 |
INTRODUCTION |
Mammalian isoforms of the classical transient receptor potential
channel (TRPC)1 subfamily,
TRPC1-7, are likely candidates for cation channels mediating
phospholipase C-dependent, receptor-operated
Ca2+ influx (for reviews, see Refs. 1-5). The properties
and activation mechanisms of TRPCs have been studied extensively in
heterologous expression systems. By contrast, relatively little
information is available on their role in native cells. Two recent
studies (6, 7) report that endogenous receptor-stimulated cation currents in vascular smooth muscle cells show properties identical to
those described for heterologously expressed TRPC6. Therefore, precise
knowledge of the properties of heterologously expressed TRPC channels
may be essential to evaluate the involvement of TRPC proteins in
receptor-operated cation conductances in native cells.
Structurally, TRP channels, like many other cation channels, have been
proposed to have six transmembrane segments (S1-S6), intracellular N
and C termini, and a pore-forming reentrant loop between S5 and
S6. Based on amino acid sequence similarity, the mammalian
members of the TRPC subfamily can be subdivided into four groups (4,
8): TRPC1 (group 1), TRPC2 (group 2), TRPC3/6/7 (group 3), and TRPC4/5
(group 4). This subdivision is also supported by functional data. One
of the major functional criteria is the mechanism of channel
activation. From studies in heterologous expression systems, it is
undisputed that receptor-mediated stimulation of phospholipase C is a
key event in the activation of all TRPC isoforms. Evidence indicates
that currents mediated by TRPC3, TRPC6, or TRPC7 can be activated by
diacylglycerol (DAG) independently of protein kinase C (9-12),
although there is some controversy regarding the
physiological significance of this stimulation (13). By contrast, TRPC4
and TRPC5 are not activated by DAG, and some evidence indicates that
unidentified components of the phospholipase C pathway other than DAG
or store depletion activate the channels (14-16). Other evidence
supports a store-dependent activation mechanism (17-20).
TRPC1 has been reported to be a store-dependent channel in
some studies (21-23), but doubts have been raised as to whether TRPC1
expression results in the formation of functional plasma membrane
channels in mammalian cells (16, 24, 25). The few data available
support a store-operated activation mechanism for TRPC2 (26, 27), but,
here again, other evidence suggests that TRPC2 does not form functional
channels in all tissues (28). Data from our group, confirmed in several
independent laboratories, indicate that several biophysical features
are also characteristic of certain groups of TRPCs. Thus, TRPC3-7 have
a characteristic doubly rectifying, or S-shaped current-voltage
relation (9, 14-16, 29-31). Furthermore, at the single channel level,
even though the amplitudes of single channel events are similar, the
openings of TRPC3 and TRPC6 are very brief (9, 32-35) compared with
those of TRPC4 and TRPC5 (15, 16, 31, 36).
In the absence of more specific pharmacological tools, the lanthanides
lanthanum (La3+) and gadolinium (Gd3+) are
commonly used blockers of nonselective cation channels and other
Ca2+-permeable channels. Interestingly, recent studies have
reported that 100 µM La3+ has potentiating
effects on mouse, rat, and human TRPC4 and mouse TRPC5 (15, 16, 31).
However, the actions of La3+ on TRPC4 and TRPC5 have not
been characterized further. By contrast, other TRPCs are inhibited by
micromolar concentrations of La3+ or Gd3+ (6,
29, 37-40).
Because the different effects of lanthanides are potentially an
important distinguishing feature of the group 4 TRPC channels, we
characterized the effect of these ions on TRPC5 in detail and compared
them with those on TRPC6, a member of group 3. For this study we chose
the rat TRPC6B slice variant (12), which lacks 54 amino acids at the
distal N terminus compared with rat TRPC6A, and has not previously been
characterized electrophysiologically. Unlike TRPC6A, TRPC6B has been
reported to be activated by agonist application but not by
1-oleoyl-2-acetyl-sn-glycerol (OAG) (12). In whole cell
patch clamp recordings, we found that TRPC5 was bimodally modulated by
lanthanides, with potentiation at micromolar concentrations being
succeeded by inhibition at millimolar concentrations. In contrast,
TRPC6 was inhibited by micromolar concentrations and showed no
potentiation. At the single channel level, the effects of
La3+ on TRPC5 are complex, affecting the single channel
conductance, the mean open time, and the frequency of channel openings.
By site-specific neutralization of extracellular negatively charged amino acids, we have identified two sites, close to the pore mouth, that are involved in potentiation of TRPC5 by La3+.
 |
EXPERIMENTAL PROCEDURES |
Molecular Biology and Stable Transfection--
The isolation of
TRPC5 from mouse brain total RNA has been described previously (15).
For cloning of TRPC6, total RNA was prepared from rat brain or A7r5
smooth muscle cells using a TriZol reagent (Invitrogen) according to
the standard protocol. For cDNA synthesis, 1 µg of total RNA was
reverse transcribed according to the protocol provided by the
manufacturer using 200 units of Superscript II reverse
transcriptase (Invitrogen) and 5 pmol of the primer
5'-CCAGTGAGCAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT. TRPC6 was
amplified by 30 cycles of PCR using an annealing temperature 57 °C
for 15 s then extension for 210 s at 72 °C with Expand-HF polymerase (Roche Molecular Biochemicals). The primer set used for
amplification was 5'-CCGGTACCGCCCTTATGAGCCGGGGTAATGAAAACAGAC (sense)
and 5'-CCGGATCCCTATCTGCGGCTTTCCTCTTGTTT (antisense). The PCR
products were subcloned into the pCR2.1 vector (Invitrogen) and the
sequences confirmed by DNA sequencing of both strands (ABI-Prism,
PerkinElmer Life Sciences). The rat TRPC6 characterized in this study
corresponds to the sequence of rTRPC6B published by Zhang and Saffen
(12) (GenBankTM accession number AB051213) with the exception of two
amino acid exchanges: M757I and S767F.
For the generation of a stably transfected TRPC6 cell line, the
KpnI/BamHI fragment of the cloned cDNA was
ligated into the tetracycline-inducible eukaryotic expression vector
pcDNA4/TO and transfected into T-REx-293 cells (both from
Invitrogen) using the transfection reagent FuGENE 6 (Roche Molecular
Biochemicals). Clonal selection was performed according to the
manufacturer's protocol with 250 µg/ml Zeocin (Invitrogen),
and 36 clones were functionally tested for TRPC6 expression with
electrophysiological methods (measuring the current response to
AlF
infusion). Two positive clones were selected and
used for further analysis.
Point mutations in TRPC5 C-terminally fused to yellow fluorescent
protein (YFP) were introduced using the QuikChange site-directed mutagenesis kit (Stratagene) and appropriate primer sets. Sequences of
the mutants were confirmed by DNA sequencing.
Cell Culture and Transient Transfection--
Human embryonic
kidney (HEK293) cells (ATCC, Manassas, VA) were maintained according to
the supplier's recommendations. For transient transfection, cells were
seeded in 35-mm culture dishes. The following day, 0.5-2 µg/dish of
pcDNA3 vector containing the cDNA for TRPC5, TRPC5-YFP, or point
mutants of TRPC5-YFP was mixed with 100 ng/dish of the rat histamine
H1 receptor (in pcDNA3) and, in the case of TRPC5, 50-100
ng/dish of pEGFP-C1 (Clontech), and transfected
into the cells using the transfection reagent FuGENE 6 (Roche Molecular
Biochemicals) according to the manufacturer's protocol. After 18-24
h, the cells were trypsinized and seeded onto glass coverslips.
T-REx cells and T-REx cells stably transfected with TRPC6 (T-REx-r6)
were cultured in Dulbecco's modified Eagle's medium (4.5 g/liter
glucose) supplemented with 10% (v/v) fetal bovine serum (Invitrogen),
4 mM L-glutamine (Fluka, Taufkirchen, Germany), 100 units/ml penicillin, and 100 µg/ml streptomycin (both from Biochrom, Berlin, Germany). For T-REx-r6 cells, 5 µg/ml
blasticidin (Invitrogen) and 250 µg/ml Zeocin were added to
the culture medium. For some experiments, cells were transiently
transfected with 100 ng/dish rat H1 receptor in pcDNA3
and 50 ng of pEGFP-C1 (Clontech) according to the
same protocol used for transient transfection of HEK293 cells.
For experiments on excised patches, coverslips were coated with
poly-L-lysine. All experiments were performed 2-3 days
after transient transfection and, in the case of T-REx-r6 cells, 1-2 days after induction with 1 µg/ml tetracycline (Roche Molecular Biochemicals).
Patch Clamp Recordings--
Whole cell and single channel
recordings were performed using an EPC-7 amplifier and Pulse software
(HEKA, Lambrecht, Germany). Patch pipettes were made from borosilicate
glass and had resistances of 3-6 megohms (whole cell recordings) or
6-16 megohms (single channel recordings) when filled with the standard
intracellular solutions.
Whole cell recordings were performed as described previously (15, 31).
To quantify current potentiation and current inhibition observed by
bath application of lanthanides, we interpolated currents before and
after application of lanthanides and normalized the values obtained in
the presence of the lanthanides to the interpolated values.
Interpolation was done to avoid errors arising from the fact that TRPC5
and TRPC6 currents decay with time. All current amplitudes were
calculated as the difference between resting and histamine-, OAG-, or
AlF
-induced current levels. The
concentration-response curve obtained for lanthanum inhibition of TRPC6
currents was fitted with Equation 1
|
(Eq. 1)
|
to determine the IC50 value. Values for the relative
Ca2+ permeability
(PCa/PNa) were calculated
from Equation 2,
|
(Eq. 2)
|
where VCa and VNa
are the reversal potentials in external solutions containing
Ca2+ and Na+, respectively, and R,
T, and F have their usual meanings.
For single channel recordings, the standard excised outside-out patch
configuration (41) was used. After filtering at 10 kHz, single channel
data were initially recorded onto digital audiotape (DAT, Biologic,
Claix, France). For offline analysis, the single channel data were
filtered at 1 kHz, subsequently digitized at 15 kHz, and analyzed with
the pClamp6 software (Axon Instruments, Foster City, CA). Single
channel amplitudes were obtained from events with open durations of
more than 2 ms. In the case of TRPC5, channel activity was expressed as
NPo, the product of the minimum number
(N) of channels in the patch (obtained from the observed number of open levels) and the open probability
(Po). NPo values were
calculated for consecutive 2-s periods. Openings with durations shorter
than 0.5 ms were excluded from the analysis. Because of extensive
overlap of individual unitary current responses after application of
La3+, we used an algorithm established by Fenwick et
al. (42) to obtain a reliable estimate of mean open times in the
absence and presence of La3+. The general applicability of
the algorithm has been confirmed (43). The overall estimate of mean
channel open time (to) was calculated according
to to = (
tj)/N, where N
is the number of all channel openings (transitions between a given
level j and a subsequent level j + 1),
tj designates the dwell time of a given level
j, and the sum extends over all levels encountered in the recording. Values for tj were extracted from
idealized traces generated with pClamp6.
The standard extracellular solution contained 140 mM NaCl,
5 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). For NMDG+
solutions, Na+ and Cs+ were replaced by
N-methyl-D-glucamine (NMDG), and
Ca2+ was omitted. In solutions with 20 mM
CaCl2, the NaCl or NMDG+ concentration was
reduced to 118 mM. The standard intracellular solution
contained 110 mM cesium methanesulfonate, 25 mM
CsCl, 2 mM MgCl2, 3.62 mM
CaCl2, 10 mM EGTA, and 30 mM HEPES
(pH 7.2 with CsOH) with a calculated [Ca2+] of 100 nM. In some experiments, a pipette solution with stronger Ca2+ buffering (30 mM BAPTA) with a calculated
[Ca2+] of 100 nM was used. It contained 50 mM cesium methanesulfonate, 25 mM CsCl, 2 mM MgCl2, 9.73 mM
CaCl2, 30 mM BAPTA, and 30 mM HEPES
(pH 7.2 with CsOH). For AlF
infusion, 1 µl of 0.5 M NaF was mixed with 0.5 µl of 3 mM
AlCl3 and diluted with 50 µl of the pipette solution. The
osmolarity of all solutions was between 290 and 310 mosmol/liter. All
experiments were performed at room temperature (20-25 °C).
Fluorometric Measurements and Confocal
Microscopy--
Fluorometric measurements of Mn2+ influx
and confocal microscopy were performed as described previously (15,
31).
Chemicals--
Histamine, OAG, lanthanum (La3+), and
gadolinium (Gd3+) were obtained from Sigma. Stock solutions
were made in water or dimethyl sulfoxide (OAG) and diluted to final
concentrations in the bath solutions.
Statistics--
All data are given as the means ± S.E. The
statistical significance of differences between mean values was
assessed using Student's t test. Differences were regarded
as statistically significant for p < 0.05 and as
highly statistically significant for p < 0.01.
 |
RESULTS |
Receptor-activated Currents through TRPC5 and TRPC6--
We
first performed whole cell voltage clamp recordings to compare currents
mediated by TRPC5 with those mediated by TRPC6. The latter isoform was
the rat TRPC6B splice variant, which has, to date, only been studied in
fluorometric experiments (12) and has not been characterized
electrophysiologically. As described previously (15), cells expressing
TRPC5 and the histamine H1 receptor displayed spontaneous
channel activity. By contrast, no constitutive activity was
observed at a holding potential of
60 mV in the T-REx-r6 cell line
24-48 h after induction with tetracycline. Currents on break-in were
in the range of
0.1 to
1.87 pA/picofarads (n = 24),
values not significantly different from those in control cells.
Characteristic currents were activated by the application of 100 µM histamine or infusion of AlF
, a
direct activator of G-proteins (44) in TRPC5- (Fig.
1A) or TRPC6-expressing cells
but not in control cells. Furthermore, TRPC6-mediated inward currents
could also be elicited by adding the membrane-permeable diacylglycerol
OAG (100 µM, n = 11) (Fig. 1B)
to the bath solution. This finding is in contrast to those of Zhang and
Saffen (12), who detected receptor- but not OAG-induced Ba2+ influx in COS cells expressing the species and splice
variant used here.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Opposite effects of bath application of
micromolar La3+ concentrations on currents mediated by
TRPC5 and TRPC6. A, TRPC5-mediated currents were rapidly and
reversibly potentiated by 10 µM La3+. Inward
currents at 60 mV were elicited by 100 µM histamine in
TRPC5-expressing HEK293 cells. Inset, whole cell currents at
100 and +100 mV obtained from voltage ramps. B,
TRPC6-mediated currents were reversibly inhibited by 100 µM La3+. Inward currents at 60 mV were
elicited by 100 µM OAG in TRPC6-expressing T-REx cells.
Inset, whole cell currents at 100 and +100 mV.
C and D, I-V relationships
in the presence and absence of La3+ recorded during the
experiments in A and B, respectively.
|
|
As reported previously for murine and human isoforms of TRPC5 and TRPC6
(9, 15), the I-V relation of currents mediated by both TRPC5
and TRPC6 displayed a characteristic doubly rectifying shape and
reversal potentials close to 0 mV, indicative of poor cation
selectivity (Fig. 1, C and D). Although the
I-V relations of currents mediated by TRPC5 and TRPC6 were
similar, currents in TRPC6-expressing cells display a slightly stronger
outward rectification.
Effects of Lanthanides on Currents Mediated by TRPC5 or
TRPC6--
We compared the effects of lanthanides on currents mediated
by TRPC5 and TRPC6. When 10 µM La3+ was
applied to HEK293 cells coexpressing mTRPC5 and the histamine H1 receptor during exposure to 100 µM
histamine, inward and outward currents were increased (Fig.
1A). Comparison of the I-V relations before and
after application of La3+ revealed that the potentiating
effect of La3+ was much more pronounced at negative
potentials (Fig. 1C). The effectiveness of very low
concentrations of La3+ and the absence of a shift in
reversal potential exclude the possibility that La3+ acts
as a charge carrier for the additional inward current. In control cells
transfected only with the histamine H1 receptor, neither
application of histamine nor subsequent addition of La3+
resulted in a current increase (n = 6). In these cells,
application of La3+ decreased basal leak currents rather
than causing potentiation. These data suggest that La3+
affects currents carried by TRPC5. By contrast, when 100 µM La3+ was applied to TRPC6-expressing
T-REx-r6 cells stimulated with 100 µM OAG, rapid
inhibition of the inward current was observed at a holding potential of
60 mV (Fig. 1B). Inhibition was almost complete at all
potentials tested (Fig. 1, B and D). For both TRPC5- and TRPC6-mediated currents, the effects of lanthanides were
readily reversible on wash-out (Fig. 1, A and
B).
The concentration dependence of the effects of lanthanides on TRPC5 has
not been studied previously. We therefore tested the effect of various
concentrations of La3+ or Gd3+ on
TRPC5-mediated currents and compared them with those on TRPC6 (Fig.
2, A and B).
Because agonist-induced currents in T-REx-r6 cells were relatively
short lived, infusion of AlF
via the pipette was
preferred. AlF
-induced currents in T-REx-r6 cells
were indistinguishable from agonist-induced currents with respect to
the I-V relation and their sensitivity to lanthanides.
However, the current response was slowed considerably, thus allowing
the effects of different concentrations of lanthanides to be
investigated in the same cell. The pooled concentration-response relationships for TRPC5- and TRPC6-mediated currents are shown in Fig.
2, C and D. Relative inhibition or potentiation
was calculated as described under "Experimental Procedures." The
inhibitory effect of lanthanides on TRPC6 began at concentrations
around 1 µM, and application of 1 mM resulted
in near complete inhibition. The concentration-inhibition relationship
for La3+ inhibition of TRPC6 was sigmoidal (Fig.
2D) with an IC50 value of 6.1 µM.
The effects of Gd3+ on currents mediated by TRPC6 were
examined at two concentrations, 10 µM and 1 mM. The values for relative inhibition by Gd3+
were similar to those obtained with La3+. As seen in Fig.
2, A and C, the effects of La3+ and
Gd3+ on currents mediated by TRPC5 were more complex.
Starting at a concentration of around 1 µM, both
La3+ and Gd3+ increased TRPC5-mediated currents
in a concentration-dependent manner. The largest
potentiation was observed between 10 µM and 1 mM and resulted, on average, in a 3-fold increase in
current. At millimolar concentrations, the potentiating effect was
reduced. In 5 mM La3+, the mean current was
less than the control value, indicative of inhibition. In contrast, in
5 mM Gd3+, the mean relative current amplitude
was ~120%. It has to be noted, however, that the effects of 5 mM Gd3+ on TRPC5-mediated currents were not
uniform. Some cells responded to this concentration with potentiation
of currents (n = 2/5), whereas others responded with
current inhibition (n = 3/5). Similarly, dual effects
of lanthanides were often observed during wash-in or wash-out of the
ions at a concentration of 5 mM. On wash-in, inhibition was
sometimes preceded by transient potentiation and on wash-out, removal
of inhibition followed by transient potentiation (see inset
in Fig. 2A). Thus, lanthanides inhibit TRPC6 but have dual
effects on TRPC5. Low concentrations potentiate TRPC5 currents, but
high concentrations are less effective and may result in
inhibition.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration dependence of lanthanide
effects. A, dual effects of lanthanides on currents through
TRPC5. Whole cell currents at 60 mV, elicited by the application of
100 µM histamine in TRPC5-expressing HEK293 cells, showed
potentiation by micromolar Gd3+ concentrations.
Inset, inhibitory effect of 5 mM
La3+ on TRPC5 currents at 60 mV. *, transient
potentiation on wash-in and wash-out of La3+. Scale
bar, 30 s and 50 pA. B, inhibition of TRPC6 by
increasing concentrations of La3+. Whole cell currents at
60 mV were elicited by infusion of AlF in
TRPC6-expressing T-REx-293 cells. C, concentration
dependence of lanthanide effects on TRPC5. Values are shown for
La3+ (filled symbols) and Gd3+
(open symbols). Relative amplitudes in the presence of
lanthanides were calculated with respect to current amplitudes in their
absence (obtained by interpolation and set to 100%). D,
concentration dependence of La3+ inhibition of rTRPC6
currents (filled symbols). Mean inhibition values for
Gd3+ are given by open symbols. The data for
La3+ inhibition were fitted with Equation 1 with an
IC50 of 6.1 µM and a cooperativity factor
x = 1.3.
|
|
Effect of Extracellular Ca2+ on Potentiation by
La3+--
To investigate whether physiological cations can
act at the same site as lanthanides, we tested the effect of
Ca2+. Raising [Ca2+]o
from 2 to 20 mM resulted in a rapid increase in TRPC5 channel currents similar to that produced by micromolar
La3+ concentrations (n = 5; data not
shown). This similarity includes a stronger potentiation of currents at
negative than at positive potentials. In addition to the rapid increase
in inward current, there was a slower increase in both inward and
outward currents which may reflect channel activation by an increase in
[Ca2+]i resulting from
Ca2+ entry through the channel (14, 15). The decay of
current upon returning [Ca2+]o to
2 mM was slow compared with that after removal of
La3+. In the presence of 20 mM
Ca2+, potentiation by 10 µM La3+
was prevented or strongly reduced (n = 5). These
results indicate that Ca2+ can compete with
La3+ for the same site.
High Concentrations of Intracellular BAPTA Do Not Reduce
Potentiation by La3+--
In general, the actions of
lanthanides on ion channel behavior are considered to be strictly
confined to the extracellular face of the plasma membrane. However, a
recent report on lanthanide-induced inhibition of currents mediated by
human TRPC3 transiently expressed in Chinese hamster ovary cells
suggested intracellular regulatory actions for both La3+
and Gd3+, although additional extracellular effects could
not be excluded (39). If the effects of La3+ on TRPC6- and
TRPC5-mediated currents result from an intracellular action of the free
cation they should depend on the trivalent cation buffering capacity of
the intracellular solution. Thus, raising the buffer capacity of the
intracellular solution by replacing 10 mM EGTA with 30 mM BAPTA, which binds both La3+ and
Gd3+ more strongly than Ca2+, should reduce the
lanthanide effects on TRPC5 and TRPC6. The free Ca2+
concentration of both pipette solutions was kept at ~100
nM. As seen in Fig. 3,
A and C, however, with BAPTA, 10 µM
La3+ was still able to increase histamine-induced TRPC5
currents strongly. The relative increase of TRPC5-mediated currents was
even more pronounced with BAPTA (7-fold, n = 4) than
with EGTA in the pipette solution. Interestingly, the amplitudes of
basal and histamine-induced (
4.5 ± 3.2 pA/picofarads,
n = 4, Cm = 21.9 ± 5, p < 0.05) TRPC5 currents were greatly reduced in
BAPTA-containing pipette solutions, confirming previous reports
indicating that TRPC5-mediated currents are dependent on
[Ca2+]i (14, 15). In the presence
of intracellular BAPTA, the inhibitory effect of La3+ on
TRPC6 (Fig. 3, B and D; inhibition by 89.5 ± 2.2%, n = 4) was similar to that in EGTA (96.8 ± 1.5%, n = 12). These results provide evidence for
an extracellular site of action for the potentiating and inhibitory
effects of lanthanides on TRPC5 and TRPC6, respectively.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of increased intracellular
Ca2+ buffering on lanthanide actions. A,
potentiating effect of 10 µM La3+ on TRPC5
currents recorded with a BAPTA-buffered pipette solution. Inward
currents at 60 mV were elicited by 100 µM histamine in
mTRPC5-expressing HEK293 cells. The pipette solution contained 30 mM BAPTA instead of 10 mM EGTA, which was used
in previous experiments. B, inhibitory effect of
La3+ on TRPC6 currents recorded with a 30 mM
BAPTA-buffered pipette solution. Currents at 60 mV were elicited by
100 µM histamine in TRPC6-expressing T-REx cells.
C and D, I-V relationships in the
presence and absence of La3+ obtained from voltage ramps
from 100 to +100 mV in the experiments in A and
B, respectively.
|
|
Effects of La3+ on Single Channel Currents through
TRPC5--
To investigate the mechanism involved in the dual effect of
La3+ on TRPC5, the single channel properties of TRPC5 were
characterized in the outside-out patch configuration. Under the same
conditions used for whole cell recordings, stimulation with 100 µM histamine activated single channel events of around
2.5 pA (Fig. 4, A and C, and Table I) in
TRPC5-expressing cells at a potential of
60 mV. To determine the
potential dependence of the single channel current amplitude for TRPC5,
the membrane potential was stepped from
100 to +80 mV (Fig.
4C). Under the conditions used, patches tended to be less
stable at positive potentials. The pooled single channel i-V
relationship for TRPC5 (Fig. 4D) closely resembled that of
whole cell currents, with a reversal potential close to 0 mV. At
60
mV, the chord conductance of TRPC5 was 41.3 ± 1.1 picosiemens
(n = 12), and the mean open time of histamine-induced TRPC5 currents 7.5 ms (cf. Table I). As reported previously
for human TRPC6 (9), single channel openings of rat TRPC6, elicited by
application of histamine or OAG, were typically of very short duration,
with more than 70% of the openings shorter than 0.5 ms. Hence, under
the recording conditions used, most openings of TRPC6 channels could
not be fully resolved. From openings longer than 2 ms, the amplitude of
single channel events at a holding potential of
60 mV was
2.81 ± 0.07 pA (n = 8, Fig. 4B and Table I), a
value that yielded a single channel chord conductance at
60 mV of
46.6 ± 1.5 picosiemens (n = 12).

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
Single channel properties of TRPC5 and TRPC6.
A and B, current traces recorded in outside-out
patches from TRPC5- and TRPC6-expressing cells at a membrane potential
of 60 mV (upper panels). c denotes the closed
level. Respective amplitude histograms are given in the lower
panels. C, voltage dependence of single channel
currents through TRPC5. Currents were recorded from one patch at the
indicated potentials. D, i-V relation of the
single channel current for TRPC5 (filled symbols) and TRPC6
(open symbols). Values are the means ± S.E.
(error bars as indicated).
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Effect of different La3+ concentrations on the single channel
properties of heterologously expressed TRPC5
HA, histamine; ND, not determined. The values in the last column
designate the number of experiments used for the calculation of
NPo, mean open time, and frequency at the respective
concentration of La3+.
|
|
When La3+ was added to the bath solution at a concentration
of 100 µM, the open probability of TRPC5 was dramatically
increased (Fig. 5A). At the
same time, the single channel current amplitude was approximately
halved compared with that recorded before the addition of
La3+ (Fig. 5A). An analysis of the
La3+ block of TRPC6 was precluded by the extremely short
events, which also made estimates of open probability difficult.
Channel activity was, however, abolished by the addition of 100 µM La3+ (Fig. 5B) and was
associated with a decrease in the frequency of channel openings (Fig.
5C).

View larger version (28K):
[in this window]
[in a new window]
|
Fig. 5.
Effects of 100 µM La3+ on single channel
currents mediated by TRPC5 and TRPC6. A, effects of 100 µM La3+ on TRPC5 channel activity in an
outside-out patch at 60 mV. Channel activity is expressed as
NPo over time (lower panel).
Upper panel, sample traces obtained from the same recording
in the absence (left) and presence (right) of
La3+ are given at two different time resolutions. Closed
levels (c) are indicated by the dotted line.
Inset, amplitude histograms obtained from the respective
current traces. B, current traces recorded in outside-out
patches from TRPC6-expressing cells at a membrane potential of 60 mV
before (upper trace) or after (lower trace)
application of 100 µM La3+. c,
closed levels. C, statistical analysis of La3+
effects on the opening frequency of TRPC6 channels.
|
|
A reduction in the single channel current of TRPC5 upon application of
100 µM La3+ was observed at all potentials
(Fig. 6, A and B),
with no increase in open channel noise. This result indicates that
La3+ may produce a very fast, flickery block of the
channel, not resolvable under our recording conditions, at a site
outside the membrane electrical field. Inhibition by an allosteric
mechanism cannot be excluded. The shape of the i-V
relationship of TRPC5-mediated currents and the reversal potential of
around 0 mV were preserved in the presence of La3+ (Fig.
6B). The effects of different La3+
concentrations on single channel amplitude are shown in Fig. 6C. Increasing the La3+ concentration from 1 µM to 5 mM resulted in a successive reduction of single channel current. The effects of different La3+
concentrations on the single channel properties including single channel current amplitude, open probability, mean open time, and opening frequency are summarized in Table I. The open probability was
roughly doubled in the presence of 1 µM La3+
and increased about 10-fold in the presence of 100 µM
La3+. The increased open probability of TRPC5 channels in
the presence of La3+ was the result of both increased open
times and higher frequency of channel openings (Table I). Because brief
events (<0.5 ms, see "Experimental Procedures") were excluded from
the analysis, and the proportion of these decreases with increasing
La3+ concentrations, the effect on mean open time will be
underestimated. The combined effect of the increase in
NPo and the decrease in single channel current
was an ~10-fold increase in the total current through the patch
(NPo·i). Interestingly, the
increase in patch current had a nearly identical dependence on the
La3+ concentration to the whole cell current. From the
single channel data, the EC50 for potentiation by
La3+ was around 3 µM, whereas a 50%
reduction of the single channel current occurred at a La3+
concentration of 100 µM. Taken together, the above data
suggest that La3+ exerts diverse effects on single channel
activity, having opposite effects on channel conductance and open
probability.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 6.
Potential dependence and concentration
dependence of La3+ effects on single channel currents
through TRPC5. A, records of single channel currents for
TRPC5 obtained at the indicated potentials in the presence of 100 µM La3+. The traces were recorded from the
same outside-out membrane patch. B, i-V relation
of the single channel currents calculated from the recordings displayed
in A (filled symbols with continuous
line). For comparison, the single channel i-V relation
from Fig. 4D is superimposed (open symbols with
broken line). C, sample traces from single
channel recordings obtained at the indicated concentrations of
La3+ in different outside-out membrane patches from
TRPC5-expressing cells at a holding potential of 60 mV. A
and C, closed levels are indicated by the dotted
lines.
|
|
Identification of Amino Acids Involved in Potentiation of TRPC5 by
La3+--
In an attempt to identify the site involved in
potentiation of TRPC5 by La3+, we searched for negatively
charged amino acids (Glu and Asp residues) in the putative
extracellular loops of the TRPC5 protein. We identified 10 residues
that are conserved in TRPC5 (Fig.
7A) and TRPC4 and generated
six point mutants in which individual residues, and two in which pairs
of close neighbors, were neutralized (Glu to Gln, or Asp to Asn). The
C-terminally YFP-tagged mutants were expressed in HEK293 cells and
their subcellular distribution and membrane targeting determined by
confocal laser microscopy. Like wild type TRPC5 (TRPC5-wt) (15), the
mutants displayed a clustered appearance in the plasma membrane and
retention in a perinuclear compartment.

View larger version (24K):
[in this window]
[in a new window]
|
Fig. 7.
Localization of negatively charged amino
acids in the extracellular loops of TRPC5 and characterization of the
mutant TRPC5-E543Q which lacks La3+-induced potentiation.
A, putative transmembrane topology of TRPC5 showing the
positions of negatively charged amino acids in the extracellular loops.
The framed amino acid numbers indicate the
positions at which neutralization led to a loss of
La3+-mediated potentiation. B, La3+
does not potentiate, but inhibits, histamine-activated currents through
TRPC5-E543Q. Currents were recorded at a holding potential of 60 mV,
and histamine and La3+ were applied at the times indicated
by the bars. The inset shows the currents at
100 (inward currents) and +100 mV (outward currents) obtained from
voltage ramps in the same experiment. C,
I-V relations from the experiment in B
before and during application of La3+ (100 µM). D, inhibition of single channel currents
from TRPC5-E543Q recorded in an outside-out patch at 60 mV.
Left, individual current traces; right, the
corresponding amplitude histograms. N indicates the number
of events.
|
|
As an initial probe for channel function, we tested for the presence of
histamine-induced Mn2+ influx in fura-2AM-loaded cells.
Five of the mutants (D392N, E404Q and E543Q, E570Q and E595Q/E598Q)
displayed robust accelerations in Mn2+ influx upon addition
of histamine, E559Q only weak responses (data not shown). In contrast,
E467Q/E470Q and E479Q did not respond. In whole cell recordings, the
mutants that showed robust accelerations in Mn2+ influx
displayed histamine-activated currents indistinguishable from those of
TRPC5-wt (n
10 for each mutant). Addition of
La3+ resulted in increases in current in the mutants D392N,
E404Q, and E570Q similar to those observed for TRPC5-wt (data not
shown). In contrast, neutralization of negatively charged amino acids at two sites in the putative pore-forming loop, E543Q, just
after transmembrane segment S5, and E595Q/E598Q, close to S6,
led to a loss of La3+-induced potentiation (Figs. 7 and
8). Both of these mutants did, however,
display inhibition. This inhibition was different for the two mutants.
Both inward and outward currents mediated by the mutant E543Q were
inhibited to a similar extent by La3+ (Fig. 7, B
and C). In contrast, inward currents mediated by the mutant
channel E595Q/E598Q were inhibited to a larger extent than outward
currents (Fig. 8, A and B).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 8.
Mutant TRPC5-E595Q/E598Q lacks
La3+-induced potentiation and shows a modified
La3+ block. A, La3+ inhibits
histamine-activated inward currents through TRPC5-E595Q/E598Q. Currents
at 60 mV and 100 mV (inward currents in inset) were more
strongly inhibited by La3+ at 100 µM and 1 mM than currents at +100 mV (outward currents in
inset). B, I-V relation in
the absence and presence of 100 µM La3+.
C, the addition of 100 µM and 1 mM
La3+ reduces single channel currents at 60 mV by a slow
flickery block. Left, current traces; right,
corresponding amplitude histograms.
|
|
We also tested whether potentiation by Ca2+ was influenced
in the mutants E543Q and E595Q/E598Q. Currents through the mutants did
not show the rapid potentiation observed upon raising
[Ca2+]o from 2 to 20 mM for TRPC5-wt (data not shown). Both inward and outward
currents mediated by E543Q were inhibited in 20 mM
Ca2+ (n = 3, data not shown). In contrast,
currents through E595Q/E598Q slowly increased to a maximum, then
declined spontaneously in the continued presence of 20 mM
Ca2+ (n = 3, data not shown). In these
experiments, there was a more noticeable shift in the current reversal
potential on raising [Ca2+]o from
2 to 20 mM for E595Q/E598Q than for TRPC5-wt or E543Q. We
therefore quantified the relative Ca2+ permeability of
E595Q/E598Q and compared it with that of TRPC5-wt. Current reversal
potentials were first measured from voltage ramps in a nominally
Ca2+-free, Na+ solution, then in a
Na+-free (NMDG+) solution containing 20 mM Ca2+. From the reversal potentials, we
calculated values for
PCa/PNa of 1.83 ± 0.18 (n = 3) for TRPC5-wt, a value close to the 1.79 in
our previous study (15), and 4.28 ± 0.08 (n = 4)
for E595Q/E598Q.
Thus, the mutants E543Q and E595Q/E598Q did not show rapid potentiation
by La3+ and Ca2+, further supporting a similar
site of action. Furthermore, mutant E595Q/E598Q had a higher relative
Ca2+ permeability than TRPC5-wt.
Effects of La3+ on the Single Channel Properties of the
Mutants E543Q and E595Q/E598Q--
In outside-out patches, the single
channel properties of the mutant E543Q were similar to those of
TRPC5-wt (Fig. 7D). The mean single channel current at
60
mV was
2.63 ± 0.10 pA (chord conductance, 43.8 picosiemens;
n = 4), a value not significantly different
(p = 0.28) from that of the wild type channel. It is noteworthy that very high levels of channel activity were observed in
patches from mutant E543Q-expressing cells, necessitating the use of
much smaller pipettes. Addition of 100 µM
La3+ to the extracellular solution reduced the current
amplitude to
1.47 ± 0.16 pA (Fig. 7D), a decrease
similar to that observed for TRPC5-wt. Like the inhibition of TRPC5-wt,
the reduction in channel current was not accompanied by an increase in
open channel noise. However, in contrast to the wild type channel,
application of 100 µM La3+ did not result in
an increase in NPo (0.50 ± 0.21 and
0.53 ± 0.14 (n = 4; p = 0.8) in
control and 100 µM La3+, respectively). The
mutant E595Q/E598Q showed more drastic changes in its single channel
properties (Fig. 8C). The single channel current of
2.98 ± 0.03 pA at
60 mV (chord conductance, 49.7 picosiemens;
n = 9) was significantly (p < 0.01)
larger than that for TRPC5-wt. Application of 100 µM or 1 mM La3+ resulted in weaker reductions in
current amplitude than in TRPC5-wt (to
2.50 ± 0.03 pA,
n = 7 and
1.25 ± 0.07 pA, n = 4, respectively) and a clear increase in open channel noise indicative
of a slower, flickery channel block (Fig. 8C). Thus, at the
single channel level, neither mutant showed potentiation. E543Q had a
similar conductance and was inhibited by La3+ in a manner
similar to TRPC5-wt. In contrast, E595Q/E598Q had a higher conductance
than TRPC5-wt or E543Q, and inhibition by La3+ was modified.
 |
DISCUSSION |
In the present study, we show that currents mediated by TRPC5 and
TRPC6 are affected differently by the lanthanides La3+ and
Gd3+. Although whole cell currents through TRPC6 were
inhibited concentration-dependently by lanthanides, those
through TRPC5 were potentiated by low concentrations but inhibited by
high concentrations. The dual effect of La3+ on TRPC5 was
also observed at the single channel level and involved a combination of
an inhibitory effect on channel amplitude and an increase in channel
open probability. By an analysis of point mutants, we identified two
sites, close to the extracellular mouth of the pore, which are involved
in La3+- and Ca2+-induced channel potentiation.
Inhibitory effects of different di- and trivalent cations, including
La3+ and Gd3+, have been described for most
Ca2+-permeable channels. Accordingly, current block by bath
application of lanthanide ions has been reported for several members of
the TRPC subfamily of TRP channels, e.g. for human and mouse
TRPC3 (29, 37-39), for mouse TRPC6 (6, 45), and for human TRPC7 (40).
There is considerable variability in the IC50 values
obtained, with higher values in Ca2+ imaging experiments
than in electrophysiological recordings. The IC50 value
obtained for rat TRPC6 in the present study was in good agreement with
the values obtained in whole cell patch clamp experiments for mouse
TRPC6 (6) and human TRPC3 (39).
The pronounced increase in agonist-induced currents in TRPC5-expressing
cells with micromolar La3+ in the present study are in
agreement with previous studies on this channel (15, 16), and we have
extended this observation to Gd3+, which is approximately
equally effective. A further novel finding of the present study is that
higher lanthanide concentrations (
1 mM) were less
effective in potentiating the current and even reversibly inhibited
currents carried by TRPC5. Millimolar concentrations of
Ca2+ also potentiated agonist-induced currents and
prevented the effects of micromolar La3+, suggesting that
physiological divalent cations may also bind at the same site.
Evidence for two different actions of lanthanides on the channel was
supported by the effects of La3+ on single channel currents
in outside-out patches. La3+ caused a
concentration-dependent decrease in single channel current amplitude, while, at the same time, increasing the channel open probability (NPo). Both effects were already
observed at a concentration of 1 µM. The concentration
dependence of the increase in current in outside-out patches
(NPo·i) closely paralleled the
increase in whole cell current, although the maximum potentiation was, on average, about 3-4-fold higher in outside-out patches than in whole
cell experiments. Because of our inability to resolve currents at
millimolar concentrations of La3+, it is not clear from the
single channel data why potentiation declines and inhibition occurs. A
decrease in single channel current is at least partly responsible for
the decrease in whole cell current.
There are few reports of potentiating effects of La3+ on
ion channel currents, and more importantly, to our knowledge, there are
no reports that describe dual effects on ion channel activity. Potentiating actions of 100 µM La3+ have been
observed in whole cell recordings for mouse, rat, and human TRPC4- and
mouse TRPC5-mediated currents (15, 16, 31) and for receptor-operated
currents in cells coexpressing mouse TRPC1 and mouse TRPC5 (16). In the
latter, heteromultimers of TRPC1 and TRPC5 are thought to be formed,
which, compared with homomeric TRPC5, have a drastically reduced single
channel current (
0.5 pA at a holding potential of
60 mV). The
single channel current amplitude was not affected by the inclusion of
La3+ in the pipette solution. For native nonselective
cation currents, there is one report of a potentiation of the native
current (Icat) in rat ileal smooth muscle cells
by La3+, with an apparent Kd of 190 µM (46). From relaxation analysis, prolonged single
channel mean open life times were suggested to be the main cause of the
augmentative effect of La3+. Interestingly, in the mouse,
TRPC4 is expressed in this tissue (47). With regard to heterologously
expressed TRPC4 and TRPC5, it should be noted that some studies
reported an inhibition by micromolar lanthanide concentrations (14,
48).
The loss of the potentiating effects of La3+ and
Ca2+ in mutants of two sites (Glu543 and
Glu595/Glu598), which, according to models of
TRP channel structure, are located opposite each other at the start and
end of the pore-forming loop between S5 and S6, strongly supports an
extracellular site of action. Importantly, identical amino acids are
present in TRPC4 at the positions corresponding to Glu543
and Glu595 in TRPC5, but acid amino acids are not present
at corresponding positions in TRPC3, TRPC6, and TRPC7. Larger
variations in structure prevent an identification of corresponding
residues in TRPC1. The differences between the TRPC isoforms provide an
explanation for the specificity of the potentiating effect for TRPC4
and TRPC5. Interestingly, these sites are analogous to those in TRPV1
(VR1) which are involved in proton-mediated channel potentiation
(Glu600) and proton-mediated channel activation
(Glu648) (49) and can modulate sensitivity to the activator
capsaicin (49, 50). Indeed, at the latter site TRPC4, TRPC5, and TRPV1 have identical EFTE motifs. Because the distal steps leading to activation of this channel and the activation mechanism are not known,
it is not clear how La3+ or Ca2+ binding to the
extracellular sites results in current potentiation. By analogy to
TRPV1, where neutralization of Glu600 and
Glu648 leads to potentiation of the capsaicin sensitivity
(49, 50), it is tempting to speculate that La3+ or
Ca2+, by neutralizing the negative charges, potentiate the
response of TRPC5 to its unknown activator.
The TRPC5 mutation E595Q/E598Q also affected inhibition by
La3+, whereas E543Q did not. For the wild type channel, the
reduction in single channel current by La3+ at all
potentials without an increase in open channel noise is indicative of a
fast block at a site outside the membrane electrical field. Similarly,
inhibition of whole cell currents in the mutant E543Q, which lacked
potentiation, was potential-independent. In contrast, the mutant
E595Q/E598Q showed a slower flickery block at the single channel level
and a clear potential dependence of whole cell current inhibition, with
inward currents being more strongly reduced than outward currents. The
loss of the fast block by mutation at the extracellular site
E595Q/E598Q and the potential dependence of the block remaining after
mutation indicate that in both cases La3+ blocks the
channel from the outside. The effect of this mutation on inhibition by
La3+, the increase in single channel current, and the
increase in PCa/PNa
suggest that this site lies close to, or in, the permeation pathway.
Considering the change in channel inhibition by La3+, it is
possible that the increase in single channel current in E595Q/E598Q
results from a reduction in block by a physiological cation. By analogy
to other channels with similar structure, the glutamates will form a
negatively charged ring around the extracellular pore mouth, with, in
tetramers, at least 12 negatively charged residues. These amino acids
may act as "gatekeepers" controlling cation entry into the pore.
Further evidence that both potentiation of TRPC5 and inhibition of
TRPC6 by lanthanides results from an extracellular action of
La3+ is provided by the presence of the effects in
experiments with intracellular EGTA buffers and their persistence in
the presence of higher concentrations of BAPTA. Both buffers have a
very high affinity for lanthanides. Recently, Halaszovich et
al. (39) suggested that La3+ and Gd3+
block human TRPC3 channels from the cytosolic side of the membrane and
that different apparent IC50 values might simply reflect
different uptake rates for lanthanide ions in different cell types (see below). Our data support an extracellular site of action on TRPC6 and
on TRPC5, although we cannot exclude additional intracellular effects.
Because of the variability in results from different laboratories, the
applicability of results from heterologous overexpression studies on
TRPC channels to native channels has recently been questioned
(e.g. 5). However, for TRPC6, at least, properties nearly
identical to those observed after overexpression are seen for native
channels in vascular smooth muscle cells (6, 7). These properties
include the doubly rectifying I-V relation, store depletion-independent activation, as well as stimulation by DAGs (13).
For the other TRPC channels, too few data are available. Nonetheless,
the elucidation and comparison of the properties of different
heterologously expressed TRPC channels may be valuable tools to help
clarify the role of TRPC channels in native cells. TRPC4 and TRPC5 (15,
16, 31) stand out from other TRPC channels and other nonselective
cation channels in that they are strongly potentiated by
La3+ in the micromolar range. This effect is a functional
property that can be assigned exclusively to group 4 TRPCs or
heteromultimers containing group 4 TRPCs and may be an important
distinguishing feature. Another characteristic property is the shape of
the I-V relation. Currents from overexpressed, presumably
homomeric, TRPC3-7 have a doubly rectifying I-V relation
with a reversal potential close to 0 mV in physiological solutions. As
also indicated in this study for TRPC6, the relative amplitude of
outward compared with inward currents is larger for TRPC3 and TRPC6
than for TRPC4 and TRPC5. Interestingly, heteromers of TRPC1 and TRPC5
display a different shape of I-V relation, with a strong
reduction in current at negative membrane potentials which results in a
U-shaped I-V relation for inward currents (16). The
activation of TRPC3, 6, and 7 by DAGs in a protein kinase C-independent
manner is a characteristic feature of this group of channels, not
shared with other TRPC channel groups (9, 29). A previous report that the TRPC6B splice variant might be an exception to this rule in not
being activated by DAGs (12) could not be confirmed in the present
study. Within this group of channels, the effect of flufenamate can
distinguish TRPC6 from TRPC3 and TRPC7. Flufenamate potentiates TRPC6
but inhibits TRPC3 and TRPC7 (6). Moreover, in the present study, the
mean open time of TRPC5 and TRPC6 single channel events were found to
be 7.5 ms and <1 ms, respectively. Similar results have been obtained
in independent studies: brief opening events appear to be common for
human TRPC3 (32-35) and human TRPC6 (9) whereas mean open life times
longer than 1 ms have been reported for mouse, human, and rat TRPC4 and
mouse TRPC5 (15, 16, 31, 36). No information is available on single
channel properties of TRPC7. Hence, differential effects of
lanthanides, activation by diacylglycerols, and single channel open
times represent promising tools to distinguish contributions of the
TRPC3/6/7 group from those of the TRPC4/5 group when endogenous
phospholipase C-dependent cation currents are studied.
In conclusion, the potentiation of TRPC5 by micromolar La3+
and Gd3+, a feature shared with TRPC4 but not with most
other nonselective cation channels, involves interactions with
negatively charged amino acids situated close to the extracellular pore mouth.