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Structural mechanisms of transient receptor potential ion channels


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Transient receptor potential (TRP) ion channels are evolutionarily ancient sensory proteins that detect and integrate a wide range of physical and chemical stimuli. TRP channels are fundamental for numerous biological processes and are therefore associated with a multitude of inherited and acquired human disorders. In contrast to many other major ion channel families, high-resolution structures of TRP channels were not available before 2013. Remarkably, however, the subsequent "resolution revolution" in cryo-EM has led to an explosion of TRP structures in the last few years. These structures have confirmed that TRP channels assemble as tetramers and resemble voltage-gated ion channels in their overall architecture. But beyond the relatively conserved transmembrane core embedded within the lipid bilayer, each TRP subtype appears to be endowed with a unique set of soluble domains that may confer diverse regulatory mechanisms. Importantly, TRP channel TR structures have revealed sites and mechanisms of action of numerous synthetic and natural compounds, as well as those for endogenous ligands such as lipids, Ca2+, and calmodulin. Here, I discuss these recent findings with a particular focus on the conserved transmembrane region and how these structures may help to rationally target this important class of ion channels for the treatment of numerous human conditions.
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Structural mechanisms of transient receptor
potential ion channels
Erhu Cao
Transient receptor potential (TRP) ion channels are evolutionarily ancient sensory proteins that detect and integrate a wide
range of physical and chemical stimuli. TRP channels are fundamental for numerous biological processes and are therefore
associated with a multitude of inherited and acquired human disorders. In contrast to many other major ion channel families,
high-resolution structures of TRP channels were not available before 2013. Remarkably,however, the subsequent resolution
revolutionin cryo-EM has led to an explosion of TRP structures in the last few years. These structures have confirmed that
TRP channels assemble as tetramers and resemble voltage-gated ion channels in their overall architecture. But beyond the
relatively conserved transmembrane core embedded within the lipid bilayer, each TRP subtype appears to be endowed with a
unique set of soluble domains that may confer diverse regulatory mechanisms. Importantly, TRP channel structures have
revealed sites and mechanisms of action of numerous synthetic and natural compounds, as well as those for endogenous
ligands such as lipids, Ca
, and calmodulin. Here, I discuss these recent findings with a particular focus on the conserved
transmembrane region and how these structures may help to rationally target this important class of ion channels for the
treatment of numerous human conditions.
The transient receptor potential (TRP) ion channels conduct
cations and, based on similarity in their primary sequences, are
grouped into seven subfamilies: TRPC (canonical), TRPM (mel-
astatin), TRPA (ankyrin), TRPV (vanilloid), TRPN (nomp; absent
in mammals), TRPML (mucolipin), and TRPP (polycystin;
Ramsey et al., 2006;Venkatachalam and Montell, 2007). The
human genome encodes 27 TRP proteins, making them the
second largest class of ion channels, surpassed only by K
channels. TRP channels exhibit very low sequence homology
across different subfamilies, reflecting their diverse physiolog-
ical roles as sensory apparatuses that detect and respond to all
manner of environmental and physiological stimuli. The first
TRP channels (TRP and TRP like [TRPL]) were identified in fly
eyes, where they operate downstream of the rhodopsin-
phospholipase C pathway and underlie phototransduction
(Hardie and Minke, 1993;Montell, 2012). For most mammalian
TRP channels, in vivo activation mechanisms remain elusive.
However, many of them, like the ancestral fly TRP channels,
retain the ability to be regulated by hormones or neuro-
transmitters that stimulate phospholipase C-coupled receptors.
TRP channels assemble as homo- or heterotetramers with a
membrane topology and subunit organization resembling that of
voltage-gated ion channels (VGICs; Fig. 1). The transmembrane
core of each TRP subunit harbors two recognizable modules
embedded within the lipid bilayer: a voltage sensorlike domain
composed of the first four helices (S1S4; VSLD) and a pore
domain formed by the last two helices (S5 and S6) plus the in-
tervening loop. These two modules are connected by the S4S5
linker, a short amphipathic helix that runs almost parallel to the
inner membrane. TRP channels exhibit fourfold symmetry along
a central ion permeation pathway formed by pore modules from
four subunits. The central pore is flanked by the peripheral
VSLDs in a domain-swaporganization, whereby each VSLD
associates with the pore domain from a neighboring subunit, as
first observed in voltage-gated K
channels (Long et al., 2005a,
2007;Liao et al., 2013). Of note, the TRPV6 channel, when
perturbed either by introducing a single mutation in the S5 helix
or by artificially shortening the S4S5 linker, deviates from this
general architecture and surprisingly adopts a non-swapped
structure (Saotome et al., 2016;Singh et al., 2017). However,
whether this non-swappedTRPV6 channel exists and func-
tions in native epithelia remains to be determined.
TRP channels generally exhibit modest voltage sensitivity,
likely due to a lack of regularly spaced arginine or lysine resi-
dues (gating charges) in their S4 helices. Gating charges are a
Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, UT.
Correspondence to Erhu Cao:
© 2020 Cao. This article is distributed under the terms of an AttributionNoncommercialShare AlikeNo MirrorSites license for the first six months after the publication
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Rockefeller University Press 1of18
J. Gen. Physiol. 2020 e201811998
hallmark of VGICs and enable movement of the S4 helix in ac-
cordance with the electric field of the membrane, which in turn
is believed to exert force on the S4S5 linker to ultimately open
or close the pore (Long et al., 2005b;Catterall, 2010;Vargas
et al., 2012). Notwithstanding this notable difference, VSLDs of
TRP channels are coupled to the pore module via the S4S5
linker (in analogy to VGICs) and, in the case ofTRPM4, TRPML1,
and TRPV4, additionally through direct association (Chen et al.,
2017;Guo et al., 2017;Deng et al., 2018). This implies that VSLDs
could affect channel gating (the process by which the pore opens
or closes) upon interacting with chemical ligands. Beyond this
relatively conserved transmembrane core, each TRP channel
subtype is defined by a collection of unique soluble domains that
may contribute to channel assembly and trafficking, and/or
serve as receptor sites for numerous endogenous cellular factors
or exogenous ligands.
Here, I review the wealth of recent structural data on TRP
channels that have resulted largely from the resolution revo-
lutionin cryo-EM, with a particular focus on the conserved
transmembrane core. I also discuss how these structures have
enriched our understanding of function, regulation, and phar-
macology of TRP channels. I apologize for any omissions of lit-
erature, due to the page limit and scope of this review.
The resolution revolutionled to breakthrough in TRPV1
structural biology
Cryo-EM was arguably born in early 1980s when Dubochet and
his colleagues invented a practical sample preparation method
that affords rapid freezing of biological specimens within a thin
layer of vitreous ice (Lepault et al., 1983;Adrian et al., 1984). This
breakthrough preserves fragile biological molecules in a frozen,
hydrated native-like state, which not only ameliorates electron
radiation damage but also protects the embedded biological
samples from evaporation in high vacuum of a typical electron
microscope. In earlier applications, cryo-EM has been impactful
in elucidating structures of large biological assemblies (e.g., ri-
bosomes and viruses) and, in some favorable cases, yielded near
atomic structures for large and symmetric icosahedral viruses
(Agrawal and Frank, 1999;Zhang et al., 2010;Grigorieff and
Harrison, 2011). However, most cryo-EM reconstructions typi-
cally show densities of blobs only useful for demarcating distinct
domains of a protein or identifying individual subunits in a
multimeric complex, but insufficient to resolve secondary
structures, letting alone side-chains of individual residues.
Nevertheless, based purely on theoretical considerations, Richard
Henderson and others presciently predicted that single-
particle cryo-EM should be able to resolve structures of bio-
logical molecules as small as 100 kD at 3 ˚
A or better resolutions
in the 1990s, when cryo-EM was considered as a fringe tech-
nique and often referred to as blobologyby x-ray crys-
tallographers (Saxton and Frank, 1977;Henderson, 1995).
Indeed, no one could foresee that, two decades later and around
the end of 2012, steady advancements in electron microscopes,
cameras, and computational algorithms finally reached a tip-
ping pointand, it appeared to many outsiders, have magically
transformed the cryo-EM field overnight, leading to the so-
called resolution revolutionthat is rapidly reshaping the
landscape of structural biology (Kühlbrandt, 2014;Callaway,
2015). Application of a new generation of direct electron de-
tectors (DEDs) has arguably contributed the lions share to this
Figure 1. Representative structures of each of the seven TRP subfamilies. TRP channels are shown in ribbon diagrams viewed parallel to the membrane,
with each subunit color-coded differently. Note, TRP channels are decorated with unique soluble domains outside the relatively conserved transmembrane
core structure embedded within the lipid bilayer delimited with two black horizontal lines. PDB accession nos. are as follows: TRPV1 (3J5P), TRPA1 (3J9P),
TRPM2 (6MIX), TRPC3 (6CUD), no mechanoreceptor potential C (NOMPC; 5VKQ), PKD2 (5T4D), and TRPML1 (5WJ5).
Cao Journal of General Physiology 2of18
TRP channel structures
quantum leap in cryo-EM (Campbell et al., 2012;Li et al., 2013;
McMullan et al., 2014,2016). DEDs exhibit drastically improved
detective quantum efficiency (a commonly used metric for
quantifying camera performance) across all spatial frequencies.
DEDs are now routinely employed to record much sharper
micrographs with signals potentially extending to the maxi-
mum resolution limit of Nyquist frequency that can then be
extracted to determine high-resolution structures using vari-
ous computational algorithms (Frank et al., 1996;Grigorieff,
2007;Tang et al., 2007;Scheres, 2012;Punjani et al., 2017).
The emergence of the first high-resolution TRP channel
structures coincided with the resolution revolutionin cryo-
EM (Fig. 2). TRPV1, the heat- and capsaicin-activated cation
channel, was also the first integral membrane protein whose
structure was determined at near atomic resolution by single-
particle cryo-EM (Cao et al., 2013;Liao et al., 2013). On the
technical front, these TRPV1 structures broke the side-chain
resolution barrier for a membrane protein without crystalliza-
tion for the first time. The Julius laboratory actually attempted to
determine TRPV1 structures by x-ray crystallography in col-
laboration with Bob Strouds group at the University of Cali-
fornia, San Francisco (San Francisco, CA). We, a team including
Julio Cordero-Morales (now at the University of Tennessee
Health Science Center, Memphis, TN), Thomas Tomasiak (now
at the University of Arizona, Tucson, AZ), and me, obtained
TRPV1 crystals growing in bicelles around early 2012, but un-
fortunately, these crystals only diffracted to 67˚
A and were
inadequate for structure determination despite extensive opti-
mization efforts. More recently, crystal structures of TRPV6,
TRPV2, and TRPV4 were reported (Saotome et al., 2016;Deng
et al., 2018;Singh et al., 2018c;Zubcevic et al., 2018b), show-
casing the strength of x-ray diffraction (even at modest reso-
lutions) in unambiguously locating ions or small molecule
modulators by exploiting their anomalous signals at special
x-ray wavelengths.
The breakthrough in determining TRPV1 structures also ex-
emplified how technical innovations can often spur discoveries
in science. Indeed, single-particle cryo-EM is increasingly be-
coming the method of choice for resolving structures of mem-
brane proteins and large protein complexes because these
targets are notoriously difficult to crystallize. On a side note, the
growing popularity of cryo-EM in the structural biology com-
munity also highlights the urgent need to establish new and to
support existing national cryo-EM centers where investigators
without access to expensive and technically demanding high-
end electron microscopes in their home institutions can still
routinely collect data for determining structures of their favorite
molecules (Stuart et al., 2016).
Ion selectivity filter of TRP channels
Most TRP channels are nonselective cation channels that con-
duct both monovalent and divalent cations (e.g., K
), with several notable exceptions: TRPV5 and TRPV6 are
-selective channels in epithelia (Vennekens et al., 2000;
Nilius et al., 2001;Yue et al., 2001), whereas TRPM4 and TRPM5
are only permeable to monovalent cations (Launay et al., 2002;
Hofmann et al., 2003;Liu and Liman, 2003;Prawitt et al., 2003).
Historically, the landmark KcsA structures were instrumental
for us to understand the origin of K
selectivity (Doyle et al.,
1998;Zhou et al., 2001b). These KcsA structures elegantly
showed that K
selectivity could stem from a size-restrictive
selectivity filter where backbone carbonyl oxygen atoms line
the pore in a geometry optimal for coordinating the dehydrated
ions but perhaps not the smaller Na
ions (Fig. 3). However,
later molecular dynamic studies showed that the selectivity fil-
ter of K
channels is flexible and thus cannot discriminate K
over Na
solely based on their size difference of 0.38 ˚
A, but
nonetheless remains optimal for K
due to electrostatic prop-
erties of carbonyl oxygen ligands (Noskov et al., 2004). Re-
gardless of the precise mechanisms of K
selectivity, all K
ion channels bear a signature selectivity filter sequence
(i.e., TVGYG), which folds into almost identical 3-D structures
that dictate K
selectivity of these channels (Kuang et al., 2015).
For most TRP channels, however, no such recognizable unify-
ing selectivity filter sequence or structural characteristics
stands out to adequately explain how ion selectivity (or lack
thereof) is achieved. TRP channel selectivity filters are not only
diverse in size, ranging from 3.2 to 10.6 ˚
A in diameter, but also
lined with divergent amino acid sequences, albeit with aspartic
acid and glycine residues commonly found. However, none of
Figure 2. Determination of TRPV1 structures by single-particle cryo-EM. (A) The 3.4 ˚
A map of TRPV1 determined by single-particle cryo-EM is shown in a
side view (left) and a cytoplasmic view (right). (B) The resolution revolutionin cryo-EM has led to a recent explosion of structures deposited at EMDB. The
plot is available at
Cao Journal of General Physiology 3of18
TRP channel structures
these features can reliably predict the level of selectivity to
various cations (Fig. 3). Ion selectivity of TRP channels is per-
haps defined by interaction energy of competing permeable
cations with dynamic selectivity filters, which is beyond what
can be gleaned from only inspecting the existing static snap-
shots of TRP channel structures. Further experiments, such as
molecular dynamic studies performed for TRPV1 (Jorgensen
et al., 2016) and TRPV6 (Sakipov et al., 2018), are needed to
fill this gap of knowledge. Nevertheless, as discussed below, the
selectivity filters of TRP channels do conform to the same basic
design principles that have emerged from structural analyses of
(Doyle et al., 1998), Na
(Payandeh et al., 2011;Zhang et al.,
2012;Shen et al., 2017;Yan et al., 2017;Pan et al., 2018;Jiang
et al., 2019), and Ca
channels (Tang et al., 2014;Wu et al.,
2016). These principles are expected to be generally applica-
ble to other cation-selective ion channels as well.
First, the selectivity filter of TRP channels is cradled by one
(in all TRPV channels) or two pore helices (perhaps in all other
TRP channels). At the outermost site of their selectivity filter
facing the extracellular vestibule, most TRP channels harbor a
negatively charged residue, predominantly aspartic acid (Fig. 3).
Four Asp residues (DDDD), one from each subunit, form a neg-
atively charged ring, offering an immediate impression that they
are strategically positioned to either attract and/or directly co-
ordinate cations; these Asp residues could alsorepel anions from
the filter. Neutralization of this negative charge affects ion se-
lectivity of many TRP channels, including TRPV1 (Chung et al.,
2008), TRPV4 (Voets et al., 2002), TRPV5 (Nilius et al., 2001),
TRPV6 (Voets et al., 2004b), TRPM7 (Duan et al., 2018b), fly TRP
channels (Liu et al., 2007), TRPA1 (Christensen et al., 2016), and
PKD2L1 (Fujimoto et al., 2011;DeCaen et al., 2016). TRPM4 and
human TRPM2 (Guo et al., 2017;Winkler et al., 2017;Autzen
et al., 2018;Duan et al., 2018c), but not Nematostella vectensis
TRPM2 (Zhang et al., 2018b), are notable exceptions in having a
glutamine at the outermost position of their selectivity filters
(Fig. 3), which appears to underlie their lack of permeability to
divalent cations (e.g., Mg
and Ca
). Indeed, substitution of this
glutamine to glutamate in TRPM4 restores negative charges in
the selectivity filter and confers moderate Ca
(Nilius et al., 2005). Such a design principle is evolutionarily
ancient, as an analogous structure (EEEE) is also present in bac-
terial homotetrameric Na
and Ca
(Ren et al., 2001;Zhang et al.,
2012;Tang et al., 2014). In some eukaryotic Na
and Ca
a lysine residue replaces the Asp/Glu at the second or third po-
sition, giving rise to alternative EKEE or EEKE arrangements
(Stephens et al., 2015). Notwithstanding variations to this basic
negatively charged ring structure, the conserved Asp or Glu, found
strength site predicted by Hille decades before atomic structures
of VGICs and TRP channels were resolved (Hille, 1971,1972).
Figure 3. Chemistry and size of the selectivity filterof representative TRP channels and VGICs. TRP channel selectivity filters are diverse in both size and
constituent residues. The diameters measured from two narrowest diagonally opposed oxygen atoms are shown at the top. The selectivity filters of Ca
selective Ca
Ab and K
-selective K
2.1 are also shown for comparison. Ions in TRPV6 (Ba
), Ca
Ab (Ca
), and K
2.1 (K
) are shown as purple spheres. PDB
accession nos. are as follows: TRPV1-Apo (3J5P), TRPV1-DkTx (3J5R), TRPM4 (6BQR), TRPV4 (6BBJ), TRPV6 (5IWR), Ca
Ab (5KLB), and K
2.1 (2R9R).
Cao Journal of General Physiology 4of18
TRP channel structures
Second, as observed in KcsA and VGICs, backbone carbonyl
oxygen atoms of some selectivity filter residues also face the
pore in TRP channels, possibly forming additional coordination
sites for cations (Fig. 3). In some cryo-EM maps of TRP channels,
non-protein densities were indeed observed within the selec-
tivity filterand thus interpreted to represent permeatingcations
(Chen et al., 2017;Guo et al., 2017;Hirschi et al., 2017;Wilkes
et al., 2017;Duan et al., 2018b,c;Hughes et al., 2018a;Zhang
et al., 2018b). In TRPV6 and TRPV4 crystal structures, the ion
coordination sites were unambiguously confirmed by exploiting
anomalous signals of more electron dense ions,such as Cs
and Ba
(Saotome et al., 2016;Deng et al., 2018). Multiple closely
spaced ion binding sites were initially observed in KcsA crystal
structures (Doyle et al., 1998), and later in voltage-gated Na
as well (Payandeh et al., 2011;Tang et al., 2014), an ar-
rangement that appears to support a knock-offmechanism
whereby repulsion between adjacent ions would overcome the
attractive interactions between ions and the channel protein so
as to achieve rapid rates of ion conduction that, in some cases,
can approach the diffusion limit (Almers and McCleskey, 1984;
Hess and Tsien, 1984;Neyton and Miller, 1988). As might have
been expected from their close kinship to VGICs, many TRP
channels also likely consist of multiple ion binding sites within
the selectivity filter, indicating that such a knock-offmecha-
nism of ion permeation may also be pervasive in the TRP
channel superfamily (Jorgensen et al., 2016;Saotome et al., 2016;
Sakipov et al., 2018). Surprisingly, only a single ion-binding site
was identified in the current TRPV4 crystal structures (Deng
et al., 2018). TRPV4 also boasts the widest selectivity filter
(~10.6 ˚
A in diameter; Fig. 3) among all TRP channels for which
structures have been determined, implying that fully hydrated
cations could diffuse freely through the filter and probably only
interact very weakly with the pore lining residues. Such a wide
selectivity filter might explain the unusually large single chan-
nel conductance of TRPV4 (~280310 pS) when Ca
is included
asachargecarrier(Liedtke et al., 2000). Of note, in a Ca
condition, single-channel conductance of TRPV4 reduces to ~100
pS, which is comparable to the related TRPV1 channel (Hui et al.,
2003). Therefore, it remains to be determined whether TRPV4
contains multiple ion binding sites in other conformational
states as do most other TRP channels, or it utilizes a distinct
permeation mechanism based on a single ion binding site.
Finally, in some TRP channels, the selectivity filter may
function as a gate (or upper gate), in addition to the more
common gate (or lower gate) formed by hydrophobic residues
located near the cytoplasmic end of the pore lining S6 helix.
TRPV1 was speculated to harbor an upper gate based on the
observations that a pore helix mutation leads to a constitutively
open channel and that spider toxins (e.g., double knot toxin
[DkTx]) activate TRPV1 by binding to and stabilizing the outer
pore region in an open conformation (Myers et al., 2008;
Bohlen et al., 2010). In addition, state-dependent accessibility
of substituted cysteine residues along the S6 helix predicted that
two constrictions exist in the TRPV1 ion permeation pathway
(Salazar et al., 2009). Indeed, TRPV1 structures captured in
three distinct pore conformations, presumably representing
apo-closed, capsaicin-bound partially open, and resiniferatoxin
(RTX)- and DkTx-bound open states (Cao et al., 2013;Gao et al.,
2016), show gating-associated structural changes within the se-
lectivityfilter region. Comparison of these structures showsthat,
upon binding DkTx, the narrowest constriction of the selectivity
filter expands from 4.4 to 7.7 ˚
A(Fig. 3). DkTx binding evokes two
salient structural changes within the selectivity filter region:
first, a methionine inthe middle of theselectivity filter rotates so
that its side chain no longer points toward the center of the pore
to obstruct ion permeation as observed in the apo state; second,
four Asp residues, one from each subunit, residing at the ex-
tracellular mouth of the selectivity filter, moving toward the
central axis of the pore, as if they are now better positioned to
coordinate and partially dehydrate cations for efficient ion
conduction. In a TRPV2 channel engineered to be sensitive to
RTX, an even more drastic dilation of the upper gate was recently
observed as the channel transitions from fourfold to twofold
symmetry upon binding RTX (Zubcevic et al., 2018b), further
confirming the existence of an upper gate within the TRPV
channels. Such flexibility within the TRPV1 outer pore region
also likely contributes to distinct single-channel conductance
states evoked by different TRPV1 agonists (Canul-S´
anchez et al.,
2018;Geron et al., 2018).
However, one potential caveat of these structural analyses is
how faithfully these TRPV structures resemble channel con-
formations in native cells. For instance, a recent study showed
that the selectivity filters of TRPV13 channels are permeable to
ions even in a closed state and thus possibly do not gate
permeation of physiological cations (Jara-Oseguera et al., 2019).
Irrespective of whether TRPV1 harbors a selectivity filter gate,
its dynamic outer pore region and potential allosteric commu-
nications with the lower conventional activation gate undoubt-
edly contribute to its complex regulation by endogenous ligands
and a wide range of pharmacological compounds.
In most non-TRPV TRP channels, the selectivity filter is
rigidly framed between two pore helices as observed in VGICs,
making conformation changes within the selectivity filter region
unlikely, if not impossible. Indeed, in the agonist ML-SA1bound
structures of TRPML1 and TRPML3 channels, the selectivity
filter region only shows negligible changes as compared with the
apo state, if there are any, whereas the lower gate undergoes
significant expansion (Schmiege et al., 2017;Zhou et al., 2017).
Similarly, TRPM2 also shows no conformational changes in the
selectivity filter region when comparing TRPM2 structures de-
termined in presumably closed, sensitized (i.e., primed for ac-
tivation), or open states (Wang et al., 2018). Moreover, in TRPV5
and TRPV6 channels, the selectivity filter also remains station-
ary during channel gating (Hughes et al., 2018b;McGoldrick
et al., 2018;Singh et al., 2018b,c;Dang et al., 2019), as might
have been anticipated for a Ca
-selective channel since a dy-
namic selectivity filter would be counterproductive for main-
taining an optimal geometry essential for strict Ca
Finally, a much wider selectivity filter, comparable in size to that
observed in the DkTx-bound TRPV1 structure, exists in many
TRP channels determined presumably in closed states (Fig. 3),
implying that, in these channels, only the lower gate controls ion
permeation, whereas the selectivity filter may be always per-
missive to ion conduction. Of course, we should be cautious not
Cao Journal of General Physiology 5of18
TRP channel structures
to overinterpret these structural data asthey may not captureall
conformational states that a TRP channel can sample as it pro-
gresses along its gating cycle on native cells. Indeed, TRPV5, a
close homologue of TRPV6, is inhibited by acidification, possibly
due to proton-induced conformational changes in the pore helix
(Yeh et al., 2005).
The hydrophobic lower gate
In all TRP channels whose structures have been determined so
far, hydrophobic residues, residing near the intracellular end of
the pore lining S6 helices, point their side chains toward the
center of the pore, forming a hydrophobic seal (or lower gate)
that blocks ion permeation. In K
channels, the S6 helix contains
a -Pro-X-Pro- motif and/or a glycine residue, located roughly at
the middle of the lipid bilayer (Jiang et al., 2002;Long et al.,
2005a). These so-called gating hinge elements can function in-
dependently or in tandem to permit splaying open of the lower
half of the S6 helix upon activation induced by membrane de-
polarization (Labro and Snyders, 2012). TRP channels appear to
lack such a gating hinge, but instead harbor a conserved π-helix
at a similar position (Fig. 4). Compared with canonical α-helices,
π-helices are high-energy and conformationally dynamic sec-
ondary structures often found in functional sites of proteins
(Cooley et al., 2010). The S6 π-helix was firstspeculated to play a
role in TRP channel gating based on comparative sequence
analysis (Palovcak et al., 2015). Later, by comparing TRPV2 and
TRPV1 structures, transition of this π-helix to a canonical
α-helix was hypothesized to mediate TRPV2 gating (Zubcevic
et al., 2016). More recently, it was directly shown that an α-to
π-helix transition opens the lower gate of TRPV6 (McGoldrick
et al., 2018). Such a transition from a low- to high-energy state,
on the surface, is counterintuitive, but it can be rationalized
that the high-energy π-helix is compensated by formation of
new hydrophilic interactions within its neighboring regions
(McGoldrick et al., 2018). Similarly, an α-toπ-helix transition in
S6 accompanies the transition from a closed to sensitized or
open state in TRPV3 (Singh et al., 2018a;Zubcevic et al., 2018a,
2019), and from a closed to desensitized state in TRPM8 (Diver
et al., 2019). We found that a gain-of-function (F604P) mutation
in PKD2 leads to an opposite π-toα-helix transition, resulting in
dilation of the lower gate that might underlie constitutive
channel activity of this mutant (Arif Pavel et al., 2016;Zheng
et al., 2018). In the TRPV1 channel, however, the π-helix struc-
ture maintains regardless of pore conformations (Cao et al.,
2013). Nevertheless, it is interesting to note that the π-helix is
precisely the point below which the distal S6 segment starts to
diverge when superimposing structures of the pore captured in
three distinct states, thus functionally analogous to the gating
hinge in K
In TRPML1 and TRPML3 channels, the S6 π-helix appears to
play no role in agonist (ML-SA1)-induced channel activation
(Schmiege et al., 2017;Zhou et al., 2017). ML-SA1 binds to a
hydrophobic pocket located above the π-helix, leading to out-
ward movement of the entire S6 helix, and consequently, ex-
pansion of the lower gate. However, it remains to be determined
whether other stimuli, such as phosphoinositides in combina-
tion with membrane depolarization, open the TRPML channels
by facilitating structural transition of the S6 π-helix. Similarly,
in TRPM2, adenosine diphosphate ribose (ADPR) and Ca
gether trigger dilation of the lower gate, which appears to not
involve the S6 π-helix (Huang et al., 2018;Wang et al., 2018).
Notably, the S6 π-helix has not been identified in structures of
PKD2L1 channels (Hulse et al., 2018;Su et al., 2018b), and future
studies will determine whether the π-helix exists in other states
of this channel. Taken together, unlike the upper selectivity
filter gate, the lower gate is present in all TRP channels, and the
prevalent S6 π-helix may act as a dynamic flexing point around
which the distal S6 segment can rotate and/or bend to open the
lower gate (Zubcevic and Lee, 2019).
The classic voltage-sensor domain (VSD) consists of four helices
(S1S4) and plays a predominant role during gating of VGICs
(Catterall, 1986,2010;Aggarwal and MacKinnon, 1996;Long
et al., 2005a;Bezanilla, 2018). In particular, the S4 helix bears
an array of four to six positively charged arginine or lysine
residues (or gating charges) at every fourth position that are
stabilized in an otherwise inhospitable hydrophobic and low
Figure 4. Structural elements that confer conformational flexibility to the pore lining S6 helix in K
and TRPV1 channels. (A) Gating hinges in KcsA
(Gly99; red) and K
2.1 (Pro-Val-Pro; shown in sticks) allow for splaying open of their activation gates. (B) Aπ-helix bulge, highlighted in sticks, likely provides a
flexing point around which the distal S6 helix can rotate and/or bend to open the lower gate in TRPV1. PDB accession nos. are as follows: KcsA (5VKE), Kv2.1
(2R9R), and TRPV1 (3J5R).
Cao Journal of General Physiology 6of18
TRP channel structures
dielectric lipid bilayer by negatively charged residues protrud-
ing from other helices (Long et al., 2007;Payandeh et al., 2011).
These residues form two charge clusters separated by a highly
conserved phenylalanine residue. Channel gating elicited by
changes in membrane potential is believed to involve transfer of
positively charged residues from the external charge cluster,
across this phenylalanine gap, to the internal charge cluster, or
vice versa (Tao et al., 2010;Vargas et al., 2012;Li et al., 2014).
Such large motion of the S4 helix isbelieved to exert force on the
S4S5 linker, leading to its outward or inward movement with
respect to the pore domain that ultimately opens or closes the
activation gate of VGICs.
In contrast to VGICs, most TRP channels only exhibit weak
voltage sensitivity generally manifested as an outwardly recti-
fying I-V relationship in electrophysiological recordings (Wu
et al., 2010), as if membrane depolarization promotes channel
opening and/or sensitizes channel to other stimuli (Brauchi
et al., 2004;Voets et al., 2004a). The VSLD of TRP channels
lacks most, if not all, of the positively charged residues along the
S4 helix, providing a simple explanation for their weak voltage
sensitivity as compared with VGICs. For instance, TRPV1 and
TRPM8 show apparent gating charges of less than 1 e as com-
pared with 13 e measured for the Shaker K
channel (Schoppa
et al., 1992;Voets et al., 2004a;Matta and Ahern, 2007). PKD2L1
represents a notable exception as it retains two gating charges,
and channel activation by disparate stimuli all requires transfer
of a total of approximately four gating charges (i.e., a single
gating charge per subunit; Ng et al., 2019). Nevertheless, the
VSLD of most TRP channels, as first observed in TRPV1 (Cao
et al., 2013), is rigid and remains static during channel gating.
For instance, the VSLD was also shown to remain relatively
stationary in TRPV6 (McGoldrick et al., 2018), PKD2 (Shen et al.,
2016;Grieben et al., 2017;Wilkes et al., 2017;Zheng et al., 2018),
TRPML1 (Schmiege et al., 2017), TRPM2 (Huang et al., 2018;
Wang et al., 2018), TRPV2 (Dosey et al., 2019), TRPV3 (Singh
et al., 2018a), and TRPML3 channels (Zhou et al., 2017), for
which structures captured in presumably open and closed states
have been determined to discern mobile structural elements
during channel gating. More recently, the VSLD of TRPA1 was
shown to undergo a rigid body rotation relative to the pore
domain upon activation by cystine reactive irritants (Zhao et al.,
2019), highlighting a vast conformational landscape accessible to
polymodal TRP channels.
In addition to a lack of gating charges along the S4 helix, the
VSLD of TRP channels often associates extensively with the pore
domain, in sharp contrast to K
channels, where only minimal
contacts exist at a small extracellular interface between the S1
helix of one subunit and the S5 helix from a neighboring subunit
(Fig. 5;Long et al., 2005a). Generally speaking, in VGICs, VSD
appears to be largely indirectly coupled to the pore domain via
the S4S5 linker, and the voltage-sensing S4 helix is freeto
move relative to the pore domain. In the Shaker K
direct interactions between the VSD and pore domain addi-
tionally contribute to electromechanical coupling (Soler-Llavina
et al., 2006;Fern´
andez-Mariño et al., 2018). In TRPM4 and
TRPML1, however, the S4 helix exhibits extensive hydrophobic
interactions with the S5 helix from an adjacent subunit for
virtually its entire length, making large translational movement
of the S4 helix unlikely in these channels (Fig. 5;Chen et al.,
2017;Guo et al., 2017). In TRPV4, the VSLD engages with the pore
domain via an even more extensive interface involving the S3
and S4 helices of one subunit and the S5 and S6 helices of a
neighboring subunit (Fig. 5;Deng et al., 2018). Additionally, the
S3 helix of TRPV4 appears to be directly coupled to the pore
lining S6 helix, suggesting that it could transduce stimuli acting
on the VSLD to impact the S6 activation gate by exerting force on
the S6 helix (Deng et al., 2018).
TRP channels and VGICs differ significantly in structures of
their S1S4 domains, which undoubtedly contributes to their
fundamentally distinct gating mechanisms. Nevertheless, the
last two or three turns of the S4 helix of TRP channel often
adopts a 3
-helical fold, as observed in VGICs. Similar to
π-helix, 3
-helix is also highly energetic and conformationally
dynamic (Enkhbayar et al., 2006;Vieira-Pires and Morais-
Cabral, 2010). Indeed, subtle conformational changes within
the 3
-helix have been observed in TRPV1 (Gao et al., 2016),
TRPV6 (McGoldrick et al., 2018;Singh et al., 2018b,c), and PKD2
(Zheng et al., 2018) to cause and/or accommodate gating-
associated structural rearrangements in the S4S5 linker and
TRP helix.
Ligand and lipid binding sites
TRP channels are versatile cellular sensors for a wide range of
physiological and environmental signals, and elucidating how
these channels interact with and respond to chemical ligands and
physical stimuli is of paramount importance for understanding
unique aspects of TRP channel pharmacology, regulation, and
function. As would have been anticipated for polymodal sensory
ion channels, each TRP channel often harbors multiple receptor
sites that are exploited by small chemical compounds, peptide
toxins, or cellular factors to regulate channel function. Here, I
highlight major regulatory sites emerged from structural anal-
yses of TRP channels.
The vanilloid binding pocket. TRPV1 was first cloned in 1997
by the Julius group, as expression of this channel confers sen-
sitivity to the pungent vanilloid compound capsaicin to other-
wise capsaicin-insensitive HEK293 host cells (Caterina et al.,
1997). Birds, however, are spared of the burningeffects of
capsaicin, and indeed, capsaicin acts as a partial agonist for
chicken TRPV1 (Jordt and Julius, 2002). By exploiting this nat-
ural difference in capsaicin sensitivity, chimeras of rat and
chicken TRPV1 were constructed to delimit the vanilloid-binding
site to a minimal region encompassing the S2 and S3 helices,
including Y511, which was hypothesized to directly engage the
vanilloid moiety of capsaicin molecule (Jordt and Julius, 2002).
Further mutagenesis studies also pinpointed residues on the S4
helix for specifying sensitivity to various vanilloid ligands (Chou
et al., 2004;Gavva et al., 2004;Phillips et al., 2004). It is grat-
ifying that TRPV1 structures bound with capsaicin, RTX, or
capsazepine (a TRPV1 antagonist) are in excellent agreement
with these mutagenesis studies. These structures showed that all
these vanilloid ligands are buried within a pocket formed by S3,
S4, and the S4S5 linker from one subunit and the pore domain
(i.e., S5 and S6 helices) of an adjacent subunit (Cao et al., 2013;
Cao Journal of General Physiology 7of18
TRP channel structures
Gao et al., 2016). The Y511 residue indeed plays a prominent role
in binding vanilloid compounds, as its aromatic ring is induced
to point toward the pocket to engage ligands, as opposed to
facing cytosol in apo state. Vanilloid agonists, such as RTX and
capsaicin, appear to directly interact with and pull the S4S5
linker away from the central pore, and consequently facilitate
opening of the lower gate (Yang et al., 2015a;Gao et al., 2016).
Remarkably, a mere introduction of four to six point muta-
tions into TRPV2 or TRPV3 so as to mimic the TRPV1 vanilloid
site confers RTX sensitivity to the otherwise insensitive chan-
nels, further validating the vanilloid binding pocket defined by
structures and computational studies (Yang et al., 2016;Zhang
et al., 2016,2019). More broadly, the vanilloid pocket also rep-
resents the site of action of other small molecule modulators for
other TRP channels, including a TRPC6 antagonist BTDM and a
TRPV5 antagonist econazole (Hughes et al., 2018a;Tang et al.,
Intriguingly, the vanilloid pocket harbors a resident lipid,
perhaps phosphoinositides, in apo state. Vanilloid ligands were
therefore hypothesized to displace this lipid to produce either
agonistic or antagonistic effects (Gao et al., 2016). However, it
was also shown that a cytoplasmic arginine residuedistant from
the vanilloid pocket specifies sensitivity of TRPV1 to different
phosphoinositide species, suggesting that multiple phosphoino-
sitide binding sites may exist in TRPV1 (Ufret-Vincenty et al.,
A regulatory site at the cytoplasmic opening of VSLD. In VGICs,
VSD is frequently targeted from the extracellular side by a large
class of gating modifier toxins found in animal venoms, re-
inforcing the notion that movement of VSD is fundamental for
gating these channels (Alabi et al., 2007;Catterall et al., 2007;
Bosmans et al., 2008;Kalia et al., 2015). In contrast, in most TRP
channels, the equivalent VSLD and its constituent S4 helix ap-
pear to remain relatively stationary during channel gating, and
so far, peptide toxins were only identified to bind to the pre-
and Swartz, 2005;Cuypers et al., 2006;Siemens et al., 2006;
Andreev et al., 2008;Bohlen et al., 2010;Yang et al., 2015b).
Nevertheless, the S4S5 linker, which relays movements of VSD
to the pore in VGICs, is similarly positioned to affect the lower
gate in TRP channels. Therefore, the S4S5 linker in TRP
channels can transduce subtle conformational changes induced
by ligands acting on the VSLD to allosterically gate the pore.
Indeed, in TRPV6, antagonist 2-APB binds to a cytoplasmic
opening of VSLD, causing the S3 helix to move toward the S4S5
linker; such subtle structural rearrangements foster formation
of a hydrophobic cluster composed of residues from the S2S3
linker, the cytoplasmic end of the S3 and S4 helices, and the
S4S5 linker, which in turn appears to disengage a presumably
activating lipids from the S4S5 linker and ultimately closes the
channel pore (Singh et al., 2018c). Similarly, an antagonist of
TRPV5 identified by in silico docking screen binds to an analo-
gous site and inhibits channel activation (Hughes et al., 2019). In
TRPM2, TRPM4, TRPM8, and TRPA1 structures, a Ca
ion is
coordinated by hydrophilic residues at an equivalent site (Guo
et al., 2017;Winkler et al., 2017;Autzen et al., 2018;Duan et al.,
2018c;Huang et al., 2018;Wang et al., 2018;Yin et al., 2018;
Zhang et al., 2018b;Yin et al., 2019a,b;Zhao et al., 2019); this
-binding site is essential for activation of TRPM8 by a
cooling compound icilin, as well as for activation and desensi-
tization of TRPA1 downstream of metabotropic receptors (Yin
et al., 2019a;Zhao et al., 2019). In TRPM8, this pocket appears to
be malleable to also accommodate both antagonists and agonists
of diverse chemical structures (Diver et al., 2019;Yin et al.,
2019a). Moreover, in TRPC4, a non-protein density at a similar
hydrophilic pocket was tentatively modeled as a Na
ion, the
predominant ion in the specimen used for structure determina-
tion (Duan et al., 2018a). Taken together, many TRP channels have
evolved a pocket of distinct chemical properties (i.e., hydrophobic
or hydrophilic) on the cytoplasmic side of VSLD to accommodate
various ligands such as 2-APB, icilin, menthol, or ions that can
allosterically regulate channel gating.
The outer pore region. The outer pore region of several TRPV
channels is known to be involved in activation by chemical li-
gands (Jordt et al., 2000;Yeh et al., 2005;Ryu et al., 2007). For
instance, two glutamate residues located at the outer pore region
of TRPV1 are essential for acid-evoked potentiation or activation
(Jordt et al., 2000). The outer pore region similarly serves as a
receptor site for environmental or physiological stimuli in other
TRP channels. For example, in both TRPC4 and TRPC5, a disul-
fide bond in the outer pore region confers redox-sensitivity as
application of reducing reagents (e.g., dithiothreitol or TCEP)
Figure 5. Association of the S1S4 domain with the pore domain in representative TRP and K
channels. TRP channel voltage-sensor like domain (VSLD;
cyan) typically associates with pore domainfrom an adjacentsubunit (orange; red) via a larger interface and thus remains relatively static during channel gating
as compared withVGICs. The potential hydrophobic interactions between the S3 and S6 helices in TRPV4 are highlighted with sticks. PDB accession nos. are as
follows: Kv2.1 (2R9R), TRPM4 (6BQR), TRPML1 (5WJ5), and TRPV4 (6BBJ).
Cao Journal of General Physiology 8of18
TRP channel structures
potentiates channel currents (Xu et al., 2008;Hong et al., 2015;
Duan et al., 2018a;Vinayagam et al., 2018). An analogous di-
sulfide bond exits in several members of the TRPM subfamily,
but whether it also regulates channel activity remains unclear.
Finally, just as plants have evolved pungent TRPV1-activating
vanilloid compounds (e.g., capsaicin and RTX) to discourage
herbivory, venomous animals, such as spiders and centipedes,
produce peptide toxins that agonize TRPV1 to elicit acute pain
perhaps for predator deterrence (Siemens et al., 2006;Bohlen
et al., 2010;Yang et al., 2015b). Remarkably, these peptide tox-
ins, despite their distinct structural fold and different origin, all
convergently target the TRPV1 outer pore region to exert their
effects, unmistakably proving that the outer pore region serves
as a critical allosteric site in TRPV1 and perhaps in other TRP
channels as well.
The TRPV1 structures bound with DkTx spider toxin showed
that the toxin inserts into a crevice formed by the outer pore
region of two neighboring subunits (Fig. 6;Cao et al., 2013;Bae
et al., 2016;Gao et al., 2016). Interestingly, several lipids were
found to mediate interactions between DkTx and TRPV1, sug-
gesting that lipids may modulate pharmacological effects of
DkTx (Bae et al., 2016;Gao et al., 2016). This observation is
consistent with the notion that some inhibitory cysteine knot
toxins, to which DkTx belongs, first partition into the lipid bi-
layer, where they are believed to be concentrated and gain un-
obstructed access to receptor sites on channel protein via free
lateral diffusion (Lee and MacKinnon, 2004). Of note, the DkTx
toxin appears to stabilize the TRPV1 outer pore region in an open
conformation without making discernable contacts with the
VSLD, settingit apart from a large class of gating modifier toxins
that target the VSD of VGICs. Generally speaking, voltage-sensor
toxins principally latch onto VSD and perhaps are further con-
strained by anchoring to adjacent extracellular loops or lipid
bilayer, thus physically impeding voltage sensor movement
(Fig. 6;Shen et al., 2018,2019;Xu et al., 2019). DkTx also differs
significantly from pore blocking peptide toxins, such as char-
ybodotoxin and μ-conotoxin KIIIA, that insert a lysine into the
entrance to selectivity filters and essentially plug the extracel-
lular ion entryway of various K
and Na
channels, respectively
(Fig. 6;Banerjee et al., 2013;Pan et al., 2019).
Pockets near the pore helix. In TRPA1, a relatively occluded
pocket surrounded by S5, S6, and pore helix 1 from a single
subunit constitutes the receptor site for antagonist A-967079,
which may act as a molecular wedge that inhibits activation-
associated conformational changes (Paulsen et al., 2015). In
TRPV2, agonist cannabidiol interacts with a topologically anal-
ogous pocket to promote channel opening (Pumroy et al., 2019).
In TRPML1 and TRPML3, the ML-SA1 agonist binds to a lipid-
facing cleft at the interface of pore modules, which is enclosed
by pore helix 1, S5, and S6 from one subunit, and S6 of a
neighboring subunit (Schmiege et al., 2017;Zhou et al., 2017).
The ML-SA1binding site likely harbors endogenous lipids in
apo state, and ML-SA1 binding appears to squeeze out these
resident lipids, pushes the S6 helix away from the central axis,
and consequently expands the lower gate. Lipids play important
structural and regulatory roles for membrane proteins, so
it should not come as a surprise that many small molecule
compounds, including ivermectin (a partial inverse agonist of
pentameric ligand-gated ion channels) and vanilloids (ligands of
the TRPV1 channel), appear to compete with lipids for the same
or overlapping binding sites to regulate ion channel activity
(Hibbs and Gouaux, 2011;Cao et al., 2013;Gao et al., 2016).
Notably, lipid-like densities are also observed in or near the ML-
SA1binding pocket in other TRP channels, including PKD2 and
TRPC3 (Shen et al., 2016;Grieben et al., 2017;Wilkes et al., 2017;
Fan et al., 2018), raising the possibility that this site could be
similarly exploited to target these channels for drug discovery
as well. More broadly, in related Ca
channels, an equivalent
pocket is the receptor site for dihydropyridines, a large class of
antagonist drugs that are widely prescribed for treating
cardiovascular disorders (Tang et al., 2016).
The cytoplasmic opening of the pore. Some TRP channels
undergo Ca
-dependent inactivation and, in TRPV5 and TRPV6,
this process is mediated through direct interaction with cal-
modulin (CaM; Nilius et al., 2003;Lambers et al., 2004). The
recently reported structures of TRPV5 or TRPV6 bound with
CaM showed that a lysine residue, located at the H6H7 loop of
CaM, inserts its side chain into the cytoplasmic entryway of
channel pore, where it establishes cation-πinteractions with a
ring of four tryptophan residues (one from each subunit),
leading to inward movement of the lower half of S6 helices, and
consequently, pore closure (Hughes et al., 2018b;Singh et al.,
2018b;Dang et al., 2019).
More broadly and beyond TRP channels, it has long been
known that the ion permeation path of many ion channels bears
receptor sites for small or medium-sized molecules. These in-
clude blocking ions (e.g., Gd
) that compete with permeating
ions for binding sites within the selectivity filter, and small
organic molecules that bind to other disparate sites along the
pore (Voets et al., 2004b;Oseguera et al., 2007;Saotome et al.,
2016). For instance, Shaker-type K
channels are blocked by
tetrabutylammonium, a small organic cation (Armstrong, 1971).
These channels also close spontaneously after opening induced
by depolarization in an inactivation process whereby inactiva-
tion gate, a stretch of ~20 amino acids ball peptidefrom the N
terminus of either α-orβ-subunit of these channels, physically
plugs the open pore from intracellular side (Hoshi et al., 1990;
Zagotta et al., 1990;Rettig et al., 1994). Structural studies lately
confirmed that pore opening provides a cytoplasmic entryway
for tetrabutylammonium and ball peptideto gain access to
central cavity sites below the selectivity filter and inner pore
sites, ultimately leading to blockage of ion permeation (Zhou
et al., 2001a). Moreover, Br-verapamil and flecainide, two
drugs widely used for treating arrhythmias, respectively reach
an analogous site in Ca
Ab and Nav1.5 via open pore and occlude
the ion-conduction pathway (Tang et al., 2016;Jiang et al., 2019).
Regulatory sites within soluble domains. From a birds-eye
view, most TRP channels are homotetramers, exhibiting
fourfold symmetry around a central ion permeation path-
way. There are several notable departures from this general
architecture: structural analyses of TRPV2, TRPV3, and
TRPM2 channels show that fourfold symmetry can be broken
and reduced to twofold symmetry (Zubcevic et al., 2018a,b;
Yin et al., 2019b), and the recently determined PKD1/PKD2
Cao Journal of General Physiology 9of18
TRP channel structures
structure, a heteromeric complex, understandably exhibits
no symmetry at all (Su et al., 2018a). All TRP channels share a
roughly similar transmembrane core composed of VSLD and
pore domain, indicative of relatedness within this large
cation channel family. However, outside the lipid bilayer,
each TRP subtype consists of a combination of unique soluble
domains located at both intracellular and extracellular sides.
These widely diverse soluble domains can serve as receptor
sites for numerous endogenous cellular factors or exogenous
ligand(s) and thus undoubtedly contribute to unique aspects
of function, regulation, and pharmacology of each TRP sub-
family. TRP channel structures allow for direct visualization
of some of these important regulatory sites located in these
soluble domains, including the IP
-binding site in TRPA1 (Paulsen
et al., 2015), a reactive pocket targeted by electrophile irritants in
TRPA1 (Suo et al., 2019;Zhao et al., 2019), ATP-binding sites in
TRPM4 (Guo et al., 2017), ADPR binding site in TRPM2 (Huang
et al., 2018;Wang et al., 2018;Yin et al., 2019b), and decavanadate-
binding site in TRPM4 (Winkler et al., 2017). Moreover, together
with mutagenesis, structures of TRPML channels also reveal how
phosphoinositides, which bind to a pocket just beneath the inner
membrane, allosterically regulate the pore of these channels (Fine
et al., 2018). Of note, TRPML and TRPP channels feature a large
luminal or extracellular domain, collectively called polycystin-
mucolipin domain (PMD), which is absent in other TRP sub-
families and related VGICs. The PMD domain sits atop and
Figure 6. Receptor sites for animal toxins targeting TRPV1 and VGICs. (A) The spider DkTx toxin latches onto the outer pore region of TRPV1 and
stabilizes an open pore conformation. (B) Dc1a wedges into a cleft between the VSD and pore domains of Na
and impedes VSD movement. (C) Char-
ybodotoxin inserts a lysine side chain into the extracellular entrance to the selectivity filter of K
and physically plugs the entryway of ion permeation. PDB
accession nos. are as follows: TRPV1/DkTx (5IRX), NavPaS (6A90), and K
2.1/charybodotoxin (4JTC).
Cao Journal of General Physiology 10 of 18
TRP channel structures
strategically interacts with the transmembrane core, giving
an immediate impression that this domain may recognize as
yet unidentified ligand(s) and/or respond to physical force to
allosterically gate these channels (Shen et al., 2016;Chen et al.,
2017;Grieben et al., 2017;Hirschi et al., 2017;Schmiege et al.,
2017;Wilkes et al., 2017;Zhou et al., 2017). Indeed, proton-
ation of the luminal loop on PMD regulates TRPML channels
(Li et al., 2017). In both TRPC3 and TRPC6, the S3 helix ex-
tends into the extracellular space and, together with the
neighboring S1S2 and S3S4 loops, forms a distinct extra-
cellular domain (Azumaya et al., 2018;Fan et al., 2018;Sierra-
Valdez et al., 2018;Tang et al., 2018). This structure interacts
with the channel pore, implying that it may regulate channel
function upon receiving external stimuli and confer drug
sensitivity. Finally, in the mechanosensitive no mechanore-
ceptor potential C channel, 29 ankyrin repeats form a helical
spring structure in cytosol, supporting the hypothesis that
these ankyrin repeats, when anchored to cytoskeleton, can
function as an elastic tether that would transduce mechanical
displacement to the ion channel gate (Jin et al., 2017).
Temperature sensing in TRP channels
In addition to sensing chemical ligands and mechanical force, a
subset of mammalian TRP channels can be directly activated by cold
(TRPA1, although debatable), cool (TRPM8 and TRPC5), warm
(TRPV3, TRPV4, TRPM2, TRPM4, and TRPM5), hot (TRPV1 and
TRPM3), or extreme hot (TRPV2) temperatures (Caterina et al., 1997,
1999;McKemy et al., 2002;Peier et al., 2002a,b;Smith et al., 2002;
Xu et al., 2002;Chung et al., 2003;Story et al., 2003;Jordtetal.,
2004;Talavera et al., 2005;Vriens et al., 2011;Zimmermann
et al., 2011;Song et al., 2016;Tan and McNaughton, 2016).
Some of these thermo-sensitive TRP channels, such as TRPV1,
TRPM8, TRPA1, TRPM2, and TRPM3, function as molecular
thermometers for detecting environment and body temper-
atures, which is essential for us to avoid damaging temperature
extremes and to maintain body temperature within a narrow
physiological range (Caterina et al., 2000;Davis et al., 2000;
Bautista et al., 2007;Colburn et al., 2007;Dhaka et al., 2007;
Vriens et al., 2011;Song et al., 2016;Tan and McNaughton, 2016;
Vandewauw et al., 2018). Interestingly, TRPV1 in ground
squirrels and camels exhibits diminished heat sensitivity, al-
lowing them to tolerate high temperatures and inhabit other-
wise inhospitable ecological niches (Laursen et al., 2016). In the
same vein, a TRPV1 splice variant in vampire bats activates in
much lower temperatures, which may underlie its role in
sensing infrared radiation (Gracheva et al., 2011). TRP channels
also play pivotal roles in thermosensation in lower organisms.
For instance, TRPA1 is expressed in the pit organ of some
snakes, where it is activated by heat in the form of infrared
radiation for detection of warm-blooded predators or prey
(Gracheva et al., 2010). Similarly, in insects such as flies and
mosquitos, TRPA1 also serves as a warm sensor and controls
their temperature preference behavior (Viswanath et al., 2003;
Hamada et al., 2008;Wang et al., 2009). Taken together, these
studies demonstrate fundamental roles of TRP channels in
thermosensation in both mammals and lower organisms and
highlight how adaptive diversification and tuning of TRP
channel thermal sensitivity can accommodate special needs and
variations in environment niches of different organisms.
However, how an ion channel acquires steep temperature
sensitivity is a much tougher nut to crack than understanding
ligand- or voltage-gated ion channels. Chemical ligands or
voltage can be at least conceptually understood to either snugly
fit into a pocket in a lock-and-key mechanism or exert electric
force on membrane-embedded gating charges, respectively. In
contrast, thermal stimuli,being ubiquitous, are not confined to a
discrete domain(s), and thus can exert global effects on protein
structures. Indeed, TRPV3 structures determined at different
temperatures show conformational changes in both transmem-
brane and cytosolic domains upon heat activation (Singh et al.,
2019). Moreover, in the most extensively studied heat-activated
TRPV1 channel, various domains dispersed throughout the en-
tire primary sequence have been identified to contribute to
thermal activation (Brauchi et al., 2006;Yao et al., 2011;Laursen
et al., 2016;Liu and Qin, 2017;Sosa-Pag´
an et al., 2017;Zhang
et al., 2018a); these domains either directly sense thermal
stimuli or couple the conformational changes in heat sensor to
subsequent gate opening. Intriguingly, repetitive heat activation
of TRPV1 leads to irreversible channel inactivation via a mech-
anism possibly analogous to partial denaturation of several re-
gions of the channel (S´
anchez-Moreno et al., 2018), in support of
a global effect of heat on channel structure.
None of the available TRPV1 structures has been determined
at different temperatures so far, so they are silent on heat-
associated conformational changes. Nonetheless, molecular dy-
namic simulation of the TRPV1 channel has yielded several
principles that are consistent with a large body of mutagenesis
and structural studies (Fig. 7 A;Zheng and Qin, 2015;Chugunov
et al., 2016;Wen et al., 2016;Melnick and Kaviany, 2018;Wen
and Zheng, 2018;Ladrón-de-Guevara et al., 2019). First, besides
serving as a substrate site for chemical ligands (e.g., ions, pro-
tons, and spider toxins), the TRPV1 outer pore region was shown
to contribute to heat activation. For instance, the TRPV1 turret
region, which immediately precedes the pore helix, was shown
to regulate channel thermal sensitivity (Yang et al., 2010;Yao
et al., 2010;Jara-Oseguera et al., 2016). In addition, mutations in
the outer pore region of TRPV3 similarly abolish heat activation
without perturbing sensitivity to various chemical agonists
(Grandl et al., 2008). Moreover, several residues in an outer
pore loop of both TRPV1 and TRPV3 change their accessibility to
solvent upon heat activation, further supporting a role for the
outer pore region in thermal activation of these channels (Kim
et al., 2013). More recently, it has been shown that grafting the
pore domain of TRPV1 into the Shaker K
channel confers heat
sensitivity to the otherwise temperature-insensitive channel
(Zhang et al., 2018a). Consistent with these mutagenesis studies,
molecular dynamic stimulation predicts heat-induced destabi-
lization of hydrophobic clusters within the outer pore region,
lending support to the heat capacity theory that hypothesizes that
exposure of 2025 hydrophobic residues per subunit to solvent
gives rise to heat sensitivity (Clapham and Miller, 2011). Second,
molecular dynamic studies also support the hypothesis that a
resident phosphoinositide lipid is ejected from the vanilloid
pocket upon heating, resulting in conformational rearrangements
Cao Journal of General Physiology 11 of 18
TRP channel structures
around the pocket that then propagate to and ultimately open
the pore (Gao et al., 2016). Finally, heat further evokes con-
formational changes within several cytoplasmic structures,
including the membrane proximal domain, C-terminal do-
main, and the S2S3 loop. Of note, protein kinase C (PKC)-
mediated phosphorylation of Ser502, which resides precisely at
the S2S3 loop, has been reported to enhance thermal sensitivity
of TRPV1 (Premkumar and Ahern, 2000;Numazaki et al., 2002).
Indeed, this may represent a major mechanism whereby in-
flammatory mediators (e.g., bradykinin) produce thermal hy-
peralgesia via PKC activation.
How the TRPM8 channel is activated by cold stimuli is even
more mysterious. Intriguingly, within the TRPM subfamily, to
which TRPM8 belongs, TRPM2 and TRPM3 are activated by
warm and hot temperatures (Vriens et al., 2011;Song et al.,
2016;Tan and McNaughton, 2016), respectively. Comparison
of TRPM8 and TRPM2 structures shows that they differ in
organization of two domains within an otherwise similar
overall architecture, including both harboring a similar Ca
regulatory site (Fig. 7 B;Huang et al., 2018;Wang et al., 2018;
Yin et al., 2018). First, TRPM8 conspicuously lacks the helical
S4S5 linker in closed state that is critical for gating of most
TRP channels and instead harbors a short loop connecting the
S4 and S5 helices. Second, the selectivity filter and outer pore
loops of TRPM8 are surprisingly invisible in a closed state.
Interestingly, both the S4S5 linker and selectivity filter are
well-defined in a desensitized state in TRPM8 (Diver et al.,
2019), hinting that these structure elements are likely more dy-
namic as compared to TRPM2 channels. Whether such structural
differences reflect a genuinely distinct design principle for cold-
and heat-sensitive TRPM channels remains to be determined.
Concluding remarks
When the TRPV1 structures were first reported in 2013, it was
impossible to imagine that, seven years later, structures have
been determined for at least one member of every subfamily of
TRP channels. With continuous improvement in single-particle
cryo-EM and development of novel amphiphiles, detergents, and
nanodisc technology for stabilizing membrane proteins, more
TRP channel structures are certainly forthcoming. Single-
particle cryo-EM, in essence, is a single-molecule method and
in theory can deliver multiple structures representing distinct
conformational states of a given protein from the same dataset.
However, in practice, conformational heterogeneity often leads
to lower-resolution structures as failure to accurately classify
particles into separate groups will blur conformational dynamic
regions. Indeed, developing novel algorithms for classifying
heterogeneous samples and defining their conformational land-
scapes is an actively pursued research frontier of single-particle
cryo-EM (Frank and Ourmazd, 2016;Nakane et al., 2018).
We now know the structural blueprint of each subtype of TRP
channels, but how most TRP channels are activated or regulated
remains enigmatic. This is an extremely challenging question to
address because, in contrast to VGICs, where rich pharmaco-
logical tools have greatly advanced our understanding of fun-
damental principles that underlie ion permeation and regulation
of these channels, pharmacology for most TRP channels is still
in its infancy. Consequently, we lack tool compounds and pep-
tide toxins to trap TRP channels in distinct functional states
for structure determination so as to fully appreciate the range
of conformations that certainly exist to account for the complex
regulatory and activation mechanisms exhibited by these poly-
modal cellular sensors. I hope that pharmacological discov-
eries from academic laboratories and drug development
campaigns of pharmaceutical companies will identify novel
small molecule compounds, peptide toxins, or conformation-
sensitive antibodies that can modulate TRP channel function.
Such pharmacological tools will prove indispensable for further
advancing TRP channel structural biology. Finally, as is true for
many other ion channels, it is often difficult to unambiguously
correlate a TRP channel structure with a certain functional state
along its gating cycle. Therefore, other methods, such as mo-
lecular dynamics simulation and single-molecule FRET (Zheng
and Qin, 2015;Zagotta et al., 2016;Gordon et al., 2018;Yang et al.,
2018), should be used to study structural dynamics of TRP
Lesley C. Anson served as guest editor.
The author is enormously grateful to Lesley Anson for her
invaluable editorial support throughout the process.
Figure 7. Structures of thermo-sensitive TRP channels. (A) Two diagonally apposed TRPV1 subunits are shown inribbon diagram, with regions proposed to
undergo heat-evoked conformational changes highlighted with red. The phosphoinositide lipid predicted to be ejected from the vanilloid pocket upon heating is
shown in sticks. (B) Comparison of structures of warm-activated TRPM2 and cold-activated TRPM8. Only the transmembrane region of one subunit is shown
here. Unlike other TRP channels, TRPM8 lacks the helical S4S5 linker and a structurally defined selectivity filter in a closed state, hinting at structural flexibility
of these structural elements. PDB accession nos. are as follows: TRPV1 (5IRX), TRPM2 (6MIX), and TRPM8 (6BPQ).
Cao Journal of General Physiology 12 of 18
TRP channel structures
This work has been supported by the National Institutes of
Health R01 DK110575 grant and the U.S. Department of Defense
W81XWH-17-1-0158 discovery award to E. Cao, and E. Cao is a
Pew Scholar supported by the Pew Charitable Trusts.
The author declares no competing financial interests.
Submitted: 29 April 2019
Accepted: 3 January 2020
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Cao Journal of General Physiology 15 of 18
TRP channel structures