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A Transient Receptor Potential Ion Channel in
Chlamydomonas Shares Key Features with Sensory
Transduction-Associated TRP Channels in Mammals
Luis Arias-Darraz,a Deny Cabezas,a Charlotte K. Colenso,a Melissa Alegría-Arcos,b Felipe Bravo-Moraga,b
Ignacio Varas-Concha,b Daniel E. Almonacid,b,c Rodolfo Madrid,d and Sebastian Brauchia,1
a Physiology
Department, Faculty of Medicine, Universidad Austral de Chile, Campus Isla Teja, Valdivia 5110566, Chile
Andres Bello, Center for Bioinformatics and Integrative Biology, Faculty of Biological Sciences, Santiago 8370146, Chile
c CINV, Faculty of Sciences, Universidad de Valparaíso, Valparaíso 2366103, Chile
d Biology Department, Faculty of Chemistry and Biology, Universidad de Santiago de Chile, Santiago 9160000, Chile
b Universidad
Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor
potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater
green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first
detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization.
Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we
describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian
TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP
channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone
showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP
channel gating characteristics evolved early in the history of eukaryotes.
INTRODUCTION
The transient receptor potential (TRP) channel family of cation
channels is diverse in terms of structure, ion selectivity, activation mechanisms, and tissue distribution (Clapham, 2009). In
mammals, the TRP family comprises 28 loosely related ion
channel proteins that are classified into six subfamilies (Latorre
et al., 2009). Mammalian TRP channel proteins are polymodal
cation channels that participate in sensory physiology at different levels. These include thermosensation, mechanosensation,
nociception (sensation of noxious stimuli), touch, taste, olfaction,
and vision (Clapham, 2009; Latorre et al., 2009). Under physiological conditions, TRP channel opening allows for the fast entrance of sodium and calcium ions into the cell (Owsianik et al.,
2006). Although originally found in Drosophila melanogaster
(Cosens and Manning, 1969; Montell and Rubin, 1989), at present, TRP channels are mostly studied in mammalian cells. TRPY,
from yeast vacuole, is the only TRP channel from a unicellular
organism that has been cloned and described to date (Martinac
et al., 2008). After the release of the Chlamydomonas reinhardtii
genome sequence, more than 60 putative ion channels, including TRP channels, have been reported as probable gene
products (Merchant et al., 2007). Commonly found in soil and
freshwater, C. reinhardtii is a single-celled chlorophyte alga
1 Address
correspondence to [email protected].
The author responsible for distribution of materials integral to the findings
presented in this article in accordance with the policy described in the
Instructions for Authors (www.plantcell.org) is: Sebastian Brauchi
([email protected]).
www.plantcell.org/cgi/doi/10.1105/tpc.114.131862
about 10 mm in diameter with two beating flagella that enable
swimming with a breast-stroke-type motion (Harris, 2001).
Navigating at ;50 mm/s (Harris, 2001), these algae must rapidly
integrate multiple external cues to adjust their orientation relative
to the source of the signal. Notably, C. reinhardtii possess a finetuned navigation system based on calcium conductances
(Hegemann, 2008). The presence of putative TRP channel
coding sequences in the C. reinhardtii genome makes them
good candidates for both the generation of the input signal and/
or the regulation of sensory input propagation. Two recent independent studies report behavioral changes after knocking
down TRP channel transcripts in Chlamydomonas. Apparently
expressed at the flagella, silencing the expression of TRPP2
reduces the phosphorylation of cyclic GMP-dependent protein
kinase and affects algae mating behavior (Huang et al., 2007).
On the other hand, TRPV-related TRP11 (also expressed at
the flagella) has been associated with the mechanosensory response (Fujiu et al., 2011). Unfortunately, in both cases, neither
channel heterologous expression nor algal electrical activity was
described, hampering the correct interpretation of the behavioral
data. Here, we present a novel functional TRP channel from
C. reinhardtii with a predicted molecular architecture that combines features from different TRP channel subfamilies. Of equal
importance, this TRP channel displays several functional properties present in TRP channels from multicellular organisms,
such as outward rectification, weak voltage dependence, phosphatidylinositol 4,5-bisphosphate (PIP2) sensitization, pharmacological block by N-(4-tert-butyl-phenyl)-4-(3-chloropyridin-2-yl)
tetrahydropyrazine-1(2H)-carboxamide (BCTC), and activation by
temperature (Latorre et al., 2009).
The Plant Cell Preview, www.aspb.org ã 2015 American Society of Plant Biologists. All rights reserved.
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RESULTS
Identification of TRP-Like Channels from C. reinhardtii
A previous report identified eight putative TRP channels in
C. reinhardtii (Fujiu et al., 2011). Our bioinformatics analysis
recognized six of these channels as aligning well with the full
lengths as well as the transmembrane regions of bona fide
TRP channels: Chlamydomonas TRP1, TRP2, TRP11, TRP13,
TRP16, and TRPP2. Of the other two channels identified previously, we found that TRP5 was homologous to some TRP
channels but did not align well with the transmembrane region of
bona fide channels, and TRP15 did not show sufficient similarity
to the known members of the TRP family. Our methods also
identified four additional TRP channels in C. reinhardtii: one
already identified in Phytozome as a TRP channel, TRP6, and
three others not annotated in Phytozome, named TRP21,
TRP22, and TRP23. From the whole subset, and based on its
particular predicted architecture, we decided to focus our
attention on TRP1.
Localization of Cr-TRP1 within the TRP Family and
Inferred Phylogeny
In order to classify Cr-TRP1 within the context of the entire TRP
group of proteins, we created a sequence similarity network
(SSN; Atkinson et al., 2009). SSNs are based on all-against-all
sequence comparisons and, like other networks, are very robust
to missing data. Furthermore, they allow easy identification of
clades and correlate well with phylogenetic trees (Atkinson et al.,
2009; Brown and Babbitt, 2012; Lukk et al., 2012). The network
was first built considering the 67 TRP channels annotated in
the International Union of Basic and Clinical Pharmacology database encompassing TRP subfamilies A (ankyrin), C (classical or canonical), M (melastatin), ML (mucolipin), P (polycystin),
and V (vanilloid) (http://www.guidetopharmacology.org/GRAC/
FamilyDisplayForward?familyId=78), with the addition of No
mechanoreceptor potential C (NompC) from Drosophila belonging to the TRPN (NompC-like) subfamily (Cheng et al.,
2010), as seed sequences. Then, homologs from the National
Center for Biotechnology Information (NCBI) database having
a significant BLASTP E-value hit against at least two of the seed
sequences, and which align with at least 70% of the transmembrane region of one of the seeds, were incorporated into
the network. We used the same procedure to identify putative
TRP channels in 23 different algae and unicellular organisms
(see Methods). In total, we identified 7126 proteins that show
high similarity to known TRP family members. Figure 1A shows
an SSN of 2841 representatives assembled at the permissive
E-value threshold of 1e218 (median alignment length of 651 residues, median identity of 35.8%). Here, all seven functional TRP
families are already clearly defined (six mammalian subfamilies
plus TRPN). At this level, TRPPs and TRPMLs are not connected
to the rest of the subfamilies or to each other. In this network, CrTRP1 remains attached to a cluster including members in the
TRPA, TRPC, TRPM, TRPN, and TRPV families. At a more
stringent E-value threshold of 1e287, an SSN of all 7126 proteins
of the TRP group (Figure 1B; median alignment length of 812
residues, median identity of 48%) shows all seven currently
characterized functional subfamilies separated from each other,
as expected. In this network, Cr-TRP1 is isolated from known
families and only connected to a TRP channel from Volvox carteri
(a close multicellular relative of C. reinhardtii; Prochnik et al.,
2010). Thus, according to the SSN using several known extant
TRP channels and their close homologs, it appears that Cr-TRP1
is a true member of the TRP ion channel superfamily (Figure 1A).
Yet, it does not show enough similarity to members of the seven
characterized subfamilies and is instead isolated inside its own
network (Figure 1B). Consequently, it seems that Cr-TRP1 belongs to a novel functional TRP family.
To investigate the evolutionary relationships between CrTRP1 and the rest of the members of the TRP family, a phylogenetic reconstruction was performed using a Bayesian method
and the Jones substitution model (Jones et al., 1992). The
phylogenetic tree includes the 68 functionally characterized
channels from the SSNs plus all 33 putative TRP channels
identified in the complete genomes of algae and unicellular organisms (Figure 2; Supplemental Data Set 1). Figure 2 shows
a clustering pattern similar to those seen in the SSNs (i.e., the
TRP family contains two evolutionarily distinct clusters: one
composed of TRPAs, TRPCs, TRPMs, TRPNs and TRPVs, to
which Cr-TRP1 belongs [cluster A/C/M/N/V], and the other
composed of TRPPs and TRPMLs [cluster P/ML]). Interestingly,
most of the 33 putative TRP channels from algae and unicellular
organisms seem to have diverged from a common ancestor
together with the TRPP and TRPML channels. The P/ML cluster
includes Cr-TRPP2, as expected, but also Cr-TRP11, which was
reported previously to be a distant relative of the TRPV family
(Fujiu et al., 2011). The phylogenetic tree shows that Cr-TRP1 is
closely related to a TRP channel in V. carteri as well as to two
other paralogous channels in C. reinhardtii. These four channels
seem to have diverged from a common ancestor, along with the
TRPC, TRPM, and TRPN channels, at a time when the TRPA
and TRPV channels had already diverged. These four channels
represent the only TRP proteins from algae or unicellular
organisms in the A/C/M/N/V cluster.
Identification of Domains in Cr-TRP1
According to the protein topology prediction method Transmembrane Helices Hidden Markov Model (Krogh et al., 2001),
the transmembrane domain in Cr-TRP1, as expected, consists
of six transmembrane helices extending from residues 454 to
743 (Figure 3A). A section of this protein comprising over 70%
of its length shares 19.2% to 23.3% sequence identity with the
transmembrane domains of TRPC1, TRPC7, and several TRPM
channels. In order to investigate the presence of domains known
to exist in the various TRP functional families (Latorre et al.,
2009), we performed a multiple sequence alignment of Cr-TRP1
using the sequences of the 68 functionally characterized TRP
channels. Then, a group of subfamily representatives with specific, characterized domains were aligned among themselves
plus Cr-TRP1, so that the domain boundaries in Cr-TRP1 could
be determined. We identified two ankyrin repeats in the N-terminal
region of Cr-TRP1 (Figure 3A; Supplemental Figure 1). While the
first ankyrin repeat (a protein-protein interaction motif, residues 126
Figure 1. Cr-TRP1 within the SSN of the TRP Family.
Nodes represent protein sequences, and edges represent the lowest reciprocal BLASTP E-values that exceed a given threshold. Colored nodes
correspond to functionally characterized TRP channels, and different shapes are used to identify channels from C. reinhardtii, V. carteri, and other algae
or unicellular organisms.
(A) SSN of 2841 representative sequences with edges filtered to E-value < 1e218, median alignment length of 651 residues, and median identity of 35.8%.
(B) SSN of 7126 sequences with edges filtered to E-value < 1e287, median alignment length of 812 residues, and median identity of 48%.
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Figure 2. Phylogenetic Inference for Selected Members of the TRP Family.
Branch confidence values are shown next to nodes. Taxa in color correspond to the 68 functionally characterized TRP subgroup members (same color
scheme as in Figure 1). Taxa in black (including Cr-TRP1 in yellow with black border) are the 33 TRP channels identified here in algae and unicellular
organisms. Proteomes obtained from Phytozome are as follows: 1, C. reinhardtii (Cr-TRP1, Cr-TRP2, Cr-TRP6, Cr-TRP11, Cr-TRP13, Cr-TRP16,
Cr-TRPP2, CrTRP21, CrTRP22, Cr-TRP23); 2, V. carteri (Vc-TRP1, Vc-TRP2, Vc-TRP3, Vc-TRP4); 3, Coccomyxa subellipsoidea C-169 (Cs-TRP1,
Cs-TRP2); 4, Micromonas pusilla CCMP1545 (Mp_ccmp-TRP1); 5, Micromonas pusilla RCC299 (Mp_rcc-TRP2). Proteomes obtained from UniProt
are as follows: 1, Dictyostelium discoideum (Dd-TRP1, Dd-TRP2); 2, Dictyostelium purpureum (Dp-TRP1, Dp-TRP2, Dp-TRP3); 3, Leishmania infantum
(Li-TRP1); 4, Leishmania major (Lm-TRP1); 5, Leishmania mexicana (strain MHOM/GT/2001/U1103) (Lmex-TRP1, Lmex-TRP2); 6, Paramecium tetraurelia
(Pt-TRP1, Pt-TRP2, Pt-TRP3); 7, Trypanosoma cruzi (strain CL Brener) (TcCL-TRP1, TcCL-TRP2); 8, Trypanosoma cruzi (Tc-TRP1).
to 157) is similar in sequence to ankyrin repeats from the TRPA,
TRPC, TRPN, and TRPV families, the second ankyrin repeat (residues 166 to 192) is similar in sequence to those from TRPAs,
TRPNs, and TRPVs (Latorre at al., 2009). Interestingly, the multiple
sequence alignment also shows the presence of two TRPM homology regions (MHRs, stretches of amino acids that share some
sequence similarity) similar to MHR2 and MHR4 from TRPM
channels (Figure 3A; Supplemental Figure 2). The former extends
from residues 82 to 132, and the latter from residues 340 to 440.
Therefore, our alignments indicate a small overlap between the end
of MHR2 and the beginning of the first ankyrin repeat. The alignments also identified the presence of a partially conserved TRP
domain (TD) in the C-terminal region of Cr-TRP1, which extends
from residues 757 to 779 (Figure 3B). The TD, an ;25-amino acid
motif located immediately after the TM6 transmembrane helix, is
a shared feature among channels from the C, M, N, and V subfamilies (Latorre et al., 2009). This domain contains the TRP-box,
a highly conserved region defined by the consensus sequence
WKFQR, in which tryptophan is the most conserved residue. The
positive charges at positions 2 and 5 of the sequence are shared by
almost all TRPs having a TD and have been suggested to be part of
the PIP2 sensor of the channel (Latorre et al., 2009). Although
lacking the signature tryptophan in the TRP-box stretch, Cr-TRP1
exhibits an aromatic residue (Phe-758) in that position, together
with charged amino acids (Arg-759 and Lys-762, respectively) at
equivalent positions to those observed in mammalian TRPMs
A Functional TRP Channel from Algae
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Figure 3. Structural Domains in Cr-TRP1.
(A) Protein illustration for Cr-TRP1 including the identified domains.
(B) Multiple sequence alignment depicting the TD in Cr-TRP1 and other TRPN, TRPC, TRPV, and TRPM family representatives. Note the conservation
of the positively charged residues in Cr-TRP1 responsible for PIP2 binding in mammalian TRPs (box). Conservation values range between 0 and 9; the
larger the number, the larger the conservation. Only conservation values above 5 are shown. Consensus_aa values are as follows: conserved amino
acid residues are boldface and uppercase; h, hydrophobic; s, small; p, polar; o, alcohol; l, aliphatic; +, positively charged. Consensus_ss values are as
follows: h, a-helix; e, b-strand.
(Figure 3B). Thus, Cr-TRP1 possesses a TD homologous to that
present in all known TRP channels, including highly conserved
aromatic and polar residues at the expected positions within the
TRP-box. Interestingly, the transmembrane prediction showed
a hydrophobic domain at the C terminus of the sequence. The
presence of such a hydrophobic stretch in the C-terminal domain of
TRPs is not novel and has been associated with the presence of
coiled-coil domains in the TRPC and TRPM channels (Wes et al.,
1995; Latorre et al., 2009). An alignment of the C-terminal region of
Cr-TRP1 with bona fide coiled coils present in the C termini of
TRPMs showed low levels of similarity and poor conservation of the
heptad repeat. Additionally, none of several algorithms used predicted a coiled-coil domain with high probability.
Recently, TRP channels from the C, M, and V families have
been proposed to be mediators of redox sensitivity (Hara et al.,
2002; Takahashi and Mori, 2011). Although reactive oxygen
species (ROS) may alter TRP channel function through a variety
of pathways, including oxidation of channel agonists of a lipidic
nature, cysteine residues appear to be good candidates for
ROS-dependent modification (Takahashi and Mori, 2011). Several intracellular cysteines located within the ankyrin repeat region and the TD helix have been described (Takahashi and Mori,
2011). Interestingly, Cr-TRP1 has intracellular cysteine residues
located in three regions of importance: the ankyrin repeats, the
linker connecting TM2 and TM3, and the TD helix. Further mutagenesis studies are necessary to explore ROS-dependent
modulation in this novel member of the TRP family.
To summarize, Cr-TRP1 contains a mosaic of features typically
segregated in different TRP families (Figure 3A): an N terminus
with two ankyrin repeats similar to TRPAs, TRPCs, TRPNs, and
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TRPVs and two TRPM homology regions; a transmembrane
region similar to TRPCs; and a C terminus containing a TD similar
to those in TRPCs, TRPMs, and TRPVs, conserving the positively
charged residues common to all TRPMs.
Cloning, Heterologous Expression, and
Biophysical Characterization
The cloned channel gene (Supplemental Figure 3) was first expressed by transfecting Human Embryonic Kidney (HEK)-293T
cells with a green fluorescent protein (GFP)-tagged version. The
GFP signal was found near or in the plasma membrane when
observed by total internal reflection fluorescence (TIRF) microscopy (Figure 4A); however, intracellular positive signals were also
observed. We interpret this as a consequence of overexpression.
In order to describe TRP1 channel activity, HEK-293T cells were
transfected with the untagged version of the channel. Ionic
currents were recorded using whole-cell patch clamp. Currentvoltage curves obtained from voltage ramps show a clear outward rectification, similar to currents described previously for
mammalian TRPM5 channels (Figure 4B; Supplemental Figure 4) (Hofmann et al., 2003; Clapham, 2009). The nonselective
blockers ruthenium red, La3+, and 2-aminoethoxydiphenyl borate and the more TRP-specific blocker BCTC (Valenzano et al.,
2003; Clapham, 2009) were tested on transfected HEK-293T
cells. TRP1 current was blocked by La3+ (500 mM; Figure 4B),
ruthenium red (1 mM; Figure 4B), and BCTC (IC50 = 1.03 mM;
Figures 4B and 4C). By contrast, 2-aminoethoxydiphenyl borate
Figure 4. Functional Characterization of TRP1 Expressed in HEK-293T Cells.
(A) Transfection of the TRP1-GFP clone in HEK-293T cells. The images present one cell in epifluorescence mode (left) and one cell in TIRF mode (right).
Bar = 10 mm.
(B) Current-voltage relations determined from voltage ramps from 2100 to +140 mV obtained from HEK-293T cells transfected with TRP1 in pTracer
vector (untagged). The curves were obtained under symmetrical Na+ conditions (box; concentrations in mM), and each represents the average of four
independent experiments. Statistics are available in Supplemental Figure 4.
(C) Dose-response curve showing the blocking effect of BCTC on the TRP1 conductance (IC50 = 1.03 mM; n = 5). Error bars correspond to SE.
(D) Representative curves showing changes in the Vrev (Erev) we used to determine the permeability ratio PX/PNa when the external solution was replaced.
The table above the plot contains the calculated permeability ratios and the corresponding Erev (n = 4).
A Functional TRP Channel from Algae
(50 mM) and the 3,5-bis(trifluoromethyl)pyrazole derivative BTP2
(10 mM; a TRPC channel blocker and activator of TRPM4) were
ineffective. To determine the ion selectivity of TRP1, we analyzed
shifts in the reversal potential (Vrev) after exchanging the bath
solution with different cations (Figure 4D). The recordings were
performed in whole-cell mode with a calcium-free NaCl-based
pipette solution. There was little change in the Vrev (toward positive Vrev and close to 0 mV) after replacement with 140 mM CsCl
or KCl. However, replacement of the bath solution with 140 mM
N-methyl-D-glucamine (NMDG+, a large monovalent cation) or
Ca2+-containing solutions resulted in a shift of Vrev toward
negative potentials (239 and 24.6 mV, respectively). Therefore,
although permeable to Ca2+, the channel showed greater preference for monovalent cations (Figure 4D, table), a feature only
observed in the calcium-activated channels TRPM4 and TRPM5
(Clapham, 2009). Cr-TRP1 is activated by membrane depolarization, as shown in Figure 5A. After a depolarizing pulse, the
instantaneous tail current follows a linear ohmic relationship
(Figures 5A and 5B). The observed outward rectification, therefore, must come from a voltage-dependent gating process similar
to other voltage-dependent TRP channels including TRPMs and
TRPVs (Clapham, 2009). A more precise quantification of open
probability and equivalent charge was obtained from the tail
currents taken at 2100 mV (Figure 5C) plotted against activation
voltage (Figure 5D). The conductance-voltage (G-V) curve was
well fit by a Boltzmann distribution with a half-maximal activation
voltage (V0.5) around 240 mV.
The sequence of Cr-TRP1 contains two conserved arginines
when compared with mammalian TRPMs (i.e., one located at
the top of TM3 and the other located at the bottom of TM4)
(Supplemental Figure 5). While the former has not been explored, the latter position has previously been associated with
the voltage-dependent gating of TRPM8 channels (Voets et al.,
2007) and PIP2 allosteric interactions in temperature-activated
TRP channels (Brauchi et al., 2007; Latorre et al., 2009). In
TRPV1, the tryptophan at position 697, located within the TRPbox, is critical for the activation of the channel and cannot be
replaced by other aromatic residues without severely affecting
functional expression (Gregorio-Teruel et al., 2014). Naturally
replaced by a phenylalanine in Cr-TRP1’s TRP-box (Figure 3),
apparently the role of Phe-758 is well compensated, since
strong currents can be observed. Polar amino acids located in
the TD helix have been implicated in PIP2-dependent channel
modulation (Clapham, 2009; Latorre et al., 2009). As expected,
incubation with wortmannin (10 nM), an inhibitor of phosphatidylinositol 3-kinase, was enough to diminish TRP1 whole-cell
currents observed in transfected HEK-293T cells (Supplemental
Figure 6). Because the TD helix has been proposed to be a negative allosteric modulator of TRP channel activation (Latorre et al.,
2009; Gregorio-Teruel et al., 2014), we tested a subtle mutation
within the conserved TRP-box sequence. The position Arg-759 is
equivalent to Lys-698 in TRPV1, a residue that was recently
suggested to be important for the regulation of the channel’s gate
(Steinberg et al., 2014). To test this directly, we replaced the
naturally occurring arginine with the conserved lysine residue
present in most mammalian channels. The mutant channel now
elicited functional characteristics of mammalian channels, with
evidently slower activation kinetics of the macroscopic current
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and the G-V curve shifted ;180 mV toward more positive potentials (V0.5 = +148 mV; Figure 5D). This result highlights the
important role of the TD helix in the regulation of channel gating
and the fine-tuned modulation that occurs in this region (Brauchi
et al., 2007; Gregorio-Teruel et al., 2014; Steinberg et al., 2014).
Thus, all our observations are in reasonable agreement with
architecture, voltage dependence, and the well-described regulatory role of the C-terminal domain in mammalian TRPM and
TRPV channels (Clapham, 2009; Latorre et al., 2009).
A subset of TRP channels, dubbed thermoTRPs, display an
exquisite thermal sensitivity, often having Q10 values over 10
(Clapham, 2009; Latorre et al., 2009; Pertusa et al., 2012). As
several channels from the C, M, and V families have been described as temperature-activated channels (Pertusa et al., 2012),
we analyzed Cr-TRP1’s temperature sensitivity. Whole-cell recordings were performed at 14, 22, and 28°C (Figure 5E), producing an evident increase in the macroscopic current (Figures
5E and 5F). It is important to note that although the calculated
temperature dependence of the channel allows it to be classified
as temperature-dependent (Q10 = 5, n = 5; Figure 5G), the activation coefficient was modest when compared with the mammalian
thermoTRP set (Q10 > 10; Pertusa et al., 2012).
DISCUSSION
We have shown that Cr-TRP1 is a functional TRP channel from
algae. Our conclusion that Cr-TRP1 encodes a member of
a novel family of polymodal receptors is based on the fact that
the channel presents sequence features that are responsible
for functional properties typical in TRP channels, such as
voltage-dependent outward rectification, cationic nonselective
permeability, BCTC blocking, TD-associated modulation, and
temperature-dependent gating. Yet, sequence similarity as well
as phylogenetic reconstruction indicate that Cr-TRP1 might be
different enough to be considered part of a novel family. Additionally, characteristic structural features that are segregated in
TRP channels from insects and mammals are present and tied
together in this novel TRP clone, which is a relative of channels
from the C, M, and N families in the A/C/M/N/V cluster identified
by phylogenetic reconstruction. The striking conservation observed for the TD and the TM3-TM4 domain underscores the
importance of these regions in the overall structure.
Under physiological conditions of the algae (i.e., low-sodium
freshwater), Ca2+ and K+ are the major contributors to the voltage changes observed in the Chlamydomonas plasma membrane (Harris, 2001; Hegemann, 2008). While light-dependent
depolarization is mainly driven by Ca2+ and H+ influx, a K+ efflux
repolarizes the algal membrane toward the K+ equilibrium potential (Hegemann, 2008). Considering algal sensory physiology
and TRP1’s permeability and voltage dependence, it is not unreasonable to speculate that TRP1 channels might function
downstream of the sensory input generation (e.g., light-induced
Channelrhodopsin activation), contributing to the regeneration of
the resting potential after sensory-dependent depolarization. An
association between photosensory and chemosensory pathways
has been suggested in the past (Govorunova and Sineshchekov,
2005), and there is experimental evidence supporting the idea
that the potassium conductance linking these pathways may
Figure 5. Voltage and Temperature Dependence of TRP1 Expressed in HEK-293T Cells.
(A) Instantaneous current protocol for TRP1 (top left). The cell was exposed to a +160-mV depolarizing potential pulse and then pulsed to voltages
between 2160 and +160 mV in 20-mV increments (bottom left). Macroscopic tail currents were fitted with a single exponential function (dotted line) to
obtain the instantaneous currents at zero time (bottom right). The box depicts the ionic concentrations (in mM) in the whole-cell configuration used. The
blue and red arrows here and in (B) indicate the steady state and instantaneous currents, respectively.
(B) The steady state curve (closed gray circles, blue arrow) rectifies while the instantaneous current-voltage curve (open orange circles, red arrow)
follows an ohmic behavior. Closed black circles correspond to the uncorrected raw data, showing a discrete rectification of the ohmic behavior.
(C) Representative traces obtained in transfected cells subjected to voltage steps from 2160 to +200 mV in 20-mV increments. The holding potential
was 0 mV, and the tail current was taken at 2100 mV. Ionic concentrations (in mM) used in the whole-cell configuration are depicted in the box.
(D) G-V curves were obtained by plotting peak tail currents at 2100 mV in response to voltage-activated steady state currents as in Figure 5C (wild type,
z = 1.09, V0.5 = 239.3 mV [n = 9]; R759K, z = 0.8, V0.5 = +148 mV [n = 5]).
(E) Representative traces obtained from transiently transfected HEK-293T cells subjected to voltage steps from 2100 to +160 mV in 20-mV increments
at three different temperatures.
(F) Current-voltage (I-V) relations were obtained by plotting steady state currents depicted in Figure 5E. Each represents the average of five independent
experiments.
(G) Current density at different temperatures taken from two fixed voltages (2100 and +100 mV). Error bars correspond to SE.
A Functional TRP Channel from Algae
correspond to a nonselective cation-permeant channel (Nonnengässer et al., 1996). The temperature dependence we describe here might help to define permissive temperatures at which
the algae can repolarize, affecting navigation accordingly. Such
TRP channel-dependent modulation of the sensory input, downstream of receptor activation, has been proposed before for
mammalian TRPM channels such as the taste-associated channel
TRPM5 (Liman, 2007) and TRPM1 channels involved in photosensation (Shen et al., 2009). As the canonical role of TRP channels is to serve as cellular sensors (Clapham, 2009), we cannot
rule out the possibility that TRP1 might function as a temperature
detector in the same way that mechanosensitivity has been associated to Chlamydomonas TRP11 channels (Fujiu et al., 2011).
Further experiments, conducted in algae, will be needed to confirm these hypotheses. We are aware that, without experimental
evidence of the subcellular localization of TRP1, it is difficult
to speculate further on its role. However, previous reports demonstrated that deflagellation upregulates TRP1 expression, suggesting a flagellar localization (Fujiu et al., 2011).
Action potentials observed in animal cells are generally based
on Na+ and/or Ca2+ conductances. However, plants lack genes
coding for important mediators found in the animal sensory response, such as voltage-dependent Ca2+ channels (Ca2+v), TRP
channels, inositol 1,4,5-trisphosphate receptors, and ryanodine
receptors (Wheeler and Brownlee, 2008). Interestingly, chlorophyte
algae such as Chlamydomonas use Ca2+V channels in signaling.
Therefore, Ca2+-dependent signaling mechanisms, while absent in
land plants, are present in these single-celled eukaryotes. The
widespread distribution of these channels in modern, extant organisms suggests that they were present in a common ancestor of
plants and animals; the most recent common ancestor existed
;1.6 billion years ago (Nei et al., 2001; Yoon et al., 2004). In the
metazoan lineage, ion channel diversification can be traced down
to choanoflagellates, a group of free-living unicellular and colonial
flagellate eukaryotes considered to be the closest living relatives of
animals, where few putative TRP channel genes have been reported (Martinac et al., 2008; Jegla et al., 2009). The presence of
a functional TRP channel in Chlamydomonas implies a deep
evolutionary origin of this gene family. Notably, the conservation
of structural-functional features through more than 1.5 billion years
of evolution underscores the robustness within this diverse family of
ion channels (Latorre et al., 2009; Jegla et al., 2009).
We envision that Cr-TRP1 was inherited from a common ancestor of plants and animals; therefore, the precursor of modern
TRP channels must have been present in a unicellular organism
predating the appearance of Chlamydomonas. In fact, several
other single-celled organisms appear to possess sequences
encoding TRP channels, including Dictyostelium, Trypanosoma,
Leishmania, and Plasmodium (Martinac et al., 2008), yet, according
to our results, these channels should belong to the evolutionarily
distinct P/ML cluster of TRP proteins.
METHODS
Identification of TRP-Like Channels from Green Algae and
Unicellular Organisms
Protein sequences corresponding to the 67 TRP ion channels functionally
annotated in the International Union of Basic and Clinical Pharmacology
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database (http://www.iuphar-db.org/DATABASE/FamilyMenuForward?
familyId=78), together with NompC, were downloaded. The data set
contains representatives of families TRPA, TRPC, TRPM, TRPML, TRPN,
TRPP, and TRPV. These 68 functionally characterized TRP channels were
clustered at 50% sequence identity using CD-HIT (Li and Godzik, 2006), resulting in 23 clusters. A representative from each cluster was then used as
a query for BLASTP searches (BLAST 2.2.28+; Altschul et al., 1990) against the
proteome of Chlamydomonas reinhardtii downloaded from Phytozome version
9.0 (Goodstein et al., 2012). From the output, we chose proteins that presented
BLASTP E-values > 1e25 with at least two of the representative sequences.
Then, we performed a new BLASTP search, this time using just the transmembrane regions of the 68 known TRP ion channels against the putative
TRPs returned from our previous search. We identified 10 gene products that
aligned well (alignment length > 70% of sequence length) with the transmembrane region of at least one TRP ion channel in our data set. The procedure was automated in a pipeline involving Python scripts and MySQL
searches and was used to identify TRP subgroup members in the proteomes of
Coccomyxa subellipsoidea C-169 (Cs), Micromonas pusilla CCMP1545
(Mp_ccmp), Micromonas pusilla RCC299 (Mp_rcc), Ostreococcus lucimarinus
(Ol), and Volvox carteri (Vc) downloaded from Phytozome. We also downloaded
from UniProt the complete proteomes of species from the following genera,
Paramecium, Dictyostelium, Trypanosoma, Leishmania, and Plasmodium,
totaling 17 complete proteomes: Paramecium tetraurelia (Pt), Dictyostelium
discoideum (Dd), Dictyostelium purpureum (Dp), Trypanosoma brucei brucei
(strain 927/4 GUTat10.1), Trypanosoma brucei gambiense (strain MHOM/CI/
86/DAL972), Trypanosoma cruzi (strain CL Brener; TcCL), Trypanosoma cruzi
(Tc), Leishmania braziliensis, Leishmania infantum (Li), Leishmania major (Lm),
Leishmania mexicana (strain MHOM/GT/2001/U1103; Lmex), Plasmodium
berghei (strain Anka), Plasmodium chabaudi, Plasmodium falciparum (isolate
3D7), Plasmodium knowlesi (strain H), Plasmodium vivax (strain Salvador I), and
Plasmodium yoelii yoelii. In total, 33 putative TRP channels were found among
algae and unicellular organisms (Figure 2).
SSN of the TRP Family
To generate the SSN, we started from the set of 68 functionally characterized
TRP ion channels (seed sequences) and aimed at identifying all potential TRP
family members present in NCBI’s nr database. To avoid redundancy in the
searches, a representative from each of the 23 clusters described previously
was used to perform BLASTP searches against a local nr database
downloaded from the NCBI ftp site on April 29, 2014. Only proteins having
hits with BLASTP E-values > 1e25 to at least two of the representative
sequences were considered further. This gave us an initial set of 38,263
sequences. As described before, the transmembrane regions of the 68 seed
sequences were used to perform searches against the 38,263 putative TRPs
returned from our previous search, and only those 7107 sequences that
aligned with at least 70% of the full length of the transmembrane domain of at
least one representative were considered further. All 7107 sequences plus
the 33 putative TRP channels from algae and unicellular organisms were
used to generate an SSN, where nodes correspond to sequences and edges
to BLASTP E-values between the sequences. We note that 17 of the 33
putative TRP channels were already included in the nr data set, so the total
number of proteins in the network is 7126. Because the SSN generated had
too many edges (over 8 million), an SSN of representative sequences from
the family was also created. For this SSN, the 7126 unique TRP subgroup
members were clustered at 90% sequence identity using CD-HIT, resulting
in 2841 clusters. The largest sequence in each cluster was used as a representative for generating an SSN. Since BLASTP E-values are not symmetrical, we performed searches using one of the sequences under
comparison as query and the other as subject, replaced the query with the
subject and vice versa, and selected the worst reciprocal BLASTP E-value for
each pair. Visualization was performed using the organic layout in Cytoscape
2.8.3 (Cline et al., 2007) at different thresholds for the E-value. The two
E-values selected for generating Figure 1 correspond as follows: Figure 1A,
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The Plant Cell
the highest E-value where cluster A/C/M/N/V is separated from cluster P/ML
(1e218); Figure 1B, the highest E-value where the seven functional families are
separated from one another (1e287).
Phylogenetic Inference for the TRP Family
A multiple sequence alignment for the 68 functionally characterized TRPs plus
the 33 sequences identified in algae and unicellular organisms was built in
MAFFT (Katoh et al., 2002) using an iterative refinement method considering
both weighted sums of pairs and consistency scores (Supplemental Data Set
1). Molecular phylogeny was inferred using the Bayesian method implemented in MrBayes version 3.2 (Ronquist and Huelsenbeck, 2003). To
estimate the best fixed-rate model for our data, we used a mixed model,
obtaining a posterior probability of 1 for the Jones model (Jones et al., 1992).
Thus, the molecular phylogeny presented here uses the Jones substitution
model and a gamma distribution model to allow rate variation among sites.
We set the number of generations to 2 3 106, sampled every 1000th generation, and discarded the initial 30% of samples before summarizing trees
and parameters. The average SD of split frequencies was less than 0.0026
upon convergence.
V0.5)/RT]), where z is the voltage dependency, V0.5 is as defined previously,
and Imax is the maximum tail current. For the permeability experiments,
current-voltage relationships were studied using a ramp protocol consisting
of a voltage step of 100 ms from the holding potential of 0 mV to 2100 mV,
followed by a 400-ms linear ramp up to +140 mV. The time interval between
each ramp was 1 s. Ramp protocols were acquired at 10 kHz and filtered at
5 kHz. Currents are presented in terms of densities (pA/pF). Data acquisition
was made using an Axopatch 200B (Axon Instruments), a 6052E acquisition
board (National Instruments), and custom-made software written in LABVIEW for all electrophysiology data acquisition.
TIRF Microscopy
HEK-293T transfected cells were imaged using an objective-based TIRF
microscope. A 473-nm diode pump laser (LaserGlow) illuminated the
sample on an Olympus IX71 microscope. Laser light passed through a highnumerical-aperture objective (603, numerical aperture 1.49, oil immersion;
Olympus) and was totally internally reflected by the glass-water interface.
Fluorescence was collected by a cooled-CCD (ORCA ER II; Hamamatsu) at
10 Hz using the micromanager plug-in (Universal Imaging) for ImageJ.
Identification of Domains in TRP1
Recording Solutions
Transmembrane Helices Hidden Markov Model was used to define
transmembrane segments. All other domains were defined from the literature
(Latorre et al., 2009) and identified in multiple sequence alignments of bona fide
TRP channels against Cr-TRP1 performed in PROMALS3D (Pei and Grishin,
2014). For clarity, the alignments presented here incorporate only one channel
per subfamily member. The protein illustration for Cr-TRP1 that includes the
identified domains was created using Protter (Omasits et al., 2014).
Cell Culture
Wild-type C. reinhardtii strain CC-124 mt2 was maintained in Sueoka’s
medium (Harris, 2001) at 18°C under constant agitation and light/dark periods of 12 h. C. reinhardtii gametes were prepared by incubating vegetative
cells with a nitrate-free medium overnight in the dark. Recordings were made
on fresh C. reinhardtii gametes in Sueoka’s bath medium. HEK-293T cells
were cultured in DMEM (Gibco) supplied with 10% FBS (Gibco). Cells were
plated on poly-L-lysine-coated cover slips and transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Recordings on HEK-293T cells were performed 24 to 48 h after transfection.
Cloning and Molecular Biology
A full-length C. reinhardtii TRP1 cDNA containing a 2703-nucleotide open
reading frame was isolated from cDNA prepared from CC-124 algae gametes
using the following cloning primers: FW (59-GCGAAGGTACCAGCGACATGAAGGTCGCTCC-39) and RV (59-CTCGCGGAATTCCTAGTGCCCCGCAGCGGC-39). TRP1 was subcloned between KpnI and EcoRI restriction sites
of pTracer-CMV2 vector (Invitrogen) and pEGFP vector. Mutations were introduced by PCR on the TRP1-pTracer background.
Unless stated otherwise, the pipette solution contained (in mM): 105 CsF,
35 NaCl, 4 EGTA, 1 Mg-ATP, and 20 HEPES, pH 7.4. The bath solution
contained (in mM): 135 NaCl, 2 CaCl2, 5 KCl, 20 HEPES, and 10 glucose, pH
7.4. The permeability of monovalent cations relative to that of Na+ was estimated from the shift in the Vrev on replacing external Na+ in a nominally
Ca2+-free bath solution (145 mM XCl, 20 mM HEPES, and 10 mM glucose,
pH 7.4, where X was Na+, K+, Cs+, or NMDG+). Ca2+ permeability was estimated from the shift in the Vrev on replacing NMDG-Cl with a solution
containing (in mM): 100 NMDG-Cl, 30 CaCl2, 20 HEPES, and 10 glucose, pH
7.4. The following equations were used to calculate the relative permeability:
Px
½Naþ o
¼
exp Vx 2 VNa F=RT
PNa
½Xo
f
f
PCa
¼ 1 þ expðVCa 2 VNa ÞðF=RTÞ
PNa
3
g
g
ð½Naþ i þ a½NMDGþ i ÞexpfðVCa 2 VNa ÞðF=RTÞg 2 ½Naþ o 2 a½NMDGþ o
4½Ca2þ o
where [X]o is defined as the extracellular concentration of the given cation,
P is defined as the permeability of the ion indicated by the subscript, F is
Faraday’s constant, R is the gas constant, T is absolute temperature, VX is
the Vrev for the ion indicated by the subscript, and VNa is the Na+ Vrev. a =
PNMDG/PNa.
Data Analysis
Unless stated otherwise, group data are presented as means 6 SE. Statistical
comparisons were made using one-way ANOVA and the Bonferroni mean
test. A value of P = 0.05 was taken to be statistically significant.
HEK-293T Electrophysiology
Whole-cell currents were obtained from transiently transfected HEK-293T
cells (ATCC). Gigaseals were formed by using 2- to 4-MV borosilicate
pipettes (Warner Instruments). Junction potentials (6 to 8 mV) were
corrected for CsF/NaCl solutions. Seal resistance was >2 GV in all cases.
Series resistance and cell capacitance were analogically compensated
directly on the amplifier. The mean maximum voltage drop was ;4 mV.
Whole-cell voltage clamp was performed, and the macroscopic currents
from voltage steps were acquired at 20 kHz and filtered at 10 kHz. The G-V
curves for the wild-type channel as well as for the mutant R759K were
fitted to a Boltzmann distribution of the form I = Imax/(1 + exp 2 [zF(V 2
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL
libraries under the following accession numbers: TRPA1_HUMAN,
NP_015628.2; TRPA1_MOUSE, NP_808449.1; TRPA1_RAT, NP_997491.1;
TRPC1_HUMAN, NP_003295.1; TRPC1_MOUSE, NP_035773.1;
TRPC1_RAT, NP_446010.1; TRPC2_MOUSE, NP_035774.2; TRPC2_RAT,
Q9R283-1; TRPC3_HUMAN, NP_001124170.1; TRPC3_MOUSE,
NP_062383.2; TRPC3_RAT, NP_068539.2; TRPC4_HUMAN, NP_057263.1;
TRPC4_MOUSE, NP_058680.1; TRPC4_RAT, NP_536321.1; TRPC5_HUMAN,
NP_036603.1; TRPC5_MOUSE, XP_033454.1; TRPC6_HUMAN, NP_004612.2;
A Functional TRP Channel from Algae
TRPC6_MOUSE, NP_038866.2; TRPC7_HUMAN, NP_065122.1;
TRPC7_MOUSE, NP_036165.1; TRPM1_HUMAN, NP_002411.3;
TRPM1_MOUSE, NP_001034193.2; TRPM1_RAT, NP_001032823.1;
TRPM2_HUMAN, NP_003298.1; TRPM2_MOUSE, NP_612174.2;
TRPM3_HUMAN, NP_066003.3; TRPM4_HUMAN, NP_060106.2;
TRPM4_MOUSE, NP_780339.2; TRPM4_RAT, NP_001129701.1;
TRPM5_HUMAN, NP_055370.1; TRPM5_MOUSE, NP_064673.2;
TRPM6_HUMAN, NP_060132.3; TRPM6_MOUSE, NP_700466.1;
TRPM7_HUMAN, NP_060142.3; TRPM7_MOUSE, NP_067425.2;
TRPM7_RAT, NP_446157.2; TRPM8_HUMAN, NP_076985.4;
TRPM8_MOUSE, NP_599013.1; TRPM8_RAT, NP_599198.2;
TRPML1_HUMAN, NP_065394.1; TRPML1_MOUSE, NP_444407.1;
TRPML2_HUMAN, NP_694991.2; TRPML2_MOUSE, NP_080932.2;
TRPML3_HUMAN, NP_060768.8; TRPML3_MOUSE, NP_598921.1;
TRPN_DROME, NP_001245891.1; TRPP1_HUMAN, NP_000288.1;
TRPP1_MOUSE, NP_032887.3; TRPP2_HUMAN, NP_057196.2;
TRPP3_HUMAN, NP_055201.2; TRPP3_MOUSE, NP_058623.2;
TRPV1_HUMAN, NP_061197.4; TRPV1_MOUSE, NP_001001445.1;
TRPV1_RAT, NP_114188.1; TRPV2_HUMAN, NP_057197.2; TRPV2_MOUSE,
NP_035836.2; TRPV2_RAT, NP_058903.2; TRPV3_HUMAN, NP_659505.1;
TRPV3_MOUSE, NP_659567.2; TRPV4_HUMAN, NP_067638.3;
TRPV4_MOUSE, NP_071300.2; TRPV4_RAT, NP_076460.1; TRPV5_HUMAN,
NP_062815.2; TRPV5_MOUSE, NP_001007573.1; TRPV5_RAT, NP_446239.2;
TRPV6_HUMAN, NP_061116.4; TRPV6_MOUSE, NP_071858.2; TRPV6_RAT,
NP_446138.1; Cr-TRP1, JX173491.1, ABG54260.1, Phytozome Cre10.
g452950.t1.3; Cr-TRP2, Phytozome g15856.t1; Cr-TRP11, AB596979.1,
Phytozome Cre07.g341350.t1.2; Cr-TRP13, Phytozome Cre03.g175050.t1.3;
Cr-TRP16 Phytozome Cre06.g278226.t1.1; Cr-TRPP2, Phytozome Cre17.
g715300.t1.2; Cr-TRP5, Phytozome Cre09.g398400.t1.2; TRP15, Phytozome
Cre10.g422750.t1.3; Cr-TRP6, Phytozome Cre07.g334300.t1.3; Cr-TRP21,
Phytozome Cre10.g434600.t1.2; Cr-TRP22, Phytozome Cre02.g112200.t1.2;
Cr-TRP23, Phytozome Cre09.g390578.t1.1; Vc-TRP1, Phytozome
Vocar20010710m; Vc-TRP2, Phytozome Vocar20007683m; Vc-TRP3,
XP_002947703.1, Phytozome Vocar20008340m; Vc-TRP4, XP_002954486.1,
Phytozome Vocar20010765m; Cs-TRP1, XP_005642695.1, Phytozome
45570; Cs-TRP2, XP_005643399.1, Phytozome 68054; Mp_ccmp-TRP1,
XP_003064374.1, Phytozome 49212; Mp_rcc-TRP2, XP_002506421.1,
Phytozome 64572; Dd-TRP1, XP_635110.1; Dd-TRP2, XP_644933.1;
Dp-TRP1, XP_003289960.1; Dp-TRP2, XP_003289259.1; Dp-TRP3,
XP_003283790.1; Li-TRP1, XP_001470439.1; Lm-TRP1, XP_001684103.1;
Lmex-TRP1, XP_003876399.1; Lmex-TRP2, XP_003872242.1; Pt-TRP1,
XP_001452370.1; Pt-TRP2, XP_001444531.1; Pt-TRP3, XP_001429751.1;
TcCL-TRP1, XP_804976.1; TcCL-TRP2, XP_804854.1; Tc-TRP1,
ADWP02012431.
Supplemental Data
Supplemental Figure 1. Multiple Sequence Alignment Depicting the
Ankyrin Domains Present in Cr-TRP1 and Other TRPN, TRPC, TRPV,
and TRPA Family Representatives.
Supplemental Figure 2. TRPM Homology Regions (MHR) in CrTRP1.
Supplemental Figure 3. Cr-TRP1 Amino Acid Sequence.
Supplemental Figure 4. The Effect of Cr-TRP1 Blockers.
Supplemental Figure 5. Alignment at the TM3-TM4 Region.
Supplemental Figure 6. Inward and Outward Whole-Cell Currents Are
Affected after 10 min Incubation with 10 nM Wortmannin.
Supplemental Data Set 1. Fasta Format Multiple Sequence Alignment of the 68 Functionally Characterized TRPs Plus the 33 Sequences Identified in Algae and Unicellular Organisms Used to Construct
Phylogeny Shown in Figure 2.
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ACKNOWLEDGMENTS
We thank H. Kurata, R. Latorre, and C. Zuker for commenting on the
article and E. Harris for providing strains and helpful information. This
work was supported by FONDECYT (Grants 11070190 and 1110906 to
S.B., Grant 1131064 to R.M., and Grant 3140233 to C.K.C.), CONICYT
(Grant ACT-1113 to D.E. and fellowship to L.A.-D.), and the Millennium
Initiative (CINV Grant P09-022-F to D.E.A.).
AUTHOR CONTRIBUTIONS
S.B. and D.E.A. designed the research. L.A.-D., D.C., M.A.-A., F.B.-M.,
R.M., and S.B. performed research. I.V.-C. and C.K.C. contributed new
analytic/computational tools. D.E.A., S.B., and C.K.C. analyzed data.
S.B., D.E.A., and C.K.C. wrote the article.
Received September 8, 2014; revised November 28, 2014; accepted
December 21, 2014; published January 16, 2015.
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A Transient Receptor Potential Ion Channel in Chlamydomonas Shares Key Features with Sensory
Transduction-Associated TRP Channels in Mammals
Luis Arias-Darraz, Deny Cabezas, Charlotte K. Colenso, Melissa Alegría-Arcos, Felipe Bravo-Moraga,
Ignacio Varas-Concha, Daniel E. Almonacid, Rodolfo Madrid and Sebastian Brauchi
Plant Cell; originally published online January 16, 2015;
DOI 10.1105/tpc.114.131862
This information is current as of June 16, 2017
Supplemental Data
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