crystal-
lography and cryo-electron microscopy). The possibility of making correlations among structural com-
ponents and functional traits in ion channels has allowed for more refined conclusions on how these
proteins work at the molecular level. With the cloning and description of the family of Transient Receptor
Potential (TRP) channels, our understanding of several sensory-related processes has also greatly moved
forward. The response of these proteins to several agonists, their regulation by signaling pathways as
well as by protein-protein and lipid-protein interactions and, in some cases, their biophysical character-
istics have been studied thoroughly and, recently, with the resolution of their structures, the field has
experienced a new boom. This review article focuses on the conformational changes in the pores,
concentrating on some members of the TRP family of ion channels (TRPV and TRPA subfamilies) that
result in changes in their single-channel conductances, a phenomenon that may lead to fine-tuning the
electrical response to a given agonist in a cell.
ARTICLE HISTORY
Received 10 April 2019
Revised 17 May 2019
Accepted 21 May 2019
KEYWORDS
TRP Channels; structure-
function; Ion channels; lipid-
protein interactions
Introduction
The appearance of the plasma membranes of cells
constituted a key event for life during evolution.
These structures represent boundaries of the units of
life, achieving pivotal functions for the cell, including
transport of molecules, communication with the
environment, and metabolic functions. The intrinsic
hydrophobic nature of these structures renders them
impermeable to the flux of charged molecules,
a process necessary for cell-cell communication, acti-
vation of signaling pathways, as well as for the ability
to respond to endogenous and exogenous signals. This
issue is resolved by the presence of certain types of
proteins in plasma membranes, called ion channels
that allow for the passage of ions from one side to the
other. The specific structure of one ion channel may
differ substantially from another; however, all these
proteins contain a pore that opens and closes to permit
the flow of ions, mediating ionic currents that, in turn,
result in the generation of signals with distinct phy-
siological consequences.
There is an intimate relationship between the
structures and the functions of ion channels, with
their different component regions serving
a specific role in the activity of these proteins. In
the 1980s, when the patch-clamp recording tech-
nique was developed, it became possible to study
the behavior of single ion channels in real time[1].
Furthermore, with the advances seen in the areas
of molecular biology, cloning and generation of
knockout animal models, we have been provided
with valuable methods[1] to carefully determine
the roles of ion channels in normal and under
pathophysiological conditions. A large variety of
ion channels have been cloned and biophysically
characterized and, in the past three decades,
CONTACT Tamara Rosenbaum trosenba@ifc.unam.mx
&
These authors contributed equally to this work
CHANNELS
2019, VOL. 13, NO. 1, 207–226
https://doi.org/10.1080/19336950.2019.1626793
© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided the original work is properly cited.

research-oriented towards determining the struc-
ture of ion channels has witnessed remarkable
progress.
TheNobelPrizeinChemistrywasawardedto
Roderick MacKinnon and Peter Agre in 2003, for
advancing the field by resolving the molecular struc-
tures K
+
channels [2–5] and aquaporins [6,7], allow-
ingustofurtherunderstandtheirfunctionalfeatures.
The present review focuses on a family of proteins
named Transient Receptor Potential (TRP) ion chan-
nels, which has fundamentally reshaped our knowl-
edge of sensory physiology and their importance is
illustrated by their pivotal roles in smell, taste, vision,
touch and our ability to detect changes in temperature.
Originally, the first trp gene was identified in
aDrosophila mutant with altered vision [8]; however,
it was not until it was cloned that the deduced amino
acid sequence led to the suggestion that trp encoded
for a cation channel [9]. Electrophysiological studies
later showed that trp indeed was an ion channel and
that its selectivity could be modified by inserting
mutations in the amino acid region that formed the
pore loop [10].
The field of study of TRP channels witnessed
a boom when mammalian homologs of the
Drosophila trp channel were cloned and when
some of these were identified as temperature sen-
sors [11,12]. A feature of these proteins is that
several of these channels are polymodal, that is,
being activated by several distinct physical stimuli
and more than one ligand. In some cases, different
biophysical properties as well as distinct confor-
mational changes in their pores, associated with
interactions with different ligands, have been
demonstrated.
We will discuss a few examples of ion channels
where conformational changes are associated with
different ion conductance states. However, we will
mainly focus on TRPV1 and other TRP ion chan-
nels for which structures have been solved, making
emphasis on the conformational changes produced
by various ligands and on their effects in the
functional properties of these proteins.
Rearrangements in the outer pore of kv2.1
lead to changes in ion conductance
For voltage-gated ion channels, modulation of
macroscopic current magnitude archetypically is
thought to occur through an effect on the gating
(opening and closing properties) of these proteins.
In this sense, most studies of gating have concen-
trated on the voltage-dependent gate at the cyto-
plasmic entrance to the pore. Moreover, there is
a large body of evidence that also supports an
important role for the selectivity filter in gating
activation [13,14]. It has been suggested that some
form of voltage-dependent gating can exist at the
selectivity filter (the region that discriminates
among types of ions passing through the pore)
[15–17], although the main closed-open transition
is controlled by the S6 bundle-crossing intracellu-
lar gate [18].
These aspects have been explored in Kv2.1 potas-
sium channels, which are slowly inactivating delayed
rectifiers present in non-neuronal excitable cells and
several neurons. In these channels, the current magni-
tude is modulated by the external K
+
concentration,
making outward currents through Kv2.1 channels
become larger when the extracellular K
+
concentra-
tion is increased [19]. Kv2.1 channels exhibit two
distinct pharmacological profiles as a function of the
K
+
concentration since they can either be sensitive to
external tetraethylammonium (TEA; IC
50
~3–5mM),
or completely insensitive to this blocker [20]. The
underlying mechanism of these effects encompasses
the opening of Kv2.1 channels into one of two differ-
ent outer vestibule conformations with different sen-
sitivities to TEA. It has been shown that the channels
that open into a TEA-sensitive conformation produce
larger macroscopic currents [19]. In contrast, channels
that open into a TEA-insensitive conformation,
aphenomenonthatoccursinthepresenceofhigher
potassium concentrations, yield smaller macroscopic
currents [19].
Trapani and collaborators examined the
mechanism by which conformational changes in
Kv2.1 channels produced changes in current mag-
nitude. By using a combination of single-channel,
macroscopic recordings, and hidden Markov mod-
eling they proposed a model in which an outer
vestibule lysine residue in position 356 (pore turret
region) interferes with the flux of K
+
through the
channel [19]. This led to the suggestion that the K
+
concentration-dependent change in orientation of
that specific K356 can modify single channel con-
ductance through a change in the level of such an
interference [19].
208 A. E. LÓPEZ-ROMERO ET AL.

In summary, in the conformation where cur-
rents are smaller in magnitude, K356 would be
oriented towards the center of the ion conduction
pathway, not easily allowing the flux of K
+
ions
and resulting in a change in the single-channel
conductance. In contrast, when K356 is oriented
away from the conduction pathway, it readily
allows the passage of ions. Thus, these results
obtained by Trapani et al., not only provided
evidence where Kv2.1 single-channel conduc-
tance is modulated by the outer end of the con-
duction pathway through the occupancy of open
states with different outer vestibule conforma-
tions, but they also showed that these occur
under physiologically-relevant K
+
concentra-
tions [19].
Hence, in a physiological scenario, it is impor-
tant to consider that with rising extracellular K
+
concentrations, the current magnitude of K
+
chan-
nels in most neuronal cells is reduced as a result of
a decrease in electrochemical driving force.
However, Kv2.1 counteracts this reduction in net
outward current in the presence of higher K
+
concentrations and, as suggested by the authors,
this would provide the cells where these channels
are expressed with a mechanism that maintains
action potential integrity when high-frequency fir-
ing conditions are present [19].
The validity of the pore dilation phenomenon
to explain changes in ion channel
conductance
Ion channels exhibit different permeability and
selectivity to various ions. Potassium, sodium,
and calcium channels contain ion binding sites in
their selectivity filters that enable them to finely
discriminate among the ions that will flow through
their pores
5
[21],. Nonetheless, some ion channels
have been shown to exhibit dynamic changes in
their ion selectivity in response to agonist activa-
tion that could, in theory, allow for changes in
their conductance. Examples of these ion channels
are P2X receptor channels [22,23], acid-sensing
ion channels (ASIC) [24,25] and TRPV1 channels
[26,27]. This phenomenon has been called pore
dilation and it will be next discussed for P2X
receptors, a well-studied example of this process,
and for TRPV1 in a later section.
P2X receptors are ion channels gated by extracel-
lular ATP [28] and it was originally thought that the
pore first opened rapidly to a conducting state selec-
tiveforsmallcationssuchasNa
+
,K
+
and Ca [2]+ (I
1
current) and gradually increased in size or dilated over
time, rendering the channel permeable to large
organic cations such as N-methyl-D-glucamine
(NMDG
+
)orI
2
current, as well as to large fluoro-
phores and dyes (i.e. YO-PRO1) [22,23]. However,
based on the original assumption that P2X receptors
experience a slow, time- and agonist-dependent pore
dilation, three mechanisms have been suggested to
explain this phenomenon: (1) it represents an intrinsic
gating property of the functional P2XR channel,
where ATP exposure results in the widening of the
pore and a change from a state that conducts Na
+
,K
+
,
Ca
2+
(I
1
) to one that conducts NMDG
+
and YO-PRO
1, a large propidium dye. Such a switch has been
proposed to be modulated by phosphorylation or
dephosphorylation events [22,23]; (2) macropores
are formed as a result of an agonist-dependent redis-
tribution and oligomerization of P2XRs. The fusion of
two or more trimeric P2XRs, as well as an enlargement
of the channel pore, or the formation of a separate but
larger pore among aggregating trimeric assemblies,
could underly macropore formation. This hypothesis
has been ruled out by some research groups since no
clustering or redistribution of channels expected dur-
ing oligomerization has been found to occur [23,29],
butsomeothersstillsupportit[30]; (3) the perme-
ability to large cations is facilitated by a structurally
separate transport pathway downstream of P2XR acti-
vation [22].
Although the phenomenon of pore dilation has
been extensively explored, accumulating evidence sug-
gests that it may be an artifact of the experimental
conditions used, as will be detailed in the Discussion
section.
Next, we will discuss some structural character-
istics of TRP ion channels that shed light into why
these proteins represent an example of conforma-
tional flexibility in response to agonists that may
result in changes in cellular excitability.
The structures of thermotrp channels reveal
conformational flexibility
The TRPV1 channel was the first mammalian TRP
channel to be cloned. In 1997, it was shown that
CHANNELS 209

TRPV1 is the receptor for capsaicin [11,12]. In this
groundbreaking study, the laboratory of David
Julius isolated a cDNA clone that reconstituted
the response to capsaicin in non-neuronal cells.
By examining the amino acid sequence of this
clone, they demonstrated TRPV1 is an integral
membrane protein, homologous to a family of
putative store-operated calcium channels and that
it is expressed by small-diameter neurons within
sensory ganglia such as the dorsal root (DRG) and
trigeminal (TG). Moreover, these authors showed
that TRPV1 is also a thermal sensor, activated in
response to temperatures in a range known to
elicit pain-associated behaviors in animals and
pain in humans [11]. It was later shown, in 1998,
that TRPV1 is also activated by low extracellular
pH (pH ≤5.9) [12].
Continued work on the study of the function of
TRPV1, one of the best studied TRP channels, has led
to the identification of several other agonists and
modulators including resiniferatoxin (RTx) [12], dia-
cylglycerol [31], hydroperoxyeicosatetraenoic acid
(HPETE) [32], anandamide [33], N-acyl ethanola-
mines (NAEs) [34], n-acyl-dopamines [35], nitric
oxide [36], cations [37,38], hydrogen sulphide (H
2
S)
[39], the double-knot toxin (DkTx) from the Earth
Tiger tarantula [40] and lysophosphatidic acid
(LPA) [41].
TRPchannelsarealltetramericstructures.The
structure of TRPV1 had been initially solved in
the year of 2008 by the group of Vera Moiseenkova-
Bell. These authors obtained a 19 Å resolution cryo-
EM structure that showed that TRPV1 exhibits
a four-fold symmetry with a large open basket-like
domain,formedbytheN-andC-terminiofthe
channel as well as a compact transmembrane region
[42]. In this first solved structure of TRPV1, the
authors described a 150 Å tall ion channel which is
divided into a “small”compact region (30% of the
total volume) and into a “large”region or the “bas-
ket-like”domain (70% of the total mass).
This seminal study was followed by the
higher resolution structures for TRPV1 obtained
by the research groups of Yifan Cheng and
David Julius. Two, back-to-back reports,
described structures of 3.4–4.2 Å resolution
obtained by cryo-EM [43,44]. They confirmed
that TRPV1 has a four-fold symmetry with
a central ion pathway that is formed by
transmembrane segments S5 and S6 and a pore-
loop, all of which are surrounded by the S1-S4
domains with a domain-swapped geometry
[43,44].Theporeregionwasdescribedasone
containing a wide extracellular mouth and
a short selectivity filter. The important TRP
domain, which is a region conserved in these
familyofionchannelsandthatisthoughtto
play an important role in their allosteric mod-
ulation, was shown to interact with the S4-S5
intracellular linker. Each of four subunits in
TRPV1 contains six ankyrin repeats that form
the ankyrin repeat domain (ARD) localized to
their N-termini [43,44](Figure 1).
These research groups also described the struc-
tures of TRPV1 obtained in the presence of different
agonists, providing insight into the mechanisms that
allow the polymodal activation of this channel. One
of these structures was obtained in the presence of
RTx (which also binds to the vanilloid pocket where
capsaicin binds through residues Y511 and S512 in
S3 and M547 and T550 in S4) and DkTx together.
The authors showed that DkTx was bound to the
extracellular loops of the outer pore region and
defined contacts of the toxin with residues at the
top of the pore helix of one subunit and the outer
pore loop in the proximal S6 region of another
nearby subunit [43,45](Figure 2).
The structures of TRPV1 obtained in the presence
of both agonists display evident rearrangements in the
ion conduction pathway and the outer pore region.
With RTx and DkTx bound to the channel, the ion
conduction pathway appears completely eased of con-
strictions.Thisisincontrasttothestructureobtained
only in the presence of capsaicin, where the lower gate
I679 is extended, but the selectivity filter G643 remains
unaltered [45](Figure 2).
When TRPV1 has RTx and DkTx bound, a shift
or tilt of the S6 helix 1.9 Å away from the central
axis of the channel is produced, as compared to
the apo or unliganded state. Moreover, an increase
in the distance between the carbonyl oxygens of
G643, which constitutes the narrowest point of the
selectivity filter, is also observed when both ligands
are present: it goes from 4.6 Å in apo (PDB 3J5P)
to 7.6 Å in RTx/DkTx (PDB 3J5Q) [45].
As mentioned above, G643 (at the upper gate)
remains unaltered between the apo structure (PDB
3J5P) and in the structure in complex with
210 A. E. LÓPEZ-ROMERO ET AL.

capsaicin, and this could be explained because, in
the absence of DkTx, the channel could be under-
going transitions to closed states. However, the
distance between the I679 residues (lower gate)
in the apo state is 5.3 Å (PDB 3J5P) and, when
in complex with RTx/DkTx (PDB 3J5Q), it shifts
to 9.3 Å, while it is at 7.6 Å with capsaicin only
(PDB 3J5R). Therefore, the authors concluded that
the structure obtained with capsaicin is likely only
a partially activated state of TRPV1 [45].
When TRPV1 is in the presence of capsaicin
only, the structure can be superimposed to that
of the apo state in the outer pore region. Hydrogen
bond interactions among side chains of the E600
residues and main-chain nitrogen atoms of the
Y653 and D654 amino acids in the outer pore
loops thought to stabilize the ion channel in the
closed state, are broken when the distances among
them are increased in response to the binding of
DkTx and RTx. The distances between residues
Figure 1. Structure of the TRPV1 channel. Only one of the subunits is highlighted for clarity.
The ankyrin repeat domain is shown in purple, the S1- S4 domain in orange, the pore forming S5-P-S6 is shown in blue. The pre-S1,
S4-S5 linker, and TRP domain, which participate in the allosteric modulation of the channel, are shown in green. The left panel is
a lateral view, the central and right panels are extracellular and intracellular views, respectively. (PDB 3J5P).
Figure 2. Relative movements of the selectivity filter and lower gate of TRPV1 in the presence of different agonists.
A. Distances between G643 (at the selectivity filter) and I679 (lower gate) in the apo (left, PDB 3J5P), RTx/DkTx (middle, PDB 3J5Q)
and capsaicin (right, PDB 3J5R) structures. Compared to the apo structure, in the presence of both RTk and DkTx the selectivity filter
and the lower gate expanded. In the structure with capsaicin only (Cap), the outer pore region remains unaltered in comparison with
the apo structure, while the lower gate widens. B. Interactions among residues in the outer pore that stabilize the closed
conformation of TRPV1. The hydrogen-bonds between E600 side chain with the main-chain nitrogen atoms of Y653 and D654
are broken in the structure with RTk/DkTx but maintained with capsaicin only.
CHANNELS 211

that stabilize the closed state change when the
channel is in the apo state or in the presence of
RTx/DkTx: D654-E600 goes from 3.6 Å to 8.4 Å,
while for Y653-E600 it goes from 4.4Å to 8.3Å,
respectively [45](Figure 2).
In summary, this ion channel displays extraor-
dinary flexibility of movement in response to the
presence of different agonists.
Another example of conformational changes
that may allow for “superactivated”states is
that of TRPV2. This channel is activated by
temperatures near 52ºC [46]andbymembrane
stretch [47]. Although structurally similar to
TRPV1, TRPV2 is not activated by vanilloid
compounds. However, the laboratory of Kenton
Swartz produced a TRPV2 channel with four-
point mutations to form a vanilloid-binding site
located in the S4-S5 linker and the base of S5
[48], rendering this channel sensitive to RTx.
These results were also confirmed by Yang
et al., who used a TRPV2-Quad channel (with
the same point mutations in mouse), where cap-
saicin competed for the same site as RTx [49].
This TRPV2_Quad channel was found to bind
RTx, and that leads the channel to an unstable
open state through a mechanism that bridges the
S4-S5 linker to the S1-domain [49].
A couple of years later, Zubcevic and collabora-
tors obtained the crystal structure of a minimal
construct of the rabbit TRPV2 channel
(miTRPV2) in the absence of agonist and com-
pared it with that of the vanilloid-sensitive
miTRPV2 (F470S, L505M, L508T, and Q528E, in
the rabbit TRPV2) in complex with RTx [50].
Interestingly, in the structure in complex with
RTx, they found that two subunits adopt distinct
orientations between the S4-S5 linker and the S5,
as compared to the other two subunits. In other
words, while two subunits (A and C) are found to
spread away from each other, the other two (B and
D) get closer. This rearrangement results in an
asymmetric pore that adopts a wider conformation
at the selectivity filter when compared to the struc-
ture without RTx (Figure 3).
The authors determined that with RTx the dis-
tance between the G604 carbonyl oxygens in con-
tracted subunits was ~7.1 Å, and in the widened
subunits the distance between the I603 carbonyls
was ~12.3 Å, while in the structure with only Ca
2+
the distances between G604 carbonyl oxygens are
6.1–6.4 Å [50,51]. The conclusion was that, when
RTx binds to the channel, it pushes the base of S5
downwards and produces a bend in the S4-S5
linker producing a different conformation that
leads to rotation and widening of the entire
involved subunit (Figure 3).
The authors also suggested that this wider con-
formation of the pore could allow for the permea-
tion of larger cations since they performed
experiments where it is observed that, upon acti-
vation of the vanilloid-sensitive TRPV2 with RTx
(250 nM), uptake of the large fluorescent molecule
YO-PRO-1 (376 Da) could be attained. With this
experiment, they confirmed the functionality of
this conformation, and suggested that it was either
a fully open state or an intermediate state that
occurred before a symmetric open state capable
of permeating large organic cations [50].
Just as in TRPV2, a vanilloid-binding site can be
introduced into the TRPV3 channel upon the
insertion of six residues in the putative vanilloid-
pocket: W521Y, H523E, F522S, L557M, A560T,
and Q580E, as shown for the mouse TRPV3 chan-
nel by Zhang and collaborators [52]. Nonetheless,
the insertion of this vanilloid-binding site is not
enough to achieve activation of TRPV3 by RTx
and the presence of other agonists, such as
2-APB (aminoethoxydiphenyl borate) or heat, is
required [52]. These results demonstrate that gat-
ing pathways are conserved among the TRPV
channels, but they also show that the energetics
for activation can dictate their sensitivity to certain
stimuli since adding a binding site for an agonist
does not necessarily result in the activation of the
ion channel [52].
Structurally, the sensitized state of the human
TRPV3 undergoes an α-helix to π-helix transition
in the S6 which, in turn, disrupts the S4-S5 linker
and S6 and the latter bends ~9° away from the
pore [53]. In a previous report, the open confor-
mation of the mouse TRPV3 obtained with 2-APB
only shows a π-helix in the S6 [54], leading the
authors to suggest that the secondary structure
transition could be a hallmark for a sensitized
channel, in other words, a state that requires less
energy to activate [53].
Additionally, Zubcevic and collaborators also
observed that during the activation of TRPV3
212 A. E. LÓPEZ-ROMERO ET AL.

with 2-APB, the intermediate states display asym-
metrical rearrangements that are not present in the
apo or sensitized states. The differently observed
deviations for the subunits are caused because
theπ-helices in the S4-S5 linkers begin at different
residues. This is interesting because it accounts for
a similar phenomenon as the one described above
for the activation of TRPV2 with RTx, which also
originates at the S4-S5 linker [50]. Together, these
results further support the hypothesis that the
gating mechanisms of TRPV channels are con-
served and a break in symmetry may be character-
istic to these channel’s gating.
The TRPV4 channel was first described as an
osmosensor, capable of activating after exposure to
extracellular hypotonic conditions [55,56].
Subsequent reports demonstrated that, like other
members of the TRPV subfamily, TRPV4 is a heat-
sensitive channel activated by temperatures above
27°C [57] and chemical agonists such as phorbol
derivates [58] and 5,6-epoxyeicosatrienoic acid,
a metabolite of arachidonic acid [59,60].
Deng and collaborators obtained the first high-
resolution cryo-EM structure (3.8 Å) of the
TRPV4 channel [61]. These authors determined
that the channel was in a closed conformation,
that the narrowest diameter in the pore (5.3 Å)
was located at residue M714, which impeded the
permeation of ions. Even in the closed state,
TRPV4 displayed a particularly wide selectivity
filter: the diameter at G675 in TRPV4 was
10.6 Å, while the diameter at G643 in TRPV1 in
complex with RTx/DkTx was 7.6 Å[45]. This evi-
dence suggested that unlike TRPV1, the TRPV4
channel lacks an upper gate [61].
The single-channel conductance of TRPV4,
when stimulated with hypotonic solutions, has
been reported to be around 310 pS, at positive
voltages [55]. However, reports for its single-
channel conductance during spontaneous activity
is near 88 pS [56], which is near the value (around
98 pS) obtained with other agonists such as 4α-
Phorbol 12,13-didecanoate (4αPDD) and with heat
(around 105 pS) [58]. Altogether these results
Figure 3. Comparison of open and closed states of TRPV2.
Extracellular views of A) a closed miTRPV2 channel (PDB 6BWM) and B) vanilloid-sensitive miTRPV2 (PDB 6BWJ) in complex with RTx
shown as yellow spheres, representing the open state. On the lower panel are side views of the closed (C) and open (D) channel.
Only two subunits are shown for simplicity. The green sphere is Ca
2+
trapped in the pore which is notably wider in the open
configuration, its also visible the rearrangement of the intracellular domain. The side chains of G604 are also depicted.
CHANNELS 213

suggest that the channel can adopt a different
functional state when it is stimulated under hypo-
tonic conditions vs. heat or phorbol, and that
differences in the conductance of TRPV4 may be
observed with other agonists, but further studies
must be performed.
TRPA1 is a channel mainly expressed in nocicep-
tive neurons which responds to multiple noxious sti-
muli, including pungent chemicals present in garlic,
mustard oil, cinnamon or wasabi [62,63]. Moreover,
TRPA1 was initially described as a channel activated
bynoxiouscoldtemperatures[64,65], but there has
been controversy around this topic since this channel
functions as a temperature receptor in a species-
specific fashion [66–68]. Chemical agonists of
TRPA1 have been divided based on their mechanism
of action into electrophilic and non-electrophilic ago-
nists. Examples of the first are allyl isothiocyanate
(AITC) [69] or allicin [62] that activate the channel
through covalent modification of cysteines at the
N-terminal domain. In fact, allicin can also activate
TRPV1throughthesamemechanism[70–72]. The
non-electrophilic agonists include carvacrol, oleo-
canthal, Δ9-tetrahydrocannabinol (THC) and acidic
pH, which activate TRPA1 by mechanisms not asso-
ciated with cysteine modifications and which remain
mostly unclarified [73–75].
Cavanaugh et al., determined, in excised TRPA1-
expressing membrane patches, that the channel needs
to be exposed to polyphosphates (PPPi) [76]inorder
to be activated by AITC, but in this AITC-insensitive
conformation (in the absence of PPPi) it can be acti-
vatedbyTHC[77]. This result indicates that the
channel shifts to a different functional conformation
unresponsive to cysteine modification but responsive
to activation by a different mechanism [77], each with
a distinguishable function and agonist dependence.
The different functional states described by
Cavanaugh et al., have not been structurally com-
pared. Unlike TRPV1, the cryo-EM structure for
TRPA1 was not obtained in the presence of differ-
ent agonists, but only with AITC (PDB 3J9P).
Nonetheless, it was possible to determine that the
pre-S1 helix, linker S4-S5, and TRP-like domain
are bound by hydrophobic interactions and repre-
sent an important site for allosteric modulation,
similar to what happens with TRPV1 [45,78].
Nevertheless, recent studies have compared the
structural rearrangements of TRPA1 in the
presence of different agonists using techniques
such as limited proteolysis combined with mass
spectrometry [75]or circular dichroism spectro-
scopy only for the N- and C- terminal domains
[79]. These robust methodologies show important
interactions between cytoplasmatic domains
involved in the gating of the channel [79], as well
as differences between the activation with electro-
philic and non-electrophilic agonists [75].
TRPV5 and TRPV6 display limited structural
rearrengements in their pores
Both TRPV5 and TRPV6 ion channels are
expressed mainly in the epithelial tissue of the
digestive tract and kidney, where they play roles
in the reabsorption and transcellular transport of
Ca
2+
[80]. Unlike other members of the TRPV
family, TRPV6 and TRPV5 show inward rectifica-
tion and these highly Ca
2+
-selective proteins are
constitutively active under physiological condi-
tions [81]. Despite the high ion selectivity, in the
absence of extracellular divalent cations, they can
permeate monovalent cations [80].
TRPV5 and TRPV6 channels share structural
traits that differentiate them from the other
known TRPV channels. Three phenylalanine resi-
dues in S6 give rigidity to the pore, and a ring of
aspartates in the selectivity filter confers its high
Ca
2+
selectivity [82]. Neutralization mutation of
the negative aspartate charge not only affects Ca
2
+
selectivity but reduces inward rectification [80].
Functionally, both channels can be inhibited by
endogenous calmodulin (CaM) [83]orbyeconazole,
an antifungal [84], while PIP
2
helps stabilize their open
state [85]. The conformational changes between open
and closed states of these channels have been explored
by comparing the cryo-EM structures of econazole-
bound TRPV5 and of both channels in the presence of
either PIP
2
or CaM [86,87].
One of the main findings of this comparison is that
the selectivity filter remains mostly the same regardless
ofthemoleculethatbindsthechannel.Thelowergate
identified in TRPV5 is formed by F574, M578, and
H582 that constrict the pore to a diameter between 4.5
and 5.9 Å in the same bundle crossing configuration
observed in other TRP channels. The closing seems to
involve a change in the position of the S4-S5 linker and
the loop connecting S6 with the TRP domain [86,87].
214 A. E. LÓPEZ-ROMERO ET AL.

The binding of PIP
2,
induce a change in the
orientation of the lateral chain of the aspartate in
the selectivity filter. This change may be explained
by an interaction of the head group of PIP
2
with
R584 in the S6 helix, analogous to what has been
observed in TRPV1, which rotates and pulls away
from the center of the pore accompanied by move-
ment of the S4-S5 linker [87]. The main difference
in the response of TRPV5 and TRPV6 was
observed in the CaM-bound structures. Both chan-
nels bind to a single CaM-molecule which is cap-
able of physically blocking the pore from the
intracellular side, but while TRPV5 shows no con-
formational changes, TRPV6 shows an α-toaπ-
helical transition of S6 which tilts the helix toward
the center, so I575 becomes an inactivation gate
[87], which has also been observed in TRPV3 [53].
Analysis of the pores of TRPV5 and TRPV6 points
to rigid structures that contrast with the more flexible
pores of TRPV1-4. To date, there is no physiological
evidence of changes in their single-channel conduc-
tances in response to different stimuli, but it has been
suggested that their activity might be regulated by the
formation of TRPV5/V6 heterotetramers, as they are
co-expressed in several tissues. Hoenderop et al. used
Xenopus laevis oocytes to express such heterotetra-
mers and found that they have Ca [2]+ -dependent
inactivation and block by ruthenium red with features
intermediate to those of TRPV5 and TRPV6 [88].
Although this is an interesting possibility, they have
not been identified in vivo,andtheirbiophysicalprop-
erties at the level of single-channels have not been
investigated.
Lysophosphatidic acid: A pain-producing
phospholipid
Another activator of TRPV1 shown by our group
to directly interact with this ion channel is lyso-
phosphatidic acid (LPA) [41]. This is the only
example for which a change in single channel
conductance in a TRP channel has been shown
to depend upon the presence of different agonists;
thus, we will discuss this phenomenon in detail.
LPA has been extensively linked to the genera-
tion of chronic neuropathic pain through its inter-
actions with G protein-coupled receptors (GPCRs)
[89–92] and to modulate the activity of several ion
channels [93–97]. Regulation of the activity of
TRPV1 by molecules of a lipidic nature has been
explored, and it has been described that PIP
2
[98–
106], polyunsaturated fatty acids (PUFAs) [107],
monounsaturated fatty acids [108] and cholesterol
[109–111] can modulate the activity of TRPV1,
either directly or indirectly. However, reports of
direct interactions of LPA with ion channels, in
general, are scarce [95,112,113].
LPA is a phospholipid constituted by an acyl
chain of saturated or unsaturated fatty acids asso-
ciated with a glycerol backbone by ester links, and
that contains a phosphate head group [114]. LPA
can be naturally found as different species that
vary in their acyl-chain length as well as in their
saturation (16:0; 18:0; 16:1, 18:1; 18:2 and 20:4).
Our group studied whether LPA could produce
acute pain in mice and showed that it is considerably
dependent upon TRPV1. These results led us to deter-
mine that LPA could, in fact, produce currents and
action potentials in DRG neurons from wild-type
mice but not in DRG neurons from Trpv1
−/−
animals.
Using a heterologous expression system of HEK293
cells transfected with TRPV1, we found that currents
could be induced when LPA was applied to the intra-
cellular or extracellular faces of the ion channels and
that the responses were dose-dependent [41].
In our studies, we ruled out the participation of
GPCR-related signaling pathways on TRPV1 activa-
tion by LPA and identified a site of interaction for this
phospholipid, residue K710 in the C-terminus of
TRPV1 [41]. Confirmation of these results was
obtained later by another group in a study where
TRPV1 was expressed in lipid bilayers and activated
by LPA [115].
Hence, we extended the aforementioned study and
determined that, for fatty acids to activate TRPV1, the
following features had to be met by these molecules:
thepresenceofachargedphosphategroup,alength
of, at least 18 carbons of the acyl chain and a mono
unsaturation [116].
LPA produces a different open
conformational state to that of capsaicin
Our first study on the effects of LPA on TRPV1
was published in 2011 [41], and since then, we
have been studying the specifics of how this mole-
cule regulates the activity of TRPV1. Our most
recent study on this subject provides functional
CHANNELS 215

evidence that LPA produces a different open con-
formational state of TRPV1 that has a larger
conductance.
In preliminary experiments, we had found that
addition of LPA (5 μM) to excised membrane
patches of TRPV1-expressing HEK293 cells
resulted in larger magnitude macroscopic currents,
as compared to those obtained in the presence of
saturating capsaicin concentrations (4 μM). We
had also observed that LPA (5 μM) produced
activation of TRPV1 single-channel currents with
an open probability (Po is 0.8) similar to that of
capsaicin (4 μM).
These data could be explained as 1) LPA either
producing an increase in the number of ion chan-
nels in the excised membrane patches (which was
highly improbable); 2) An increase in the mem-
brane negative surface charge near the channel
leading to an accumulation of positively-charged
ions near the pore mouth of TRPV1, 3) Producing
a phenomenon termed “pore-dilation”in which
the permeability to large ions occurs in the pre-
sence of prolonged exposures to agonists and that
had only been described for activation with cap-
saicin [26] and/or 4) a change in the single-
channel conductance.
In this last study, we performed a set of experi-
ments in order to discern among all of these pos-
sibilities. We started by studying how LPA affected
the unitary TRPV1 currents by first applying cap-
saicin (4 μM), washing off the agonist and then
applying LPA (5 μM) to the same membrane
patch. The results showed that LPA produced
a 41% increase in the magnitude of single-
channel currents at a single voltage (60 mV),
which was also observed at other voltages
(Figure 4).
Furthermore, our experiments ruled out all of
the other possibilities enlisted above and showed
that coapplication of subsaturating concentrations
of both agonists together resulted in two different
types of openings in a single channel, with con-
ductances corresponding to those achieved either
by adding capsaicin or LPA alone [117]. This was
the strongest indication of these two agonists pro-
ducing distinct open conformations that varied in
their conductance. Finally, we defined that the
presence of K710, the residue that is pivotal for
the activation of TRPV1 by LPA, was also neces-
sary for the generation of the higher-conductance
open state [117].
TRPC and TRPM channels
Until just recently, finely defined structural char-
acteristics of TRPC channels have remained elu-
sive, mainly because of the lack of high-resolution
structures. The cryo-EM structures obtained to
date are those for the TRPC3, TRPC4, TRPC5
and TRPC6 channels [118–123]. However, no
functional studies reporting changes in the con-
ductance of these channels in response to different
agonists are available.
These channels exhibit four ankyrin repeats and
linker helices at their N-terminal domains and at
the C-terminal domains they possess a TRP
domain that connects a helix and a coiled-coil
domain. Inside the pore, all TRPC members have
a conserved “LFW”motif that maintains the struc-
ture of the pore [124].
Figure 4. LPA increases single channel currents.
Current amplitude for a TRPV1-expressing membrane patch in the presence of capsaicin (4 µM; left) and in the presence of LPA
(5 µM; right). Traces were obtained at +60 mV.
216 A. E. LÓPEZ-ROMERO ET AL.

The TRPC subfamily can be divided into two
groups that share some distinctive characteristics.
The first group is constituted by the TRPC3/C6/C7
channels are activated by DAG. Structurally, both
TRPC3 and TRPC6 display a pre-S1 elbow, a TRP
re-entrant helix embedded in the membrane
bilayer after the TRP domain, and an unusually
long S3 that forms a “pseudo”extra cellular
domain (ECD) [121,122,124]. Furthermore, unlike
most TRP channels, TRPC3 channels possess
a linker that connects the TRP domain to the S6.
This results in a more flexible TRP domain that
exhibits a higher coupling with the S4-S5 linker
and a lipid binding site near the pre-S1. All of
these peculiar interplays of domains may provide
a molecular basis for TRPC3 lipid gating [121].
The second group includes the TRPC4/C5
channels that are modulated by G
q,
G
i/o
GPCRs,
membrane lipids, and intracellular Ca
2+
[119,125].
These channels share the common feature of
a disulfide bond between two cysteines at the
loop linking S5 and the pore domain that main-
tains the residue E555 in proper position and thus
stabilize the upper region of their selectivity filters
[119,120,123,124]. As in other TRP channels, the
TRP domain of TRPC4 is an important molecular
effector for gating in this protein since it interacts
with the S4-S5 linker and other proteins [123].
The structures of TRPC3/C6 and TRPC4 (both
human and mouse) show a closed pore conformation.
For TRPC3 the lower gate is located at residues I658
and L654 where they present a radius of less than 1 Å
[121]. As for the human TRPC4, the residues I617 and
N621 were identified as the lower gate where the
constriction of the diameter was around 1.6 and
0.7 Å (defined by opposing van der Waals surfaces)
[123], and for the mouse TRPC4 the narrowest point
is located at N621 (3.6 Å) and constitutes the lower
gate along with I617 and Q625 [119].
However, the TRPC5 is considered at least par-
tially open since the constriction at the lower gate
formed by the residues I621, N625 and Q629 was
around 4.9 Å [120]. Comparing this structure with
the closed TRPC4, Duan and collaborators suggest
that the extracellular disulfide bond is a transducer
of conformational changes. Nonetheless, more
structures of TRPC channels at their open confor-
mation will help to elucidate if there are differ-
ences in the pore during their gating [120].
The TRPM subfamily is composed of eight
members with diverse features and functions
[126]. The TRPM members for which cryo-EM
structures have been solved in the closed or par-
tially open conformations are TRPM2, TRPM4,
TRPM7 and TRPM8 [127–135]. Their subunits
are composed of six transmembranal segments,
but at their N-terminal domains, they present
four TRPM homology regions (MHR) and
a C-terminal coiled-coil domain [127–135].
The C-termini of TRPM2, TRPM6, and TRPM7
channels possess enzymatic domains and have
been named “chanzymes”[136–139]. Similar to
TRPC4/C5 channels, TRPM channels possess dis-
ulfide bonds at their extracellular pore that stabi-
lize the pore domain, but these disulfide-bonds
form between cysteines at the pore loop and the
pore helix S6 [119].
TRPM2 is a non-selective cation channel sensitive
to temperature [140] that is activated when ADP-
Ribose (ADPR) and Ca
2+
are co-applied [141].
Wang and collaborators obtained the structures of
human apo-, ADPR-bound, and ADPR-Ca
2+
-bound
TRPM2 channels. They observed that the binding of
ADPR alone produced a closed conformation, con-
sidering it a “primed state”[128].
The lower gate was identified at residues I1045
and Q1053 in the pore and shown to be enlarged
in the ADPR/Ca
2+
structure. Although a change
distance of the residues that form the gate was
observed, it was not considered large enough to
allow passage of Ca
2+
(~4 Å radius) [128].
For zebrafish TRPM2 channels it was shown
that, once ADPR binds to the MHR1/2 domain,
a displacement of MHR4 is transmitted to the TRP
helix, pushing it towards the S4-S5 linker.
Consequently, the S4-S5 relocates the S5 and S6
and finally produces the opening of the channel.
Again, as for other TRP channels, the TRP helix is
shown to act as a transducer between the intracel-
lular and transmembranal domains during gating
[129]. Moreover, the authors propose putative
binding sites for Ca
2+
in the S3, leading them to
speculate that Ca
2+
facilitates the opening due to
the movement of the S3 upon the binding of this
ion which allows for the relocation of the S4-S5
linker [129].
TRPM4 and TRPM5 channels are among the
only TRP members that are not permeable to
CHANNELS 217

divalent cations [132,142,143]. Despite their ion
selectivity, TRPM4/5 channels are activated upon
intracellular Ca
2+
increases [144] and can be
modulated by PI(4,5)P
2
[145,146]. However,
TRPM4 is inhibited by intracellular ATP and
nucleotides, while TRPM5 is insensitive to these
molecules [147,148].
The pore of TRPM4 has two gates: one located
at the selectivity filter and another formed by the
intracellular portion of the S6. In the human chan-
nel, the selectivity filter gate is formed by residues
975-FGQ-977 [130–132]. Likewise, the intracellu-
lar gate is formed by the I1040 residue [131,132].
Strikingly, both gates remain unchanged in all
reported Ca
2+
-bound structures, and it has been sug-
gested that Ca
2+
- binding is required for the opening
of the channel in response to voltage [131].
In the structure obtained in complex with ATP,
Guo and collaborators located a nucleotide binding
domain along with twelve helices and two ankyrin
repeats in the N-terminal [149]. The ATP-bound
structure is an inhibited state of TRPM4, and there
were no overall changes in the pore compared to the
apo structure. The TRP domain forms hydrophobic
interactions with the S4-S5 linker and forms a cavity
between the S1-S4 domain, which harbors
apotentialCa
2+
-binding site [149].
The protein kinase domain in TRPM7 induces
phosphorylation of receptor tyrosine kinase
(RTK)-signaling intermediates and chromatin
modifications [150,151]. TRPM7 is permeable
to Mg
2+
,Zn
2+
and Ca
2+
[152], and the cryo-EM
structure of TRPM7 was obtained in the presence
and absence of Mg
2+
[133].
There were no notable changes on the pore
domain of TRPM7 under different ionic condi-
tions. Interestingly, the selectivity filter identified
in TRPM7 (1045-FGE-1047), varies only on one
residue to the selectivity filter described in TRPM4
(971-FGQ-973 in mouse TRPM4 channel), which
is crucial to confer the monovalent selectivity of
the latter. The lower gate was determined to be
formed by I1093 and N1097 residues [133].
The TRP domain of TRPM7 interacts with dif-
ferent regions of the channel, highlighting the
importance of this region for gating in this pro-
tein. For example, the TRP domain interacts with
the N-terminal residues S744 and Q740, with the
S4-S5 linker by forming hydrogen bonds between
W1111 and R1115 (both located to the TRP
domain) and with A981 and V982 (in the S4-S5
linker) [133]. Additionally, it establishes π-πstack-
ing (Phe1118/Tyr1122) and cation- πinteractions
(Arg1115/Trp1111) among its α-helical structure
[133]. However, unlike what has been observed in
other TRP structures, the TRP domain of TRPM7
does not contact the pre-S1 helix [133].
Another region that may produce important
conformational rearrangements in TRPM7 is the
pore helix. It has been shown that a disulfide-bond
established between residues C1056 and C1066, is
only formed in the presence of Mg
2+
. Thus, it has
been proposed that this cation could stabilize the
bond and impact directly on the structure of the
pore [133].
Finally, the TRPM8 is a non-selective cationic
channel permeable to Ca
2+
and activated by cold
temperatures (below 25°C) and menthol [153].
The channel is modulated by PI(4,5)P
2
[153,154].
In the same report, the cryo-EM structures for
TRPM8 have been obtained in complex with PIP
2
,
Ca
2+,
and icilin, and with WS-12 (a menthol ana-
log) together with PIP
2
[134]. These structures
demonstrate that both, icilin and WS-12, bind to
a cavity formed between S1-S4 and the TRP
domain [134].
The structures reveal a different binding site of
PIP
2
, as compared to other TRP channels. This site
is formed by the pre-S1 domain, the S4-S5 junc-
tion, the TRP domain and the MHR4 of an adja-
cent subunit. Interestingly, upon the binding of
PIP
2
, the S4 undergoes a change from a complete
α-helical to a 3
10
helical conformation [134]. This
produces the movement of the TRP domain and
the S5 toward the cavity. At the same time, it
disrupts the interaction among the S1-S4 domain
and the S6-pore domain, enabling gating [134].
Additionally, during gating, the S1-S4 domain of
TRPM8 suffers a rigid rotation away from the pore
[134], unlike what happens to the S1-S4 domain in
TRPV1 which remains static [45].
The lower gate of TRPM8 is formed by L973
where the tightest constriction along the pore
was found in a previous structure
134135
,Yin
and collaborators determined that both struc-
tures (PIP
2
-Ca
2+
-icilin and WS-12-PIP
2
)were
non-conducting states. Although, in the icilin-
PIP
2
-Ca
2+
structure the S6 is curved, which
218 A. E. LÓPEZ-ROMERO ET AL.

suggested that there may be a πhelical turn
along the S6 as previously described for the
sensitized state of TRPV3
53
. Therefore, the
structures may represent sensitized or presensi-
tized states of TRPM8 [134].
Although conformational changes in TRPM
channels in response to agonists or modulators of
their activities have been clarified, changes in their
conductance levels in response to these molecules
have not been reported.
Discussion
Here we have summarized some phenomena that
could potentially lead to changes in the conduc-
tance of single ion channels. For example, pore
dilation is a phenomenon suggested to occur in
TRPV1 [26,155] and other channels such as the
P2X receptors, and it is characterized by a change
in the permeability of the ion channel to larger ions,
induced by long times in the presence of the ago-
nist. However, the results that we have obtained, as
well as those of other groups, suggest that perme-
ability changes may result from changes in the
cytoskeleton organization or from time-dependent
local changes in the concentrations of ions in the
vicinity of the channel, a phenomenon known as
ion accumulation, that is at play in whole-cell
experiments [156] and is further discussed below.
Li and collaborators have examined this care-
fully and have shown that P2X receptors readily
activate and do not display a slow phase of activa-
tion under symmetrical ionic conditions [156].
These authors also show that changes in the equi-
librium potential are observed with NMDG
+
in the
external solution during sustained activation and
that this prolonged activation in bi-ionic solutions
primes the depletion of internal Na
+
and accumu-
lation of NMDG
+
in the cell. Li et al., also discuss
that P2X receptors are exceptional in that they
demonstrate significant permeability to large
cations such as NMDG
+
and that, even in the
absence of pore dilation, would allow the conduc-
tion of relatively large molecules to enter or exit
cells [156]. Finally, and most importantly, these
authors point out that changes in ion concentra-
tion, produced by ion accumulation and revealed
during experiments with P2X receptors, are an
inherent problem in voltage-clamp recordings,
where the flow of ionic currents across the mem-
brane may alter the concentrations of intracellular
ions [157,158]. In other words, when symmetrical
ionic conditions are used, ion concentrations can
be maintained by working under conditions in
which current amplitudes are moderate and cur-
rent measurements will always be equal to or
greater than the flux of any individual ionic spe-
cies. This is in contrast to experiments performed
with asymmetric ionic conditions, which require
a membrane conductance of an order of magni-
tude smaller than the access conductance to ade-
quately control ion concentrations.
These results are prompting a reexamination of
the concepts of pore dilation and dynamic ion
selectivity for all ion channels [156], and highlight
the importance of further studying the molecular
mechanisms underlying different conformational
changes induced by ligands in these proteins.
TRPV1 has been shown to contribute to several
physiological and pathophysiological processes,
importantly. Among these, the participation of
TRPV1 in pain-related processes is an aspect that
has attracted the attention of several research
groups. Understanding cellular mechanisms lead-
ing to the generation of pain constitutes the basis
for designing tools in order to attack them. Hence,
research directedtoward defining the molecular
mechanisms that underlie the production of pain
is of invaluable importance. This requires a great
effort from many researchers and from several
points of view, including that of understanding
various particularities of the modulation of the
effectors involved in pain processes.
As for changes in the conductance of TRPV1
due to the actions of agonists, it is interesting to
note that, it has been recently reported that DkTx
diminishes the unitary conductance by 32% of
TRPV1, as compared to that observed with capsai-
cin [159]. DkTx is a molecule composed of two
moieties joined by a short linker, which bind in
the outer pore region of the channel so that each
motif sits at a subunit interface [45]. Moreover, the
effect was eliminated when the linker of DkTx was
either absent or elongated and also when the pore
turret of TRPV1 was removed. The lack of pore
turret in the construct used for the cryo-EM of
TRPV1 explains why the structure with DkTx/RTx
is observed to be fully open. In fact, the unitary
CHANNELS 219

conductance of the construct used to obtain the
TRPV1 structure with bound DkTx was similar to
that obtained with capsaicin [159].
With this, Geron et al. confirmed that DkTx
and capsaicin elicit distinct gating mechanisms
that are dependent upon the pore turret of
TRPV1. They argued that the binding of DkTx
directly restricts the movement of the pore helix
and consequently the conductance.
In terms of the physiological relevance of LPA’s
actions on the conductance of the TRPV1 channel,
one can only hypothesize that in the presence of
this agonist, as compared to capsaicin, depolariza-
tion of the neuron where it is expressed will occur
more efficiently. Since the change in the conduc-
tance of the channel also occurs at subsaturating
concentrations of LPA (i.e., 1 μM), this would
mean that such an efficient depolarization could
also occur even when lower levels of the phospho-
lipid are present.
TRP channels share conserved sequences of
amino acids in some regions of their structures.
One such region is the TRP box (Figure 5), the
region where the K710 residue that interacts with
LPA lies. We have tested the effects of LPA on
some TRP ion channels and have found that
TRPV2, TRPV3, or TRPA1 channels are not acti-
vated by this phospholipid [41]. Although TRPV3
and TRPA1 have a lysine in this position, just as
TRPV1, LPA is not capable of activating these
channels. Thus, this phospholipid seems incapable
of providing the energy necessary to drive the
transition from the closed to the open state in
this particular ion channels and the question of
why this occurs remains open. Moreover, since
other members of the TRPV subfamily of ion
channels exhibit a lysine in the corresponding
position to that of K710, it will be interesting to
test whether their activities can be modulated by
LPA. If so, this would constitute the identification
of an agonist for these other ion channels for
which there are few endogenous activators
known (i.e., TRPV4, TRPV5, and TRPV6).
Since achieving different conductance states can
modulate the response of the cell where ion chan-
nels are expressed, not only will it be interesting to
determine if LPA activates these other TRP chan-
nels, but also whether it can produce different con-
formational changes as what happens with TRPV1.
This would further demonstrate the conformational
versatility of this family of ion channels.
Several ion channels have been studied at great
detail with respect to their responses to different
agonists. In particular, the idea that LPA produces
a conformational change that results in a different
conductance of TRPV1 at the single-channel level
compared to capsaicin challenges the notion in the
field of study of ion channels in general.
Here we have discussed that LPA leads the
TRPV1 ion channel to open with a single-
channel conductance level that is higher than
that attained with capsaicin. Both ligands promote
nearly maximal open probabilities [117]; hence,
this is most probably not a mechanism in which
different subconductance levels can be attained in
the presence of partial and full agonists.
Thus, it will be interesting to reflect upon and
determine what mechanisms or structural rearran-
gements allow TRPV1, and maybe other TRP
channels, to exhibit a higher conductance level in
the presence of one agonist or another.
Acknowledgments
This work was supported by grants from Dirección General
de Asuntos del Personal Académico (DGAPA)-Programa de
Apoyo a Proyectos de Investigación e Innovación
Figure 5. Sequence alignment corresponding to the trp box in
rat and human TRPV and TRPA1 channels.
Identical residues are highlighted in yellow and similar residues
in blue. Tryptophan and glutamine residues in the middle of
the trp box are only present in TRPV channels. The residue K710
in hTRPV1 and the corresponding residue in other TRP channels
is highlighted in pink, notice that most channels, including
TRPA1 have a positively charged residue in that position.
220 A. E. LÓPEZ-ROMERO ET AL.

Tecnológica (PAPIIT) IN200717 and Consejo Nacional de
Ciencia y Tecnología (CONACyT) A1-S-8760 to T.R.
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
This work was supported by the Consejo Nacional de Ciencia
y Tecnología [A1-S-8760];Dirección General de Asuntos del
Personal Académico, Universidad Nacional Autónoma
de México [IN200717];
ORCID
Ileana Hernández-Araiza http://orcid.org/0000-0003-4102-
5282
Francisco Torres-Quiroz http://orcid.org/0000-0002-3073-
8546
Luis B. Tovar-Y-Romo http://orcid.org/0000-0003-2605-
1378
León D. Islas http://orcid.org/0000-0002-7461-5214
Tamara Rosenbaum http://orcid.org/0000-0002-4791-3195
References
[1] Hamill OP, Marty A, Neher E, et al. Improved patch-
clamp techniques for high-resolution current record-
ing from cells and cell-free membrane patches.
Pflugers Arch. 1981;391(2):85–100.
[2] MacKinnon R. New insights into the structure and
function of potassium channels. Curr Opin
Neurobiol. 1991;1(1):14–19.
[3] Pore Loops: MR. An emerging theme in ion channel
structure. Neuron. 1995;14(5):889–892.
[4] Potassium MR. Channels and the atomic basis of
selective ion conduction (nobel Lecture). Angew
Chem Int Ed Engl. 2004;43(33):4265–4277.
[5] Doyle DA, Morais Cabral J, Pfuetzner RA, et al. The
structure of the potassium channel: molecular basis of
K+ conduction and selectivity. Science. 1998;280
(5360):69–77.
[6] Agre P. Aquaporin water channels (nobel lecture).
Angew Chem Int Ed Engl. 2004;43(33):4278–4290.
[7] Yasui M, Kwon TH, Knepper MA, et al. Aquaporin-6: an
intracellular vesicle water channel protein in renal epithe-
lia. Proc Natl Acad Sci U S A. 1999;96(10):5808–5813.
[8] Cosens DJ, Manning A. Abnormal electroretinogram
from a drosophila mutant. Nature. 1969;224
(5216):285–287.
[9] Montell C, Rubin GM. Molecular characterization of
the drosophila trp locus: A putative integral membrane
protein required for phototransduction. Neuron.
1989;2(4):1313–1323.
[10] Hardie RC, Minke B. The Trp gene is essential for a
light-activated Ca2+ channel in drosophila photore-
ceptors. Neuron. 1992;8(4):643–651.
[11] Caterina MJ, Schumacher MA, Tominaga M, et al. The
capsaicin receptor: A heat-activated ion channel in the
pain pathway. Nature. 1997;389(6653):816–824.
[12] Tominaga M, Caterina MJ, Malmberg AB, et al. The
cloned capsaicin receptor integrates multiple pain-pro-
ducing stimuli. Neuron. 1998;21(3):531–543.
[13] Perozo E, Cortes DM, Cuello LG. Structural rearran-
gements underlying K+-channel activation gating.
Science. 1999;285(5424):73–78.
[14] VanDongen AMJ. K channel gating by an affinity-
switching selectivity filter. Proc Natl Acad Sci U S A.
2004;101(9):3248–3252.
[15] Schoppa NE, Sigworth FJ. Activation of shaker potas-
sium channels iii. An activation gating model for wild-
type and V2 mutant channels. J Gen Physiol. 1998;111
(2):313–342.
[16] Zheng J, Sigworth FJ. Intermediate conductances dur-
ing deactivation of heteromultimeric shaker potassium
channels. J Gen Physiol. 1998;112(4):457–474.
[17] Bao H, Hakeem A, Henteleff M, et al. Voltage-
insensitive gating after charge-neutralizing mutations
in the S4 segment of shaker channels. J Gen Physiol.
1999;113(1):139–151.
[18] Yifrach O, MacKinnon R. Energetics of pore opening
in a voltage-gated K(+) channel. Cell. 2002;111
(2):231–239.
[19] Trapani JG, Andalib P, Consiglio JF, et al. Control of
single channel conductance in the outer vestibule of
the Kv2.1 potassium channel. J Gen Physiol. 2006;128
(2):231–246.
[20] Immke D, Korn SJ. Ion-ion interactions at the selec-
tivity filter evidence from K(+)-dependent modulation
of tetraethylammonium efficacy in Kv2.1 potassium
channels. J Gen Physiol. 2000;115(4):509–518.
[21] Tang L, Gamal El-Din TM, Payandeh J, et al.
Structural basis for Ca2+ Selectivity of a voltage-
gated calcium channel. Nature. 2014;505(7481):56–
61.
[22] Virginio C, MacKenzie A, Rassendren FA, et al. Pore
dilation of neuronal P2X receptor channels. Nat
Neurosci. 1999;2(4):315–321.
[23] Khakh BS, Lester HA. Dynamic selectivity filters in ion
channels. Neuron. 1999;23(4):653–658.
[24] Lingueglia E, de Weille JR, Bassilana F, et al. A mod-
ulatory subunit of acid sensing ion channels in brain
and dorsal root ganglion cells. J Biol Chem. 1997;272
(47):29778–29783.
[25] de Weille JR, Bassilana F, Lazdunski M, et al.
Identification, functional expression and chromosomal
localisation of a sustained human proton-gated cation
channel. FEBS Lett. 1998;433(3):257–260.
[26] Chung M-K, Güler AD, Caterina MJ. TRPV1 shows
dynamic ionic selectivity during agonist stimulation.
Nat Neurosci. 2008;11(5):555–564.
CHANNELS 221

[27] Munns CH, Chung M-K, Sanchez YE, et al. Role of the
outer pore domain in transient receptor potential
vanilloid 1 dynamic permeability to large cations. J
Biol Chem. 2015;290(9):5707–5724.
[28] Brake AJ, Wagenbach MJ, Julius D. New structural motif
for ligand-gated ion channels defined by an ionotropic
ATP receptor. Nature. 1994;371(6497):519–523.
[29] Khakh BS, Egan TM. Contribution of transmembrane
regions to ATP-gated P2X2 channel permeability
dynamics. J Biol Chem. 2005;280(7):6118–6129.
[30] Di Virgilio F, Schmalzing G, Markwardt F. The elusive
P2X7 macropore. Trends Cell Biol. 2018;28(5):392–404.
[31] Woo DH, Jung SJ, Zhu MH, et al. Direct activation of
transient receptor potential vanilloid 1(TRPV1) by
diacylglycerol (DAG). Mol Pain. 2008;4:42.
[32] Wen H, Östman J, Bubb KJ, et al. 20-
hydroxyeicosatetraenoic acid (20-HETE) is a novel
activator of transient receptor potential vanilloid 1
(TRPV1) channel. J Biol Chem. 2012;287(17):13868–
13876.
[33] Smart D, Gunthorpe MJ, Jerman JC, et al. The endo-
genous lipid anandamide is a full agonist at the human
vanilloid receptor (HVR1). Br J Pharmacol. 2000;129
(2):227–230.
[34] Movahed P, Jönsson BAG, Birnir B, et al. Endogenous
unsaturated C18 N-acylethanolamines are vanilloid
receptor (TRPV1) agonists. J Biol Chem. 2005;280
(46):38496–38504.
[35] Chu CJ, Huang SM, De Petrocellis L, et al. N-oleoyl-
dopamine, a novel endogenous capsaicin-like lipid that
produces hyperalgesia. J Biol Chem. 2003;278
(16):13633–13639.
[36] Yoshida T, Inoue R, Morii T, et al. Nitric oxide acti-
vates TRP channels by cysteine s-nitrosylation. Nat
Chem Biol. 2006;2(11):596–607.
[37] Yang F, Ma L, Cao X, et al. Divalent cations activate
TRPV1 through promoting conformational change of
the extracellular region. J Gen Physiol. 2014;143(1):91–
103.
[38] Jara-Oseguera A, Bae C, Swartz KJ. An external
sodium ion binding site controls allosteric gating in
TRPV1 channels. eLife. 2016;5. DOI:10.7554/
eLife.13356.
[39] Patacchini R, Santicioli P, Giuliani S, et al.
Pharmacological investigation of hydrogen sulfide
(H2S) contractile activity in rat detrusor muscle. Eur
J Pharmacol. 2005;509(2–3):171–177.
[40] Bohlen CJ, Priel A, Zhou S, et al. A bivalent tarantula
toxin activates the capsaicin receptor, TRPV1, by target-
ing the outer pore domain. Cell. 2010;141(5):834–845.
[41] Nieto-Posadas A, Picazo-Juárez G, Llorente I, et al.
Lysophosphatidic acid directly activates TRPV1
through a C-terminal binding site. Nat Chem Biol.
2011;8(1):78–85.
[42] Moiseenkova-Bell VY, Stanciu LA, Serysheva II, et al.
Structure of TRPV1 channel revealed by electron cryo-
microscopy. Proc Natl Acad Sci U S A. 2008;105
(21):7451–7455.
[43] Liao M, Cao E, Julius D, et al. Structure of the TRPV1
ion channel determined by electron cryo-microscopy.
Nature. 2013;504(7478):107–112.
[44] Lishko PV, Procko E, Jin X, et al. The ankyrin repeats
of TRPV1 bind multiple ligands and modulate channel
sensitivity. Neuron. 2007;54(6):905–918.
[45] Cao E, Liao M, Cheng Y, et al. TRPV1 structures in
distinct conformations reveal activation mechanisms.
Nature. 2013;504(7478):113–118.
[46] Caterina MJ, Rosen TA, Tominaga M, et al. A capsai-
cin-receptor homologue with a high threshold for
noxious heat. Nature. 1999;398(6726):436–441.
[47] Shibasaki K. Physiological Significance of TRPV2 as a
mechanosensor, thermosensor and lipid sensor. J
Physiol Sci JPS. 2016;66(5):359–365.
[48] Zhang F, Hanson SM, Jara-Oseguera A, et al.
Engineering vanilloid-sensitivity into the rat TRPV2
channel. eLife. 2016;5. DOI:10.7554/eLife.16409.
[49] Yang F, Vu S, Yarov-Yarovoy V, et al. Rational design
and validation of a vanilloid-sensitive TRPV2 ion
channel. Proc Natl Acad Sci U S A. 2016;113(26):
E3657–3666.
[50] Zubcevic L, Le S, Yang H, et al. Conformational
Plasticity in the Selectivity Filter of the TRPV2 Ion
Channel. Nat Struct Mol Biol. 2018;25(5):405–415.
[51] Zubcevic L, Herzik MA, Chung BC, et al. Cryo-
Electron Microscopy Structure of the TRPV2 Ion
Channel.NatStructMolBiol.2016;23(2):180–186.
[52] Zhang F, Swartz KJ, Jara-Oseguera A. Conserved allos-
teric pathways for activation of TRPV3 revealed
through engineering vanilloid-sensitivity. eLife.
2019;8. DOI:10.7554/eLife.42756.
[53] Zubcevic L, Herzik MA, Wu M, et al. Conformational
ensemble of the human TRPV3 ion channel. Nat
Commun. 2018;9(1):4773.
[54] Singh AK, McGoldrick LL, Sobolevsky AI. Structure
and gating mechanism of the transient receptor poten-
tial channel TRPV3. Nat Struct Mol Biol. 2018;25
(9):805–813.
[55] Liedtke W, Choe Y, Martí-Renom MA, et al. Vanilloid
receptor-related osmotically activated channel (VR-
OAC), a candidate vertebrate osmoreceptor. Cell.
2000;103(3):525–535.
[56] Strotmann R, Harteneck C, Nunnenmacher K, et al.
OTRPC4, a nonselective cation channel that confers
sensitivity to extracellular osmolarity. Nat Cell Bio.
2000;2(10):695–702.
[57] Güler AD, Lee H, Iida T, et al. Heat-evoked activation
of the ion channel, TRPV4. J Neurosci Off J Soc
Neurosci. 2002;22(15): 6408–6414. 20026679.
222 A. E. LÓPEZ-ROMERO ET AL.

[58] Watanabe H, Davis JB, Smart D, et al. Activation of
TRPV4 Channels (HVRL-2/MTRP12) by Phorbol
Derivatives. J Biol Chem. 2002;277(16):13569–13577.
[59] Watanabe H, Vriens J, Prenen J, et al. Anandamide
and arachidonic acid use epoxyeicosatrienoic acids to
activate TRPV4 channels. Nature. 2003;424
(6947):434–438.
[60] Vriens J, Watanabe H, Janssens A, et al. Cell swelling,
heat, and chemical agonists use distinct pathways for
the activation of the cation channel TRPV4. Proc Natl
Acad Sci U S A. 2004;101(1):396–401.
[61] Deng Z, Paknejad N, Maksaev G, et al. Cryo-EM and
X-ray structures of TRPV4 Reveal Insight into Ion
Permeation and Gating Mechanisms. Nat Struct Mol
Biol. 2018;25(3):252–260.
[62] Macpherson LJ, Geierstanger BH, Viswanath V, et al. The
pungency of garlic: activation of TRPA1 and TRPV1 in
response to allicin. Curr Biol CB. 2005;15(10):929–934.
[63] Bautista DM, Movahed P, Hinman A, et al. Pungent
products from garlic activate the sensory ion channel
TRPA1. Proc Natl Acad Sci U S A. 2005;102
(34):12248–12252.
[64] Story GM, Peier AM, Reeve AJ, et al. ANKTM1, a TRP-
like channel expressed in nociceptive neurons, is activated
by cold temperatures. Cell. 2003;112(6):819–829.
[65] Viswanath V, Story GM, Peier AM, et al. Opposite
thermosensor in fruitfly and mouse. Nature. 2003;423
(6942):822–823.
[66] Caspani O, Heppenstall PA. TRPA1 and cold transduc-
tion: an unresolved issue?. J Gen Physiol. 2009;133(3):245–
249.
[67] Karashima Y, Talavera K, Everaerts W, et al. TRPA1
acts as a cold sensor in vitro and in vivo. Proc Natl
Acad Sci U S A. 2009;106(4):1273–1278.
[68] Vandewauw I, De Clercq K, Mulier M, et al. A TRP
channel trio mediates acute noxious heat sensing.
Nature. 2018;555(7698):662–666.
[69] Bandell M, Story GM, Hwang SW, et al. Noxious cold
ion channel TRPA1 is activated by pungent com-
pounds and bradykinin. Neuron. 2004;41(6):849–857.
[70] Hinman A, Chuang -H-H, Bautista DM, et al. TRP
channel activation by reversible covalent modification.
Proc Natl Acad Sci U S A. 2006;103(51):19564–19568.
[71] Salazar H, Llorente I, Jara-Oseguera A, et al. A single
n-terminal cysteine in trpv1 determines activation by
pungent compounds from onion and garlic. Nat
Neurosci. 2008;11(3):255–261.
[72] Salazar H, Jara-Oseguera A, Hernández-García E, et al.
Structural determinants of gating in the TRPV1 chan-
nel. Nat Struct Mol Biol. 2009;16(7):704–710.
[73] Peyrot Des Gachons C, Uchida K, Bryant B, et al. Unusual
pungency from extra-virgin olive oil is attributable to
restricted spatial expression of the receptor of oleocanthal.
J Neurosci Off J Soc Neurosci. 2011;31(3):999–1009.
[74] Laursen WJ, Bagriantsev SN, Gracheva EO. TRPA1
channels: chemical and temperature sensitivity. Curr
Top Membr. 2014;74:89–112.
[75] Samanta A, Kiselar J, Pumroy RA, et al. Structural
insights into the molecular mechanism of mouse
TRPA1 Activation and inhibition. J Gen Physiol.
2018;150(5):751–762.
[76] Jordt S-E, Bautista DM, Chuang -H-H, et al. Mustard
oils and cannabinoids excite sensory nerve fibres
through the TRP channel ANKTM1. Nature.
2004;427(6971):260–265.
[77] Cavanaugh EJ, Simkin D, Kim D. Activation of tran-
sient receptor potential a1 channels by mustard oil,
tetrahydrocannabinol and Ca2+ reveals different func-
tional channel states. Neuroscience. 2008;154(4):1467–
1476.
[78] Paulsen CE, Armache J-P, Gao Y, et al. Structure of
the TRPA1 ion channel suggests regulatory mechan-
isms. Nature. 2015;525(7570):552.
[79] Martinez GQ, Gordon SE. Multimerization of homo
sapiens TRPA1 ion channel cytoplasmic domains. PloS
One. 2019;14(2):e0207835.
[80] Voets T, Janssens A, Prenen J, et al. Mg2+-dependent
gating and strong inward rectification of the cation
channel TRPV6. J Gen Physiol. 2003;121(3):245–260.
[81] Den Dekker E, Hoenderop JGJ, Nilius B, et al. The
epithelial calcium channels, TRPV5 & TRPV6: from
identification towards regulation. Cell Calcium.
2003;33(5–6):497–507.
[82] McGoldrick LL, Singh AK, Saotome K, et al. Opening
of the human epithelial calcium channel TRPV6.
Nature. 2018;553(7687):233–237.
[83] Kovalevskaya NV, Bokhovchuk FM, Vuister GW. The
TRPV5/6 calcium channels contain multiple calmodu-
lin binding sites with differential binding properties. J
Struct Funct Genomics. 2012;13(2):91–100.
[84] Nilius B, Prenen J, Vennekens R, et al.
Pharmacological modulation of monovalent cation
currents through the epithelial Ca2+ channel ECaC1.
BrJ Pharmacol. 2001;134(3):453–462.
[85] van Goor MKC, Hoenderop JGJ, van der Wijst J. TRP
channels in calcium homeostasis: from hormonal con-
trol to structure-function relationship of TRPV5 and
TRPV6. Biochim Biophys Acta Mol Cell Res.
2017;1864(6):883–893.
[86] Hughes TET, Lodowski DT, Huynh KW, et al.
Structural basis of TRPV5 channel inhibition by eco-
nazole revealed by Cryo-EM. Nat Struct Mol Biol.
2018;25(1):53–60.
[87] Hughes TET, Pumroy RA, Yazici AT, et al. Structural
insights on TRPV5 gating by endogenous modulators.
Nat Commun. 2018;9(1):4198.
[88] Hoenderop JGJ, Voets T, Hoefs S, et al. Homo- and
heterotetrameric architecture of the epithelial Ca2+
channels TRPV5 and TRPV6. Embo J. 2003;22
(4):776–785.
[89] Ohsawa M, Miyabe Y, Katsu H, et al. Identification of
the sensory nerve fiber responsible for lysophosphati-
dic acid-induced allodynia in mice. Neuroscience.
2013;247:65–74.
CHANNELS 223

[90] Ueda H, Matsunaga H, Olaposi OI, et al. Chemical
signature of neuropathic pain. Biochim Biophys Acta.
2013;1831(1):61–73.
[91] Inoue M, Rashid MH, Fujita R, et al. Initiation of
neuropathic pain requires lysophosphatidic acid recep-
tor signaling. Nat Med. 2004;10(7):712–718.
[92] Inoue M, Ma L, Aoki J, et al. Autotaxin, a synthetic enzyme
of lysophosphatidic acid (LPA), mediates the induction of
nerve-injured neuropathic pain. Mol Pain. 2008;4:6.
[93] Chemin J, Patel A, Duprat F, et al. Lysophosphatidic
Acid-Operated K+ Channels. J Biol Chem. 2005;280
(6):4415–4421.
[94] Stirling L, Williams MR, Morielli AD. Dual Roles for
RHOA/RHO-kinase in the regulated trafficking of a
voltage-sensitive potassium channel. Mol Biol Cell.
2009;20(12):2991–3002.
[95] Kittaka H, Uchida K, Fukuta N, et al.
Lysophosphatidic acid-induced itch is mediated by
signalling of LPA5 receptor, phospholipase D and
TRPA1/TRPV1. J Physiol. 2017;595(8):2681–2698.
[96] Hernández-Araiza I, Morales-Lázaro SL, Canul-
Sánchez JA, et al. Role of lysophosphatidic acid in
ion channel function and disease. J Neurophysiol.
2018;120(3):1198–1211.
[97] Czirják G, Tóth ZE, Enyedi P. The two-pore domain K
+ channel, TRESK, is activated by the cytoplasmic
calcium signal through calcineurin. J Biol Chem.
2004;279(18):18550–18558.
[98] Chuang HH, Prescott ED, Kong H, et al. Bradykinin
and nerve growth factor release the capsaicin receptor
from PtdIns(4,5)P2-mediated inhibition. Nature.
2001;411(6840):957–962.
[99] Prescott ED, Julius D. A modular PIP2 binding site as
a determinant of capsaicin receptor sensitivity.
Science. 2003;300(5623):1284–1288.
[100] Klein RM, Ufret-Vincenty CA, Hua L, et al. Determinants
of molecular specificity in phosphoinositide regulation.
phosphatidylinositol (4,5)-Bisphosphate (PI(4,5)P2) is
the endogenous lipid regulating TRPV1. J Biol Chem.
2008;283(38):26208–26216.
[101] Rosasco MG, Gordon SE, Bajjalieh SM. Characterization
of the functional domains of a mammalian voltage-sensi-
tive phosphatase. Biophys J. 2015;109(12):2480–2491.
[102] Senning EN, Collins MD, Stratiievska A, et al.
Regulation of TRPV1 ion channel by phosphoinositide
(4,5)-bisphosphate: the role of membrane asymmetry.
J Biol Chem. 2014;289(16):10999–11006.
[103] Stein AT, Ufret-Vincenty CA, Hua L, et al.
Phosphoinositide 3-kinase binds to TRPV1 and med-
iates NGF-stimulated TRPV1 trafficking to the plasma
membrane. J Gen Physiol. 2006;128(5):509–522.
[104] Stratiievska A, Nelson S, Senning EN, et al. Reciprocal
regulation among TRPV1 channels and phosphoinosi-
tide 3-kinase in response to nerve growth factor. eLife.
2018;7. DOI:10.7554/eLife.38869.
[105] Ufret-Vincenty CA, Klein RM, Collins MD, et al.
Mechanism for phosphoinositide selectivity and acti-
vation of TRPV1 Ion Channels. J Gen Physiol.
2015;145(5):431–442.
[106] Ufret-Vincenty CA, Klein RM, Hua L, et al.
Localization of the PIP2 Sensor of TRPV1 Ion
Channels. J Biol Chem. 2011;286(11):9688–9698.
[107] Matta JA, Miyares RL, Ahern GP. TRPV1 Is a Novel
Target for Omega-3 Polyunsaturated Fatty Acids. J
Physiol. 2007;578(Pt 2):397–411.
[108] Morales-Lázaro SL, Llorente I, Sierra-Ramírez F, et al.
Inhibition of TRPV1 channels by a naturally occurring
omega-9 fatty acid reduces pain and itch. Nat
Commun. 2016;7:13092.
[109] Picazo-Juárez G, Romero-Suárez S, Nieto-Posadas A,
et al. Identification of a binding motif in the S5 helix
that confers cholesterol sensitivity to the TRPV1 Ion
Channel. J Biol Chem. 2011;286(28):24966–24976.
[110] Liu M, Huang W, Wu D, et al. TRPV1, but not P2X,
requires cholesterol for its function and membrane
expression in rat nociceptors. Eur J Neurosci. 2006;24
(1):1–6.
[111] Szoke E, Börzsei R, Tóth DM, et al. Effect of lipid raft
disruption on TRPV1 receptor activation of trigeminal
sensory neurons and transfected cell line. Eur J
Pharmacol. 2010;628(1–3):67–74.
[112] Telezhkin V, Reilly JM, Thomas AM, et al. Structural
requirements of membrane phospholipids for M-Type
potassium channel activation and binding. J Biol
Chem. 2012;287(13):10001–10012.
[113] Jang Y, Lee MH, Lee J, et al. TRPM2 Mediates the
Lysophosphatidic Acid-Induced Neurite Retraction in
the Developing Brain. Pflugers Arch. 2014;466
(10):1987–1998.
[114] Yung YC, Stoddard NC, Chun J, et al.
Pathophysiology. J Lipid Res. 2014;55(7):1192–1214.
[115] Cao E, Cordero-Morales JF, Liu B, et al. TRPV1 chan-
nels are intrinsically heat sensitive and negatively regu-
lated by phosphoinositide lipids. Neuron. 2013;77
(4):667–679.
[116] Morales-Lázaro SL, Serrano-Flores B, Llorente I, et al.
Structural determinants of the transient receptor
potential 1 (TRPV1) channel activation by phospholi-
pid analogs. J Biol Chem. 2014;289(35):24079–24090.
[117] Canul-Sánchez JA, Hernández-Araiza I, Hernández-
García E, et al. Different agonists induce distinct sin-
gle-channel conductance states in TRPV1 channels. J
Gen Physiol. 2018;150(12):1735–1746.
[118] Madej MG, Ziegler CM. Dawning of a new era in TRP
channel structural biology by cryo-electron micro-
scopy. Pflugers Arch. 2018;470(2):213–225.
[119] Duan J, Li J, Zeng B, et al. Structure of the mouse
TRPC4 ion channel. Nat Commun. 2018;9(1):3102.
[120] Duan J, Li J, Chen G-L, et al. Cryo-EM structure of the
receptor-activated TRPC5 ion channel at 2.9 angstrom
224 A. E. LÓPEZ-ROMERO ET AL.

resolution. bioRxiv. 2018;467969. DOI:10.1101/
467969.
[121] Fan C, Choi W, Sun W, et al. Structure of the human
lipid-gated cation channel TRPC3. eLife. 2018;7.
DOI:10.7554/eLife.36852.
[122] Tang Q, Guo W, Zheng L, et al. Structure of the
receptor-activated human TRPC6 and TRPC3 ion
channels. Cell Res. 2018;28(7):746–755.
[123] Vinayagam D, Mager T, Apelbaum A, et al. Electron
cryo-microscopy structure of the canonical TRPC4 ion
channel. eLife. 2018;7. DOI:10.7554/eLife.36615.
[124] Li J, Zhang X, Song X, et al. The structure of TRPC ion
channels. Cell Calcium. 2019;80:25–28.
[125] Thakur DP, Tian J, Jeon J, et al. Critical roles of Gi/o
proteins and phospholipase C-Δ1 in the activation of
receptor-operated TRPC4 channels. Proc Natl Acad
Sci U S A. 2016;113(4):1092–1097.
[126] Kraft R, Harteneck C. The mammalian melastatin-
related transient receptor potential cation channels:
an overview. Pflugers Arch. 2005;451(1):204–211.
[127] Zhang Z, Tóth B, Szollosi A, et al. Structure of a
TRPM2 channel in complex with Ca2+ explains
unique gating regulation. eLife. 2018;7. DOI:10.7554/
eLife.36409.
[128] Wang L, Fu T-M, Zhou Y, et al. Structures and gating
mechanism of human TRPM2. Science. 2018;362:6421.
[129] Huang Y, Winkler PA, Sun W, et al. Architecture of the
TRPM2 channel and its activation mechanism by ADP-
ribose and calcium. Nature. 2018;562(7725):145–149.
[130] Duan J, Li Z, Li J, et al. Structure of full-length human
TRPM4. Proc Natl Acad Sci U S A. 2018;115
(10):2377–2382.
[131] Autzen HE, Myasnikov AG, Campbell MG, et al.
Structure of the human TRPM4 ion channel in a
lipid nanodisc. Science. 2018;359(6372):228–232.
[132] Winkler PA, Huang Y, Sun W, et al. Electron cryo-
microscopy structure of a human TRPM4 channel.
Nature. 2017;552(7684):200–204.
[133] Duan J, Li Z, Li J, et al. Structure of the mammalian
TRPM7, a magnesium channel required during
embryonic development. Proc Natl Acad Sci U S A.
2018;115(35):E8201–E8210.
[134] Yin Y, Le SC, Hsu AL, et al. Structural basis of cooling
agent and lipid sensing by the cold-activated TRPM8
channel. Science. 2019;363(6430):eaav9334.
[135] Yin Y, Wu M, Zubcevic L, et al. Structure of the cold-
and menthol-sensing ion channel TRPM8. Science.
2018;359(6372):237–241.
[136] Perraud AL, Fleig A, Dunn CA, et al. ADP-ribose
gating of the calcium-permeable LTRPC2 channel
revealed by nudix motif homology. Nature. 2001;411
(6837):595–599.
[137] Runnels LW, Yue L, Clapham DE. TRP-PLIK, a
bifunctional protein with kinase and ion channel activ-
ities. Science. 2001;291(5506):1043–1047.
[138] Tóth B, Iordanov I, Csanády L. Putative chanzyme
activity of TRPM2 cation channel is unrelated to
pore gating. Proc Natl Acad Sci U S A. 2014;111
(47):16949–16954.
[139] Iordanov I, Tóth B, Szollosi A, et al. Enzyme activity
and selectivity filter stability of ancient TRPM2 chan-
nels were simultaneously lost in early vertebrates.
eLife. 2019;8. DOI:10.7554/eLife.44556.
[140] Tan C-H, McNaughton PA. The TRPM2 ion channel
is required for sensitivity to warmth. Nature. 2016;536
(7617):460–463.
[141] Csanády L, Törocsik B. Four Ca2+ ions activate
TRPM2 Channels by binding in deep crevices near
the pore but intracellularly of the gate. J Gen Physiol.
2009;133(2):189–203.
[142] Launay P, Fleig A, Perraud AL, et al. TRPM4 is a Ca2
+-activated nonselective cation channel mediating cell
membrane depolarization. Cell. 2002;109(3):397–407.
[143] Hofmann T, Chubanov V, Gudermann T, et al.
TRPM5 is a voltage-modulated and Ca(2+)-activated
monovalent selective cation channel. Curr Biol CB.
2003;13(13):1153–1158.
[144] Ullrich ND, Voets T, Prenen J, et al. Comparison of
functional properties of the Ca2+-activated cation
channels TRPM4 and TRPM5 from mice. Cell
Calcium. 2005;37(3):267–278.
[145] Liu D, Liman ER. Intracellular Ca2+ and the phospho-
lipid PIP2 regulate the taste transduction ion channel
TRPM5. Proc Natl Acad Sci U S A. 2003;100
(25):15160–15165.
[146] Zhang Z, Okawa H, Wang Y, et al.
Phosphatidylinositol 4,5-bisphosphate rescues
TRPM4 channels from desensitization. J Biol Chem.
2005;280(47):39185–39192.
[147] Nilius B, Prenen J, Voets T, et al. Intracellular nucleo-
tides and polyamines inhibit the Ca2+-activated cation
channel TRPM4b. Pflugers Arch. 2004;448(1):70–75.
[148] Liman ER. The Ca2+-Activated TRP Channels:
TRPM4 and TRPM5. In: Liedtke WB, Heller S, editors.
TRP ion channel function in sensory transduction and
cellular signaling cascades. Boca Raton (FL): Frontiers
in Neuroscience; CRC Press/Taylor & Francis;
2007:203–211.
[149] Guo J, She J, Zeng W, et al. Structures of the calcium-
activated, non-selective cation channel TRPM4.
Nature. 2017;552(7684):205–209.
[150] Krapivinsky G, Krapivinsky L, Manasian Y, et al. The
TRPM7 chanzyme is cleaved to release a chromatin-
modifying kinase. Cell. 2014;157(5):1061–1072.
[151] Zou Z-G, Rios FJ, Montezano AC, et al. TRPM7,
Magnesium, and Signaling. Int J Mol Sci. 2019;20(8).
DOI:10.3390/ijms20081877
[152] Numata T, Okada Y. Molecular determinants of sen-
sitivity and conductivity of human TRPM7 to Mg2+
and Ca2+. Channels Austin Tex. 2008;2(4):283–286.
CHANNELS 225

[153] Yudin Y, Rohacs T. Regulation of TRPM8 Channel
Activity. Mol Cell Endocrinol. 2012;353(1–2):68–74.
[154] Rohács T, Lopes CMB, Michailidis I, et al. PI(4,5)P2
regulates the activation and desensitization of trpm8
channels through the TRP domain. Nat Neurosci.
2005;8(5):626–634.
[155] Bautista D, Julius D. Fire in the hole: pore dilation of the
capsaicin receptor TRPV1. Nat Neurosci. 2008;11(5):528–
529.
[156] Li M, Toombes GES, Silberberg SD, et al. Physical
basis of apparent pore dilation of ATP-activated P2X
receptor channels. Nat Neurosci. 2015;18(11):1577–
1583.
[157] Mathias RT, Cohen IS, Oliva C. Limitations of the whole
cell patch clamp technique in the control of intracellular
concentrations. Biophys J. 1990;58(3):759–770.
[158] Frazier CJ, George EG, Jones SW. Apparent change in ion
selectivity caused by changes in intracellular K(+) during
whole-cell recording. Biophys J. 2000;78(4):1872–1880.
[159] Geron M, Kumar R, Zhou W, et al. TRPV1 pore turret
dictates distinct DKTX and capsaicin gating. Proc Natl
Acad Sci U S A. 2018;115(50):E11837–E11846.
226 A. E. LÓPEZ-ROMERO ET AL.