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The Journal of Neuroscience, February 15, 2003, 23(4):1340
Functional Role of C-Terminal Cytoplasmic Tail of Rat Vanilloid
Receptor 1
Viktorie
Vlachová1,
Jan
Teisinger1,
Klára
Su ánková1,
Alla
Lyfenko1,
Rüdiger
Ettrich2, 3, and
Ladislav
Vyklický1
1 Institute of Physiology, Academy of Sciences, 142 20 Prague 4, Czech Republic, 2 Institute of Physical Biology,
University of South Bohemia, 373 33 Nové Hrady, Czech Republic,
and 3 Department of Biochemistry, Faculty of Science,
Charles University, 128 40 Prague 2, Czech Republic
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ABSTRACT |
The vanilloid receptor [transient receptor potential (TRP)V1, also
known as VR1] is a member of the TRP channel family. These receptors
share a significant sequence homology, a similar predicted structure
with six transmembrane-spanning domains (S1-S6), a pore-forming region
between S5 and S6, and the cytoplasmically oriented C- and N-terminal
regions. Although structural/functional studies have identified some of
the key amino acids influencing the gating of the TRPV1 ion channel,
the possible contributions of terminal regions to vanilloid receptor
function remain elusive. In the present study, C-terminal truncations
of rat TRPV1 have been constructed to characterize the contribution of
the cytoplasmic C-terminal region to TRPV1 function and to delineate
the minimum amount of C tail necessary to form a functional channel.
The truncation of 31 residues was sufficient to induce changes in
functional properties of TRPV1 channel. More pronounced effects of
C-terminal truncation were seen in mutants lacking the final 72 aa.
These changes were characterized by a decline of capsaicin-,
pH-, and heat-sensitivity; progressive reduction of the
activation thermal threshold (from 41.5 to 28.6°C); and slowing of
the activation rate of heat-evoked membrane currents
(Q10 from 25.6 to 4.7). The
voltage-induced currents of the truncated mutants exhibited a slower
onset, markedly reduced outward rectification, and significantly smaller peak tail current amplitudes. Truncation of the entire TRPV1
C-terminal domain (155 residues) resulted in a nonfunctional channel.
These results indicate that the cytoplasmic COOH-terminal domain
strongly influences the TRPV1 channel activity, and that the distal
half of this structural domain confers specific thermal sensitivity.
Key words:
capsaicin receptor; TRPV1; C-terminal domain; heat
transduction; heat sensitivity; voltage-gated cation channel
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Introduction |
Vanilloid receptor 1 [transient
receptor potential (TRP)V1, formerly VR1] has been suggested to
function as a multimodal signal transducer of noxious stimuli in the
mammalian somatosensory system (Caterina et al., 1997 ). Noxious thermal
stimuli (>43°C), acidic pH (<6.8), or the alkaloid irritant
capsaicin are required to open the TRPV1 channel. At room temperature
and a pH of 7.3, TRPV1 behaves as a voltage-gated outwardly rectifying
nonselective cation channel, because it can be activated strongly by
depolarizing voltage steps in the absence of any agonist (Chuang et
al., 2001; Reeve and Bevan, 2002; Vlachová et al., 2002).
Although much knowledge of the structure and function of the TRPV
channel subfamily has accumulated recently (for review, see Clapham et
al., 2001; Gunthorpe et al., 2002), the critical structural domains and
the mechanisms by which various external stimuli translate into channel gating remain poorly understood.
The finding that disruption of the TRPV1 gene has minimal
effects on thermal nociception (Caterina et al., 2000; Davis et al.,
2000) and the identification of TRPV2 as a heat receptor with a high
temperature threshold (Caterina et al., 1999) suggest that other
receptors homologous to TRPV1 may be implicated in thermosensation.
Indeed, TRPV3 (Smith et al., 2002; Xu et al., 2002) (~39°C) and
TRPV4 (Liedtke et al., 2000; Guler et al., 2002) have been found to
show thermal sensitivity. A TRPV1 variant, the stretch-inactivated
channel (SIC), has a truncated N-terminal domain and shares identical
transmembrane-spanning domains with TRPV1 (Schumacher et al., 2000a).
The C-terminal domain of SIC is identical to the corresponding
C-terminal domain of TRPV4 except for aspartate 494. This channel can
be activated by hypertonic solutions; however, it is insensitive to
capsaicin. Another TRPV1 variant, the VR 5' splice variant, which is
also insensitive to vanilloids, low pH, and thermal stimuli, encodes a
C-terminal domain identical to TRPV1 but lacks the major part of the N
terminal (Schumacher et al., 2000b). The above results suggest that the N-terminal domain of TRPV1 is essential for the formation of functional ion channels, rather than specifically lacking an important functional region. It has been suggested that the C-terminal domains of the TRP
receptors act to ensure the uniqueness of their function (Xue et al.,
2001).
In the predicted TRPV1 C terminal, a serine residue S800 that can be
directly phosphorylated by protein kinase C (Numazaki et al., 2002) and
a putative Walker-type nucleotide-binding site (L726-W740) have been
identified to date (Kwak et al., 2000). However, how the C-terminal
domain contributes to the conformational stability of TRPV1 channel and
the extent to which it influences its function still remain to be
determined. In this study, we demonstrate that the cytosolic C-terminal
region of the TRPV1 channel contains a domain or domains responsible
for a steep temperature dependence of the TRPV1 heat-evoked responses.
We hypothesize that this region is also important for the regulation of
the capsaicin-, low pH-, and voltage-induced channel activity.
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Materials and Methods |
Cell culture and expression of cDNA. Human embryonic
kidney (HEK) 293T cells (SD 3515; American Type Culture Collection,
Manassas, VA) were cultured in Opti-MEM I (Invitrogen,
Gaithersburg, MD) supplemented with 5% fetal bovine serum.
Cells were plated onto dishes coated with
poly-L-lysine at a density of ~180,000
cells/cm2. HEK293T cells were
transiently transfected with 300-500 ng per dish of recombinant
plasmid DNA encoding wild-type (WT) or mutant rat TRPV1 in pcDNA3
vector (wild type provided by D. Julius, University of California, San
Francisco, CA) using the Lipofectamine 2000 method according to the
manufacturer's protocol (Invitrogen, Paisley, UK). To identify the
transfected cells in the electrophysiological experiments, DNA plasmid
encoding green fluorescent protein (GFP) in pQBI 25 vector (TaKaRa,
Tokyo, Japan) was cotransfected at a concentration of 500 ng per dish.
Transfected cells were replated onto glass coverslips (three 12 mm
coverslips per 35 mm dish) coated with
poly-L-lysine. Electrophysiological and
immunohistochemical examination was performed 24-48 hr after
transfection. For each experimental group, five to eight GFP-positive
cells per coverslip were studied from at least four different
(independent) transfections.
Construction of C-terminal deletions of TRPV1. Rat TRPV1
C-terminal deletions were constructed by PCR amplification using rat
TRPV1-specific primers synthesized to contain a point mutation converting the respective nucleotides at positions Q808, R797, E767,
E761, K735, and E684 to a stop codon (Fig.
1). The QuikChange XL site-directed
mutagenesis kit (Stratagene, La Jolla, CA) was used according to the
manufacturer's protocol to perform point mutations in TRPV1. The
overlapping primer pairs were as follows: TRPV1-C 31, 5'-GA GAT AGA
CAT GCC ACC TAG CAG GAA GAA GTT CAA CTG-3'
(sense) and 5'-CAG TTG AAC TTC TTC CTG CTA GGT GGC ATG
TCT ATC TC-3' (antisense); TRPV1-C42, (double mutation), 5'-CC CTG
GTT CCC CTT CTG TGA GAT GCA AGC ACT CGA GAT
AG-3' (sense) and 5'-CT ATC TCG AGT GCT TGC ATC
TCA GAG AAG GGG AAC CAG GG-3' (antisense);
TRPV1-C72, 5'-G GAC CCA GGC AAC TGT TAG GGC
GTC AAG CGC ACC CTG AGC-3' (sense) and 5'-GCT CAG GGT GCG CTT GAC GCC
CTA ACA GTT GCC TGG GTC C-3' (antisense); TRPV1-C78, 5'-GTG GGT ATC ATC AAC TAG GAC CCA
GGC AAC TGT GAG GGC G-3' (sense) and 5'-C GCC CTC ACA GTT GCC TGG GTC CTA GTT GAT GAT ACC CAC-3' (antisense);
TRPV1-C104, 5'-G GTG GGG TTC ACT CCT GAC GGC
TAG GAT GAC TAC CGG TGG-3' (sense) and 5'-CCA CCG
GTA GTC ATC CTA GCC GTC AGG AGT GAA CCC CAC C-3'
(antisense); TRPV1-C155, 5'-CTC ATT GCT CTC ATG GGT
TAG ACC GTC AAC AAG ATT GC-3' (sense) and 5'-GC AAT CTT GTT GAC GGT CTA ACC CAT GAG AGC AAT GAG-3'
(antisense). Base changes introducing the stop codon are in bold type;
italic type indicates the inserted point mutation. All site-directed mutated constructs were confirmed by DNA sequencing using an automated sequencer (ABI PRISM 3100; Applied Biosystems, Foster City, CA).
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Figure 1.
C-terminal truncations of vanilloid receptor
TRPV1. A, Schematic representation of TRPV1 C-terminal
deletions showing the six transmembrane-spanning domains,
S1-S6, and a pore-forming region (hatched bars).
B, Putative membrane topology and amino acids
(in circles) of the C-terminal half of the rat TRPV1
receptor with indicated locations of the amino acids at which the stop
codon has been introduced. C31, C42, C72, C78, C104, and
C155 correspond to the mutated Q808, R797, E767, E761, K735, and
E684, respectively.
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Immunohistochemistry. Polyclonal anti-VR1 raised against the
peptide corresponding to the N terminal of rat VR1 (Affinity BioReagents, Golden, CO) was diluted 1:1000 in PBS. FITC-conjugated donkey anti-rabbit IgG (Abcam, Cambridge, UK) was used in a 1:100 dilution in PBS as the secondary antibody. Before seeding and transfection of the cells, three coverslips were placed in the culture
dish. Twenty-four hours after transfection, the cells on the coverslips
were washed once with PBS, fixed with 4% paraformaldehyde for 30 min,
and permeabilized with 0.5% Triton X-100 in PBS for 5 min. After
permeabilization, the cells were washed three times for 10 min in PBS
and then incubated for 30 min in PBS solution with 0.25% BSA
and 0.25% gelatin to eliminate nonspecific binding. Cells were
incubated overnight at 4°C with primary antibody, washed three times
in PBS, incubated with secondary antibody for 1 hr, and washed again
three times in PBS. Immunostained HEK293T cells were visualized using
confocal laser scanning microscopy (MRC 600; Bio-Rad, Hercules, CA)
attached to an inverted microscope (Nikon Diaphot; Nikon, Tokyo, Japan).
Electrophysiology. Whole-cell membrane currents were
recorded using an Axopatch-1D amplifier and pClamp8 software (Axon
Instruments, Foster City, CA). Electrodes were pulled from borosilicate
glass; after fire polishing and filling, they had a resistance of 2-4 M . The series resistance was usually <10 M and was compensated to ~80%. A system for fast superfusion of the neurons was used for
drug and heat application. It consisted of a manifold of seven fused
silica capillaries connected to a common outlet made from a glass
capillary around which insulated copper wire (20 µm thick) was coiled
to pass direct current for heating the solutions superfusing the neuron
under investigation (Dittert et al., 1998). The temperature of the
superfusing solution was measured by a miniature thermocouple inserted
into the outlet capillary near its orifice that was placed <100 µm
from the cell under investigation. Before and after the test solutions,
the cells were superfused with control extracellular solution of
the following composition (in mM): 160 NaCl, 2.5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, and 10 glucose, pH adjusted to 7.3 with NaOH. The intracellular
pipette solution contained (in mM): 125 Cs-gluconate, 15 CsCl, 5 EGTA, 10 HEPES, 2 NaCl, and 0.5 CaCl2, pH adjusted to 7.3 with CsOH. Capsaicin
was dissolved in 100 µl of DMSO and diluted with 0.9 ml of distilled
water to make a stock solution of 1 mM.
Molecular modeling of the TRPV1 C-terminal structure. The
C-terminal sequence from 690 to 838 was aligned with the known x-ray structure of the fragile histidine triad (FHIT) protein, extracted from
the Protein Data Bank (entry 1FIT; http://www.rcsb.org/pdb/). The primary structure of this protein shows a high degree of similarity (44%) with the TRPV1 C terminal. The primary structures were aligned with the template structures by ClustalX (Thompson et al., 1997) and
adjusted manually when necessary. The alignment used for additional modeling is shown in Figure 9A. Three-dimensional models of
the C tail constituted by all nonhydrogen atoms were generated by the
Modeller 6 package (Sali and Blundell, 1993). All the obtained models
were subjected to a short simulated annealing refinement protocol
available in Modeller. The representative model was that with the
lowest Modeller target function value. For additional refinement,
hydrogen atoms were added, and the generated structure was minimized to
convergence of the energy gradient less a 0.01 kcal/mol using the
Powell minimizer and the Tripos force field included in the
Sybyl/Maximin2 module (Tripos, St. Louis, MO). Finally, the tertiary
structure models were checked with Procheck (Laskowski et al.,
1993).
Statistical analysis. Data are expressed as means ± SEM. Overall statistical significance was determined by ANOVA,
if not stated otherwise. In cases of significance
(p < 0.05), statistical comparisons were
performed by Student's t test for individual groups. A
rectification index (RI) of voltage-gated TRPV1-mediated whole-cell
membrane currents was calculated using the following equation: RI = [I+60/(60  Vrev)]/[I 40/(40 Vrev)]. Here,
I+60 and
I40 are the steady-state amplitudes of the responses recorded at holding potentials of +60 and 40 mV, and
Vrev is the estimated reversal potential.
The temperature coefficient Q10 was
used to characterize the temperature dependence of the membrane current
(Vyklický et al., 1999). Data sampled at the rising phase of the
temperature ramp were pooled every 0.5°C. The absolute current values
were plotted on a log scale against the reciprocal of the absolute temperature (Arrhenius plot). Q10
values were determined by using the following formula:
Q10 = exp[T × Ea/(R × T1 × T2)], where R is
the gas constant, T = 10 Kelvin, and
Ea is an apparent activation energy
estimated from the slope of the Arrhenius plot between absolute
temperatures T1 and
T2. The thermal thresholds for
wild-type and TRPV1 mutant activation were measured from the
temperatures at which the inward currents plotted on a logarithmic
scale declined significantly from a straight line. For this purpose,
piecewise linear regression was adapted using SigmaPlot 2000 (SPSS
Inc., Chicago, IL). Dose-response data were fitted to the Hill
equation, as follows: I = Imax[(capsaicin)n/([capsaicin]n + EC50n)], where I
is the normalized current at a given concentration of capsaicin,
Imax is the maximal current,
EC50 is the concentration of half-maximal
activation, and n is the Hill coefficient.
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Results |
C-terminal truncations reduce the TRPV1 responsiveness to capsaicin
and low pH
To delineate the functional role of the C-terminal domain in TRPV1
activation, the whole-cell membrane currents elicited by capsaicin, low
pH, and heat stimuli (47°C) in HEK293T cells transfected with either
wild-type rat TRPV1 receptor or the C-terminal truncated mutants were
recorded. Interactions between chemical and thermal stimuli were
measured by applying a 3 sec temperature ramp from 24 to 47°C at the
steady state of the capsaicin- or low pH-induced responses (Fig.
2A). At room
temperature and a holding potential of 70 mV, capsaicin (1 µM) induced significant inward membrane currents in all cells expressing wild type; however, the magnitudes of
the responses showed a large variation: a median of 1.5 nA (mean,
1.5 ± 0.4 nA; n = 29) and a range between 20 and
7 nA. The proton-induced currents were measured in the steady state to
remove the contribution of the fast inactivating human acid-sensing ion
channels, which are constitutively expressed in HEK293 cells (Gunthorpe
et al., 2001). As with capsaicin, the variations of the magnitudes of
the responses induced by extracellular exposure to pH 5 were large
(from 0.4 to 7 nA; 2.3 ± 0.4 nA; n = 15).
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Figure 2.
Whole-cell membrane currents induced by heat,
protons, and capsaicin in wild-type TRPV1 and the C-terminal truncated
mutants expressed in HEK293T cells. A, Heat-evoked
currents (IHEAT) were induced by a 3 sec ramp of temperature increase to 49°C in standard extracellular
solution (left), acidic pH (pH 5;
middle), and capsaicin (1 µM;
right). The cells clamped at 70 mV were superfused for
the time indicated by the open bars shown above the
records. Dashed lines indicate a zero membrane current.
B, Quantitative analysis was performed by first
stimulating cells by heat (from 25 to 49°C) in the extracellular
solution, followed by a 30 sec washout, and then by stimulating
them by heat in the presence of pH 5 or capsaicin. Mean ± SEM
responses evoked by heat, capsaicin, and low pH measured at room
temperature, 25°C, are shown (p < 0.05;
indicated with an asterisk). The numbers
above each bar indicate the number of cells measured.
C, Relative increase in capsaicin-induced peak currents
at 47°C versus steady-state capsaicin current amplitudes measured at
25°C are plotted for individual cells transfected with wild-type and
C31 (top) and C42, C72, and C78
(bottom). D, Analysis of proton-induced
potentiation of heat-evoked currents. Mean ratios of peak currents
induced by heat (47°C) at pH 6 to those measured at pH 7.3 are shown
at the top. Average amplitudes of heat-induced currents
recorded at pH 6 are shown at the bottom. Data represent
means ± SEM from 8 to 21 cells.
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Deletions of 31 and 42 residues from the C terminal (C31 and
C42) did not cause significant changes in capsaicin- and
proton-induced maximum currents at room temperature (Fig.
2B), whereas in mutant receptors C72 and C78,
the sensitivities to these two activators were substantially reduced.
The C104 mutation displayed no sensitivity to capsaicin over the
entire temperature range examined (Table 1). In this mutant, the average amplitude
of the membrane current measured in the presence of capsaicin at 47°C
was not different from that induced by heat in the control
extracellular solution (n = 11; p = 0.18; Student's paired t test), suggesting the absence of
any effective capsaicin-receptor interaction. Sequential deletions of
72, 78, and 104 residues from the C terminal also reduced the membrane
currents elicited by protons; however, responses to pH 5 were still
present in all of these mutants (Table 1).
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Table 1.
Current responses of wild-type and C-terminal truncated
mutants of TRPV1 channel to heat, capsaicin, and pH 5
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Interactions of heat, capsaicin, and protons in C-terminal
truncated TRPV1
We found that the capsaicin-elicited currents increased as the
temperature was raised in both the wild type and in TRPV1 with truncated C tails (Fig. 2C). Similarly to DRG neurons
(Vlachová et al., 2001), the smaller capsaicin responses at room
temperature were potentiated considerably more by heat than were the
large responses to capsaicin. In addition, there was an inverse
correlation between the current amplitudes measured at room temperature
and the ratios of the currents at 47°C to those at 25°C. The degree of potentiation decreased with decreasing length of the C-terminal tail
[Spearman correlation (number of cells measured): r = 0.93 (27), 0.94 (21), 0.74 (28), and 0.68 (21) for WT, C31,
C72, and C78, respectively].
Acidification of the extracellular bath solution (from 7.3 to 6.0) has
two main effects on wild-type TRPV1 function. First, increased proton
concentration markedly potentiates capsaicin and heat-induced
responses. Second, at higher concentrations (less than pH 6.0) protons
directly activate TRPV1 at room temperature. In our experiments,
acidification of the extracellular solution from 7.3 to 6.0 facilitated
the heat-induced responses in TRPV1-C31, TRPV1-C42, and
TRPV1-C78 mutants similarly to the way it did in the wild type
(Fig. 2D). In the TRPV1-C72 and TRPV1-C104 constructs, the extracellular application of pH 6 did not produce a
consistent increase in IHEAT. However,
note that these results cannot be interpreted unequivocally,
because it was difficult to exclude the possibility that some
proton-induced sensitization was masked by receptor desensitization.
Dose-response relationships for activation by capsaicin in
C-terminally truncated TRPV1 mutants
The heat- or capsaicin-induced responses in mutants C31 and
C42 displayed a slight, insignificant increase
(p = 0.1 vs wild-type controls). This finding
led us to compare the affinities for capsaicin in the C-terminally
truncated mutants with that of the wild-type TRPV1. In the wild type,
fits of dose-response relationships with the Hill equation (Fig.
3A,B) (see Materials and
Methods) yielded values of EC50 = 0.55 ± 0.08 at 30°C and EC50 = 0.24 ± 0.02 at 45°C (n = 5; p < 0.01; paired
t test), whereas the Hill coefficient appeared to be
independent of temperature. From the dose-response relationships, it
is evident that only the mutant C31 displayed differences in the
apparent affinity for capsaicin at both 30 and 45°C. Moreover, at
45°C, the agonist efficacy of the mutant C31 was strongly
increased (Fig. 3D). Actually, it is difficult to ascertain
from the experimental protocol whether significant changes in
dose-response relationships reflect increases in capsaicin potency and
efficacy (i.e., agonist-induced current), or whether they occur because
of changes in the activity mode of the channel (i.e., heat-induced
current). Note that although the variability of the currents, the
considerable tachyphylaxis, and the wide range of cooperativity (Fig.
2C) complicate the interpretation, these data
indicate that the distal region of the C-terminal domain could
participate in the allosteric conformational changes producing opening
after ligand binding.
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Figure 3.
The effects of TRPV1 C-terminal truncation on
receptor capsaicin efficacy and potency. A,
Representative traces of whole-cell currents in WT TRPV1 activated by a
heat ramp from 24 to 47°C in normal extracellular solution
(left), 0.1 µM capsaicin
(middle), and 1 µM capsaicin
(right). The holding potential (HP) is
70 mV. B, The HEK293T cell transiently transfected
with mutant C31 was exposed to heat ramps from 24 to 47°C in
normal extracellular solution (left), 0.1 µM capsaicin (middle), and 1 µM capsaicin (right). The cells clamped at
70 mV were superfused for the time indicated by the open
bars shown above the records. Dashed lines
indicate a zero membrane current. C, The
currents were normalized to the current produced by the application of
1 µM capsaicin at 30°C and the mutants C31, C42,
and C72 were tested at agonist concentrations of 0.1, 0.3, and 1 µM. At 30°C, capsaicin dose-response curves for
wild-type receptor exhibited the average half-maximal concentration
0.55 µM and Hill slope 2.4 (n = 5). The Hill equation was used for fitting control wild-type
data (solid line). Before averaging
across cells, data from each recording were normalized to the
current induced by 1 µM capsaicin measured at
30°C. Data for each construct are shown as the means ± SEM for
four independent measurements. D, At 45°C, capsaicin
dose-response curves for wild-type receptor exhibited the average
half-maximal concentration 0.24 µM and Hill slope 2.6 (n = 5). Data from each recording were normalized
to the current induced by 1 µM capsaicin measured at
30°C. C-terminal truncation had the most pronounced effects on both
agonist efficacy and agonist potency in mutant C31.
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C-terminal deletion mutants exhibit a lower temperature threshold
for IHEAT
The thermal sensitivity of TRPV1 mutants was examined using
standard ramps of increasing temperature from 25 to 47°C
(8.5°C/sec). As expected, wild-type receptors responded vigorously to
the heat stimulation within the noxious temperature range (43-47°C)
(Fig. 4A). At a
membrane potential of 70 mV, the mean thermal threshold of the
heat-induced membrane currents (IHEAT)
was 41.5 ± 0.4°C (n = 25), which is in good
agreement with the values measured by other authors (Caterina et al.,
1997; Tominaga et al., 1998). In the wild-type TRPV1, the average peak
amplitude of the noxious heat-induced response was 5.7 ± 0.6 nA
at 47°C (n = 47), and the median value of the
temperature coefficient (Q10) was 25.6 (range, 6.3-143 between 44 and 47°C, n = 26).
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Figure 4.
Sensitivity to heat in C-terminally truncated
mutants. A, Representative heat-induced responses
recorded from HEK293T cells transiently transfected with wild-type
TRPV1, C31, C42, C72, C78, and C104 mutants. Heat-evoked
currents were induced by a 3 sec temperature ramp (24-49°C) in
control extracellular solution. Dashed lines indicate a
zero membrane current. HP, Holding potential.
B, Temperature response profiles of the heat-induced
currents shown in A. The threshold for
IHEAT was 43.8, 38.2, and 28.9°C for
wild-type, C31, and C42, respectively (left). In
C104 mutant, a pronounced resting membrane current was observed at
room temperature (right). C, Arrhenius
plot in which the current (y-axis, log
scale) was plotted against the reciprocal of the absolute
temperature (x-axis). The temperature coefficient,
Q10, was estimated for each mutant by
linear regression from the slope of the Arrhenius plot in the
temperature range indicated by respective symbols.
Q10 was 104.1, 22.1, 20.5, 5.9, 1.8, and 1.2 for wild-type TRPV1, C31, C42, C72, C78, and C104
mutants, respectively. D, Thermal threshold values
plotted versus the number of residues deleted from the C-terminal tail
of TRPV1 receptor. Sequential deletions of 31, 42, and 72 residues from
the C-terminal end resulted in a significant shift of the thermal
threshold to 38.6 ± 0.7°C (SEM; n = 22),
32.5 ± 0.5°C (n = 21), and 28.6 ± 0.5 (n = 50), respectively. Deletions of 78 and 104 residues from the C-terminal end caused a shift of the TRPV1 thermal
threshold toward temperatures close to that in the bath.
E, Q10 distribution in
wild-type exhibits a median value of 25.6 (n = 26);
the histogram shows that this distribution is similar to the
distribution of Q10 in the C31 mutant
(also see Table 1). F, The median
Q10 plotted versus the number of residues
deleted from the C-terminal tail of TRPV1 receptor. The
ends of the boxes define the 25th and
75th percentiles, with a line at the median; error bars
define the 10th and 90th percentiles. The asterisk indicates
statistically significant difference from the wild type
(p < 0.05).
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In the mutants C31 and C42, the magnitude of
IHEAT at 47°C was not significantly
different from that of the wild-type TRPV1. In contrast, sequential
deletions of 72, 78, and 104 residues from the C-terminal end reduced
the amplitudes of the heat-induced current markedly (Fig.
2B). Sequential deletions of 31, 42, and 72 residues
from the C-terminal end also shifted significantly the thermal
threshold to 38.6 ± 0.7°C (n = 22), 32.5 ± 0.5°C (n = 21), and 28.6 ± 0.5 (n = 50), respectively. Deletions of 78 and 104 residues from the C-terminal end shifted the thermal threshold toward
temperatures close to that in the bath (Fig.
4A,B).
To determine the extent to which the C-terminal tail participates in
the regulation of TRPV1 channel activity induced by heat stimulation,
the temperature coefficient Q10 was
estimated for each mutant in the respective temperature range, in which
the Arrhenius plot was linear (correlation coefficient, >0.99) (Table 1). Interestingly, the distribution of
Q10 in the truncated mutant channel
lacking the final 31 aa from the C terminal was similar to the wild
type (Fig. 4E); however, the activation threshold was
decreased significantly (Fig. 4B,D). This suggests
that the distalmost C-tail region does not directly influence a high
free-energy barrier between the resting and activated states of the
TRPV1 channel; however, it shifts the thermal threshold for channel activation. The temperature coefficients were markedly reduced in all
of the other mutants examined (Fig. 4F, Table 1). In
both the wild-type and the truncated mutants, repeated applications of
capsaicin or low pH were characterized by a substantial desensitization (tachyphylaxis); in contrast, in the case of repeated heat stimuli to
47°C, the responses were reproducible for at least the first three exposures.
In contrast to the wild-type TRPV1, the mutants C42, C72, C78,
and C104 frequently became saturated above ~39°C. The large resting membrane currents (521 ± 76 pA at 25°C;
n = 16) were observed at 70 mV in all cells
expressing the C104 mutant channel from the start of the whole-cell
recording. This was in contrast to the wild type and other constructs
with less extensive deletions of the residues at the C end, which
exhibited only negligible resting currents at 70 mV. Because HEK293T
cells were routinely maintained at 37°C, the increased leak current
might reflect a constitutive activation of mutant channels by the
ambient temperature. However, two arguments suggest that it was
unlikely that the decreased maximum responses reflected a low
expression density: (1) We did not observe a reduced viability of the
HEK239T cells expressing truncated mutants. (2) The maximum amplitudes
induced by depolarizing voltage steps to +80 were lower by only 30% in
the 72 mutant (which was still not significantly different from wild
type), thus suggesting that the activation capacity of channels (or the number of functional channels on the cell membrane) was only slightly reduced. It was difficult to distinguish a specific heat response from
the nonspecific effects of the temperature on the leakage current in
the mutant C104, because Q10
estimated over the entire temperature range was close to ~1.5.
However, after leak subtraction, the amplitudes of the heat-induced
currents in this construct were still significantly larger than
maximum amplitudes recorded at 47°C in mock-transfected HEK293T
cells held at 70 mV (370 ± 59 pA; n = 13 vs 122 ± 32 pA; n = 12).
Effects of the protein kinase C activator PMA on a mutant receptor
C42 that lacks a Ser residue at position 800
It has been reported that the Ser residues S502 and S800 function
as substrates for PKC-dependent phosphorylation of the TRPV1 receptor
(Numazaki et al., 2002). To explore the role of the C terminal (S800)
in the process of direct phosphorylation, we examined the effects of 1 µM PMA, a specific membrane-permeable PKC activator, on
the membrane currents evoked by heat in the wild-type TRPV1, the C31
mutant, and the construct lacking the Ser residue at position 800, C42 (Fig. 5A-C). In the
C31 mutant, PMA potentiated the heat-evoked currents by a factor of
~1.5 ± 0.2 at 47°C (n = 9). The potentiating
effect was smaller, although still not significantly different from
that of the wild-type TRPV1 (2.0 ± 0.3; n = 7).
In contrast, PMA-mediated potentiation in the C42 mutant was
significantly smaller (1.2 ± 0.1; n = 7) compared
with the wild-type TRPV1. These findings support the study by Numazaki et al. (2002), indicating that Ser residue S800 participates in the
PKC-dependent sensitization of heat-evoked currents in TRPV1.
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Figure 5.
Effects of PMA on heat-evoked current in C31
mutant and the construct lacking the Ser residue at position 800, C42. A, Heat-evoked currents were induced by ramps of
increasing temperature from 25 to 47°C (8°C/sec) in control
extracellular solution (Control) and in the
presence of 1 µM PMA followed by a wash-off with control
(Wash). The cells clamped at 70 mV were superfused for
the time indicated by the open bars shown above the
records. Dashed lines indicate a zero membrane
current. B, Membrane currents recorded from
TRPV1-C42-transfected HEK293T cells. C, PMA-induced
potentiation of the heat-evoked currents at 47°C in C42 compared
with the wild-type and C31 mutant (p < 0.05; indicated with an asterisk). Data represent
means ± SEM; the numbers above each
bar indicate the number of cells measured.
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Voltage-dependent activation of the wild-type and C-terminal
truncated TRPV1
At room temperature and at normal pH 7.3, the rat cloned TRPV1 is
activated by depolarizing voltages in the absence of any agonists
(Reeve and Bevan, 2002; Vlachová et al., 2002). To determine the
role of the C terminal in voltage-dependent TRPV1 activation, the
current-voltage relationships of HEK293T cells transfected with either
wild-type or C-terminal truncated TRPV1 were examined (Fig.
6A). In the wild-type
TRPV1 (n = 36), depolarizing voltage steps elicited
large outward membrane currents with a maximum amplitude of 1.8 ± 0.1 nA at +80 mV. The interpolated reversal potential was +6.1 ± 0.7 mV, and the current-voltage relationships showed clear outward
rectification. The mean RI (expressed as the ratio of current induced
at +60 and 40 mV; see Materials and Methods) was 9.2 ± 1. A
region of negative slope conductance was recognized at potentials more
negative than 50 mV in the cells with a very small resting current at
a holding potential of more than 80 pA at 70 mV. Untransfected or
mock-transfected HEK293T cells exhibited only negligible
voltage-induced currents, which were linearly related to membrane
potential over 25-48°C (from 50 to +50 pA; n = 12)
(Fig. 6B).
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Figure 6.
Voltage-activated currents in wild-type TRPV1 and
C-terminal truncated mutants. A, Representative
whole-cell membrane currents induced by a sequentially applied series
of 50 msec voltage steps ranging from 140 to +80 mV, in +20 mV
increments. The currents were measured at the end of each pulse
(arrow). B, The voltage protocol was
completely ineffective in a mock-transfected HEK293T cell.
C, Superimposed example responses induced by a
depolarizing step from 80 to +80 mV in wild-type and the C72 and
C78 mutants. D, The ratio between the outward current
at +80 mV and the tail current amplitude in C31 and C42
is not statistically different from wild-type TRPV1 (Spearman
correlation; r = 0.98 and 0.95 vs 0.96 for
wild-type); on the contrary, it is markedly lower in C72 and C78
mutants (0.86 and 0.68, respectively). E, Kinetic
analysis of the outward currents recorded from 36, 24, 12, 17, and 14 cells transfected with wild-type and C31, C42, C72, and C78
mutants, respectively. The onset currents induced by the depolarizing
step from 80 to +80 mV were best fitted by the sum of two exponential
functions with the slow component
[ on(slow);
open symbols], and the fast component
[on(fast);
filled symbols]. Averages represent means ± SEM.
Linear regression analysis of the data yielded slopes of 0.0031 (r = 0.94) and 0.0033 (r = 0.93) for the slow and the fast components, respectively.
F, The tail currents were best fitted by the sum of two
exponential functions: f (t) = Afast
exp[t/tail (fast)] + Aslow
exp[t/tail (slow)]. The regression
lines (dashed) were shallow, possessing slopes of 0.0011 (r = 0.27) and 0.0011 (r = 0.33) for the slow and the fast components, respectively.
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The activation kinetics of wild-type TRPV1-mediated currents
elicited by a depolarizing step from 80 to +80 mV was best described by the sum of two exponential functions in 31 of 36 cells
(slow = 19.5 ± 0.9 msec, 70.0 ± 2.1%; fast = 2.8 ± 0.1 msec;
n = 32). In five cells, the activation process was best
fitted by one exponential function (9.0 ± 1.1 msec;
n = 5). Repolarization to the holding membrane
potential (70 mV) produced pronounced inward "tail currents." Detailed analysis of the TRPV1-mediated deactivation kinetics revealed
a fast (2.2 ± 0.1 msec) and a slow (11.9 ± 0.97 msec) time
constant, with the relative contribution of 66.9 ± 2.7% of the
fast deactivating component.
A comparison of the outwardly rectifying currents induced by
depolarizing voltage steps to +80 mV in the wild-type and in the
C-terminal truncated TRPV1 constructs shows that there was a decrease
in the outward current and a significant slowing of both the fast and
the slow onset kinetic constants (Fig, 6A,C,E,F, Table 2) as the number of C-terminal
amino acid residues deleted was increased. Mutations TRPV1-C31 and
TRPV1-C42 were less affected, whereas in mutants C72 and C78,
the differences in voltage-activated currents were pronounced. The
latter two mutants displayed an instantaneous component of the outward
current at the onset and a significantly smaller tail current when
normalized to the maximum outward current at +80 mV (Fig.
6C,D). To quantify this observation, we analyzed the tail
current amplitude in relation to the maximum current at +80 mV. The
amplitudes of the tail currents that were markedly lower in the C72
and C78 mutants were markedly reduced, suggesting that once
activated, the mutant channels entered an inactive state more readily.
These findings, together with the data showing that the mean time
constants (and the proportions) of the tail currents were not
significantly changed (Fig. 6E,F, Table 2), indicate
that shortening of the TRPV1 C terminal decreased significantly the
fraction of channels that retain their activity after
repolarization.
A comparison of the current-voltage relationships of the mutants
indicates that the current amplitudes at +80 mV were reduced with
shortening of the TRPV1 C terminal (Fig. 6D),
suggesting that the proximal regions of the C-terminal tail were
required for efficient voltage gating of TRPV1. The mutant
TRPV1-C104 showed no sign of outward rectifying currents, and the
leakage current (see above) was linearly related to membrane potential (data not shown).
To study the voltage-dependent gating of TRPV1 truncated mutants in
more detail, the RIs of the I-V relationships for mutant constructs were compared (Fig.
7A). The RI of the C31
mutant was significantly higher than that of the wild-type receptor
(Fig. 7B) (median 9.08 vs 6.42 in wild-type). In contrast,
voltage-dependent rectification in the C72 and C78 mutants was
significantly reduced (medians, 3.97 and 2.85).
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Figure 7.
Outward currents in HEK293T cells transiently
transfected with wild-type TRPV1 and C-terminal truncated mutants.
A, Current-voltage relationship for wild-type and
C31, C42, C72, and C78 mutants. Data were normalized to the
value of the current at 40 mV. B, For C31, RI was
significantly higher than that for wild-type, whereas significantly
less RI was observed for C72 and C78 mutants (Table 2). The
ends of the boxes define the 25th and
75th percentiles, with a line at the median; error bars
define the 10th and 90th percentiles. The asterisks indicate
significant differences from wild type (p < 0.05).
C, The effect of temperature on the outward current
rectification. Representative voltage-gated responses, recorded at
24°C (inset, bottom record) and at
42°C (inset, top record). Dashed
lines indicate a zero membrane current in all records in this
figure. Data were normalized to the value of the current measured at
+60 mV and at 42°C. D, Effect of temperature on
outward current rectification properties in C72 mutant.
Representative recordings of voltage-evoked currents at 24, 30, and
40°C (inset). E, Note a considerable
steady-state inward current induced at 31°C, a temperature at which
the mutant C78 is thermally activated.
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Because the threshold of heat-evoked currents was close to room
temperature in C72 and C78, we explored the effects of higher temperatures on the rectification properties of these constructs (Fig.
7C). In the wild-type channel, increasing the temperature from 24 to 42°C shifted the inflection point of the current-voltage curve to more negative values (from approximately 40 to approximately 80 mV) and decreased the RI from 5.4 ± 0.5 to 1.34 ± 0.05 (n = 4) (Fig. 7C). In contrast to the
wild-type, the mean RI in C78 was not significantly affected by
temperature (3.31 ± 0.47, 2.37 ± 0.14, and 2.50 ± 0.21 at 24, 30, and 40°C, respectively; n = 4;
p = 0.10 and p = 0.29; Student's
paired t test). These data also confirm that the
permeability of the TRPV1 channel is affected by the C-terminal tail.
Immunostaining of HEK293T cells transiently transfected with
C-terminal truncated TRPV1 construct C155
Truncation of the entire TRPV1 C-terminal domain was found
to produce a nonfunctional channel. HEK293T cells expressing the C-terminal truncated mutant C155 did not respond to either capsaicin or heat (data not shown). Exposure of TRPV1-C155 expressing cells to pH 5 produced only transient currents, which inactivated within ~2
sec, indicating the presence of acid-sensing ion channels expressed constitutively in HEK293 cells (Gunthorpe et al., 2001). To explore whether the fully truncated mutant can insert into the cell membrane, we used a polyclonal antibody against the first 21 aa residues of the
rat TRPV1 N-terminal end and confocal laser scanning microscopy to
compare the surface expression of wild type and the C155 mutant in
transiently transfected HEK293T cells. Nonspecific binding of the
primary antibody was determined by using nontransfected HEK293T cells.
The nonspecific binding of the secondary antibody was determined in
each experiment in the absence of primary antibody (data not shown).
There were no significant differences between the distribution patterns
of anti-TRPV1 immunoreactivity in the wild type and the C155 mutant
(Fig. 8). This assay indicated that the
population of truncated construct C155 was processed and not
retained in the intracellular compartments, and that the C terminal was
not essential for trafficking the receptor to the HEK293T cell
surface.
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Figure 8.
Surface expression of C-terminally truncated TRPV1
construct in transiently transfected HEK293T cells. A,
Confocal microscope image of wild-type transfected HEK293T cells.
Permeabilized cells were immunohistochemically labeled using a
polyclonal antibody against the first 21 aa residues of the rat TRPV1
N-terminal end. Cells were labeled with FITC-conjugated donkey
anti-rabbit IgG secondary antibody and visualized with standard FITC
filters. B, Four HEK293T cells transiently transfected
with truncated construct lacking the entire C-terminal end C155.
Scale bar, 5 µm.
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Predicting the secondary and tertiary structures of the C terminal
of TRPV1
To explain and predict the involvement of the C-terminal domain in
TRPV1 channel function, the sequence of the TRPV1 C terminal from A690
to K838 was used for homology modeling. This section of the TRPV1
receptor shows a high sequence homology (44%) to the FHIT protein,
whose tertiary structure has been solved at 1.85 Å resolution (Lima et
al., 1997). The overall predicted structure of the TRPV1 C tail can be
described as a general  + -type and can be further subclassed as an
+-meander fold. The C tail contains two helices, H1 and H2, and
seven -strands (Fig. 9B).
Strands 3-7 form a five-stranded antiparallel sheet. The antiparallel strands 1 and 2 form a -hairpin across from and at an angle to the
other sheet. Helix H1 packs on one side of the five-stranded antiparallel -sheet, and helix H2 packs on the same side and primarily interacts with strand 3 and the loop connecting strands 2 and
3. The template structure shows a disordered gap from residue 107 to
residue 127. Therefore, the structure of the large loop between
-strand 7 and the second helix was generated only from a loop
database and is not based on homology. Probably this loop is highly
flexible in reality; thus, the structure shown must be taken as only
one speculative possibility for its conformation. The model of the C
tail shows a high-quality stereochemistry, revealed by the Ramachandran
plot calculated with Procheck (Laskowski et al., 1993). The values of
the torsion angles of 88.6% of the residues were within the most
favored regions, and 10.6% of the residue values were within
additionally allowed regions of the Ramachandran plot. Only one residue
was found to be in a generously allowed region; no residues were found
in disallowed regions. The overall g-factor of the obtained
structures showed a value of 0.16, suggesting that the model
presented here is of general validity. (Ideally, the value of the
g-factor should be above 0.5; values below 1.0 may need
investigation.) The three-dimensional representations of the C-terminal
structure of truncated constructs C31, C72, and C104 are
demonstrated in the Figure 9B.
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Figure 9.
Molecular modeling of the TRPV1 C terminal.
A, Sequence alignment of the cytoplasmic C terminal of
TRPV1 from A690 to K838 and the template protein FHIT. Identical and
similar amino acids of the stronger groups are indicated with an
asterisk and colon, respectively. These
amino acids should conserve the structure with a probability of >95%.
Dots indicate similar amino acids of the lower groups
that should conserve the structure with a lower probability.
B, Predicted structure of the complete C terminal of
TRPV1 and the truncated mutants. WT, Ribbon diagram of
the wild-type C terminus (residues A690-K838). Homology modeling
predicts two -helices (H1, H2) and seven -strands
(1-7). Antiparallel strands 1 and 2 form a
-hairpin, and strands 3-7 form a five-stranded antiparallel sheet.
C31, This mutant (residues A690-T807) lacks the
-helix H2. C72, In this construct (A690-C766),
secondary structural elements H2 and -strands 6 and 7 are missing.
C104, The predicted structure of the truncated
construct (A690-G734) consists only of -strands 1-5.
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Discussion |
Our results demonstrate that the intracellular C-terminal tail of
the vanilloid receptor TRPV1 in HEK293T cells is required for
functional channel activation. Sequential deletions of the residues
from the C terminal decrease channel sensitivity to capsaicin, low pH,
and depolarizing voltage stimuli. However, the most striking effect of
shortening the C terminal of TRPV1 was the altered sensitivity to heat.
The threshold of heat-evoked currents was progressively shifted to
lower temperatures with the decreasing length of the C-terminal tail.
Deletions of 31 and 42 aa residues from the C terminal decreased the
temperature threshold for channel activation by 3 and 9°C, with
little or no effect on capsaicin or proton responsiveness.
A general role for TRP channels in thermosensation has been supported
by recent studies, which demonstrated that TRPV3 (Peier et al., 2002;
Smith et al., 2002; Xu et al., 2002) and TRPV4 (Guler et al., 2002) are
activated by warm temperatures. Our results indicate that the
biophysical basis of thermosensation depends on the flexibility of the
C-terminal peptide chain and the presence of residues that regulate
the thermostability of the TRPV1 channel complex. The data provide
evidence that the cytoplasmic C-terminal region is involved in
conformational changes leading to channel activation. In the wild-type
TRPV1 and the C31 mutant, the strong temperature dependence
(Q10 = 26 and 22) suggests a very
complex structural rearrangement during exposure to noxious heat; in
contrast, the mild effects on gating in C78 and C104 truncation
mutants (Q10, ~1.7) are
compatible with a very simple conformational change.
TRPV1 is a nonselective cation channel that can be activated by a
number of chemical agents of chemical or physical nature known to
produce pain in humans (Caterina et al., 1997; Tominaga et al., 1998).
TRPV1 most likely assembles into a homotetrameric complex with a
centrally located channel pore (Jahnel et al., 2001; Kedei et al.,
2001). It is evident that there are several distinct domains on this
receptor that can open the channel. The protein domains responsible for
gating of the channel by protons can be modulated independently from
the domain(s) involved in the gating by capsaicin or by heat (Welch et
al., 2000). Two negatively charged glutamic acid residues, E600 and
E648, located within the extracellular loop linking the
transmembrane-spanning domain S5 with S6, appear to play a key role in
sensing increased proton concentration (Jordt et al., 2000). The
vanilloid binding site has been suggested to be located on the
intracellular side of the membrane (Jung et al., 1999; Jordt and
Julius, 2002).
Heterologously expressed TRPV1 can be activated by large depolarizing
steps in the absence of exogenous agonists (Reeve and Bevan, 2002;
Vlachová et al., 2002). This suggests that TRPV1 contains a
transmembrane domain that senses membrane potential, and that positive
potentials induce conformational changes of the protein leading to the
channel opening. The existence of a domain sensing the depolarization
of the membrane explains pronounced outward rectification of
capsaicin-induced membrane currents that has been observed in native
vanilloid receptor (Piper et al., 1999) and analyzed in great detail in
the recombinant receptor (Gunthorpe et al., 2000). Our data demonstrate
that the TRPV1 C terminal influences both permeation and gating (i.e.,
degree of rectification and deactivation kinetics). Therefore, it is possible that the functional effects of deleting the distal half of the
C terminal suggest its close association with the putative voltage-sensing module, whose movement is coupled to the opening of the
channel. An alternative explanation is that the conformational stability of the entire tetrameric protein complex may be altered in
C-terminally truncated mutants, which display a significant decrease in
voltage-gated outward currents.
The C-terminal region of TRPV1 participates in PKC-dependent
phosphorylation, probably at S800. In agreement with the study by
Numazaki et al. (2002), sequential deletions showed that the heat-evoked currents in the construct lacking the Ser residue at
position 800, C42, are potentiated by a specific PKC activator, PMA,
but to a lesser extent than C31. This result suggests that the
effects of PMA are likely to be influenced by the incomplete consensus
site for PKC-dependent phosphorylation, and also that another region of
TRPV1 has to be involved in PMA-induced sensitization.
Deletion of the entire TRPV1 C terminal was found to cause loss of
channel sensitivity to all chemical, thermal, and voltage stimuli.
Although the fully truncated mutant likely inserts into the cell
membrane, as demonstrated by immunostaining with anti-N-terminal polyclonal antibody, it is not clear whether the mutant receptor is
expressed as a functional channel in HEK293T cells. The TRPV1 5' splice
variant is insensitive to noxious chemical stimuli (vanilloids, low pH)
and thermal stimuli (Schumacher et al., 2000b). Also, truncation of the
entire TRPV1 C-terminal domain (155 residues) results in a loss of
channel sensitivity to all chemical (capsaicin and pH), thermal, and
voltage stimuli. Therefore, the loss of TRPV1 sensitivity cannot be
unambiguously interpreted as a loss of a specific site(s), because it
could equally be caused by a large structural disturbance. Both the
reduced sensitivity and the loss of function might be associated with
impaired intrasubunit or intersubunit interactions among terminal
domains during (or leading to) channel opening. For example, a close
association between the N-terminal (tetramerization domain T1) and the
C terminal has been described for the structurally related Shaker
K+ channel (Schulteis et al., 1996).
The cytoplasmic C terminal has been reported to be critical for
expression and/or gating of various channels, including the subunit
of the human cardiac voltage-gated sodium channel SCN5A (Cormier et
al., 2002), the two-pore domain mechano-gated
K+ channel TREK-1 (Maingret et al., 2000),
the bacterial Streptomyces lividans
K+ channel KcsA (Perozo et al., 1999), the
voltage-gated K+ channel human
ether-à-go-go-related gene cardiac K+ channel (Aydar and
Palmer, 2001), the Kv2.1 potassium channel with delayed rectifier
properties (Bentley et al., 1999), the Drosophila Shaker
K+ channel (Schulteis et al., 1996), and
the small conductance Ca2+-activated
K+ channel (Bruening-Wright et al.,
2002). It is also important for the cyclic nucleotide-gated
channel, in which direct binding of cyclic nucleotides in
the C-terminal region is transduced to open a gate that presumably
resides in the channel selectivity filter (Flynn and Zagotta, 2001).
Importantly, these latter five tetrameric channels belong to a
superfamily of structurally related six-transmembrane domain proteins
that also includes the TRP channel family; this suggests the possible
existence of physical and structural analogies between the gating mechanisms.
In the molecular model based on homology with the fragile histidine
triad protein presented here, the distalmost 31 aa residues of the
TRPV1 C terminal (Q808-K838) correspond to the -helical structure
H2 and the large flexible loop connecting it with -sheet 7 (Fig.
9B). Removal of this region is sufficient to shift the thermal threshold for activation from 42 to 39°C. This structural part seems also to modulate the sensitivity to capsaicin as the mutant
C31 exhibits increased agonist efficacy (Fig. 3). Deletion of the
remaining short part of the connecting loop (R797-K838) markedly
decreased the thermal threshold (to 33°C) for receptor activation.
The large loop seems to be anchored between helix H2 and -strand 7 to form itself a highly flexible structure that regulates steric
accessibility to the core -sheet. The effects of the deletion of the
remaining 11 loop amino acids in the mutant C42 suggest the
importance of -sheets 6 and 7 in channel activation. This view is
also supported by construct C72, which lacks -strands 6 and 7 (E767-K838) and displays profound changes in channel function. The
thermal threshold dropped from 41.5 to 28.6°C,
Q10 decreased from 25.6 to 4.7, and
the currents induced by capsaicin, pH 5, heat, and voltage decreased
significantly, suggesting a distinct role of these two -strands in
the C terminal of TRPV1. In the model of the mutant C104, the helix
H1 is lost. This helix is a relatively long helical structure (18 residues) on the surface of the protein. Accessibility for
multimerization in its entire length and its distribution of
hydrophobic residues makes it a good candidate for taking part in a
multimerization module. This could either be a coiled coil controlling
the tetrameric organization of the channel or an interaction with
another receptor region (e.g., of the N terminal). The -hairpin
formed by the first two antiparallel strands does not seem to exhibit
any functional role; however, it could stabilize the proper position of
the C tail toward the membrane.
In conclusion, our results provide evidence that the structural basis
of the thermal sensitivity of the TRPV1 channel resides in the distal
half of the C terminal, and that this terminal region contributes to
the regulation of chemically induced, thermally induced, and
voltage-induced activity of the TRPV1 channel.
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FOOTNOTES |
Received July 23, 2002; revised Nov. 25, 2002; accepted Dec. 4, 2002.
This work was supported by Grants 305/00/1639 and 305/03/0802 from the
Grant Agency of the Czech Republic, by Research Project 5011922 of the
Academy of Sciences of the Czech Republic, and by Grants LN00B122,
MSM 113100001, MSM 12310001, and MSM 113100003 of the Ministry of
Education, Youth, and Sports of the Czech Republic. We thank Charles
Edwards for a critical reading of this manuscript.
Correspondence should be addressed to Dr. Vlachová Viktorie,
Institute of Physiology, Academy of Sciences of the Czech
Republic, Vídenská 1083, 142 20 Prague 4, Czech
Republic. E-mail: vlachova{at}biomed.cas.cz.
| |
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TOP
ABSTRACT
Introduction
