Institut für Pharmakologie, Thielallee 69-73, Freie Universität Berlin, D-14195 Berlin, Germany
TRPC3 (or Htrp3) is a human member of the trp family of Ca2+-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca2+ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661-671), we tested whether TRPC3 might represent a Ca2+ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca2+ over Na+. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca2+ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca2+. Likewise, infusion of Ca2+ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca2+-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca2+ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 µM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 µM). We conclude that TRPC3 codes for a Ca2+-permeable channel that supports Ca2+-induced Ca2+-entry but should not be considered store operated.
VARIOUS mechanisms exist by which Ca2+ influx may
be evoked in response to stimulation of membrane
receptors that are coupled to phospholipase C and
mediate production of inositol-1,4,5-trisphosphate (InsP3).1
Ca2+ entry pathways have been described that are activated by intracellular Ca2+ (von Tscharner et al., 1986 Independently of Wes et al. (1995) Cell Culture and Microinjection of Expression Plasmids
CHO cells (subclone CHO-K1) were cultured in Ham's F12 medium supplemented with 10% FCS, 60 U/ml penicillin, 60 µg/ml streptomycin, and
1 mM glutamine. Cells were seeded at a density of ~103 cells/mm2 on coverslips imprinted with squares for localization of injected cells. Intranuclear microinjection was performed with a manual injection system (Eppendorf, Hamburg, Germany). The aqueous injection solution contained
0.25 µg/µl of the reporter plasmid pS65T-C1 (Clontech Laboratories, Palo
Alto, CA) that codes for a modified green fluorescent protein from Aequora victoria and either 1.2 µg/µl of the eukaryotic expression plasmid
pcDNA3 (Invitrogen, Leek, Netherlands) (control) or 1.2 µg/µl of the
plasmid carrying the TRPC3 cDNA insert. Cells that coexpressed the angiotensin II receptor AT1A were injected with a solution that also contained the cDNA of the receptor in the plasmid pcDNA3 (1.2 µg/µl). Approximately 10-20 fl were injected with microcapillaries with an outlet
diameter of ~0.5 µm. The pressure was 20-40 hPa, and the injection time was 0.3 s. After injection, the cells were kept in culture medium for 15-24 h.
Measurement of [Ca2+]i in CHO Cells
Measurement of [Ca2+]i in single CHO cells loaded with fura-2 by incubation in fura-2/acetoxymethylester (MoBiTec, Göttingen, Germany) was
performed with a digital imaging system (T.I.L.L. Photonics, München,
Germany) as described, including the calibration procedure (Dippel et al.,
1996 Electrophysiology
Whole-cell currents in CHO cells were measured with the patch-clamp
technique (Hamill et al., 1981 Single-channel analysis was performed in the cell-attached or inside-out mode. Cell-attached patches were studied with the following solutions
(mM): bath, CsCl 140, MgCl2 1, CaCl2 1.8, glucose 10, Hepes 10, pH 7.4;
EGTA 1 was present instead of CaCl2 when a Ca2+-free bath was desired.
The pipette contained (mM): CsCl 140, MgCl2 1, CaCl2 1.8, glucose 10, Hepes 10, pH 7.4.
For inside-out experiments, the bath solution facing the cytosolic side
of the patches contained (mM): Na gluconate 140, Mg gluconate 1, EGTA
1, glucose 10, Hepes 10, pH 7.4, or NMDG 140, Mg gluconate 1, EGTA 1, glucose 10, Hepes 10, pH 7.4 (adjusted with methanesulfonic acid). Various Ca2+ concentrations in the first solutions were obtained by the addition of Ca gluconate, according to a computer program (Schubert, 1996 Using a DNA fragment of the expressed sequence tag
R34716 amplified by the PCR as a probe, we isolated a
full-length cDNA of TRPC3 from human brain cDNA libraries by plaque hybridization. In comparison to the sequence reported by Zhu et al. (1996) Under the assumption that TRPC3 codes for a store-operated cation channel, we first tried to demonstrate expression of TRPC3 under the same conditions we previously used for the characterization of the store-operated
channel TRPC1A. For patch-clamp measurements, we
kept the cells in Ca2+-free solution for 10-30 min and then
measured the whole-cell currents with pipette solutions
containing InsP3 (10 µM), thapsigargin (3 µM), and low
Ca2+/high Ca2+ buffer capacity (10 mM EGTA). In our
previous study (Zitt et al., 1996 We then changed the conditions by keeping the cells in
Ca2+-containing (1.2 or 10 mM) solution throughout, lowering the Ca2+ buffer capacity of the intracellular solution
(only 1 mM EGTA), and omitting InsP3 and thapsigargin
from the pipette solution. Furthermore, the cells were injected with the cDNA of TRPC3 and, additionally, the reporter plasmid pS65T-C1 that codes for a modified green
fluorescent protein (GFP) from A. victoria (Cubitt et al., 1995
TRPC3 currents were characterized by a reversal potential close to 0 mV, with a slight shift in the reversal potential to the right (by 3-6 mV, n = 4) when the Ca2+ concentration in the bath was increased from 1.2 to 10 mM, with a
corresponding reduction of the Na+ concentration (Fig. 1
B). Inward currents were almost completely abolished
when NMDG was the only extracellular cation. Thus, expression of TRPC3 results in nonselective cation currents
permeant to Na+, Cs+, and Ca2+, with little selectivity for
Ca2+ over Na+.
As soon as we had demonstrated expression of TRPC3,
we wished to obtain evidence in our expression system
that TRPC3 leads to enhanced increases in [Ca2+]i after
stimulation with an agonist. Therefore, we coinjected cells
with cDNAs for GFP, the angiotensin II receptor AT1A,
and either TRPC3 or pcDNA3 (control) and measured
[Ca2+]i responses with the fura-2 method. Fig. 2 shows that
control cells responded to angiotensin II (1 µM) with a
transient rise of [Ca2+]i. [Ca2+]i returned to baseline after
less than 1 min. TRPC3-expressing cells, in contrast, exhibited sustained increases in [Ca2+]i. Although already
the peak [Ca2+]i values were higher than in control cells,
the most striking difference was that [Ca2+]i remained elevated for more than 2 min. 100 s after stimulation with angiotensin II, the mean [Ca2+]i exceeded the baseline by
525 ± 174 nM (n = 12 cells in three separate experiments).
In controls, the corresponding value was
Whole-cell currents were observed as soon as the whole-cell configuration was obtained, as if the currents were
constitutively active. A steady decline of the current was
observed in the course of the experiments (Fig. 3 A). This
decline was not prevented when the pipette solution contained either InsP3 (10 µM, n = 4), thapsigargin (3 µM,
n = 3), or both (n = 2) (data not shown). In cells additionally expressing the angiotensin II receptor, angiotensin II
(1 µM) induced increases in the current (see Fig. 3 A, n = 4 out of 6 cells, mean increase 36 ± 20 pA), but these increases were always transient. No such current increases
were observed in controls (expressing angiotensin II receptor and GFP, n = 6, Fig. 3 B). The calcium ionophore
ionomycin induced strong current increases (n = 8) in a
concentration-dependent manner (Fig. 4). Specifically,
ionomycin (0.1 µM) enhanced currents by 120 ± 45 pA
(n = 7) and at a higher concentration (1 µM) by 580 ± 166 pA (n = 8). In controls, ionomycin (1 µM) did not enhance NMDG-blockable currents by more than 10 pA in
any cell (n = 8).
Since ionomycin might exert its effect by Ca2+ influx as
well as by release of Ca2+ from intracellular stores and
store depletion, we also tested ionomycin in the absence of
extracellular Ca2+ (n = 4). No stimulation of the current
was observed. Next, we performed experiments (Fig. 5) in
which ionomycin was applied first in the absence of Ca2+
in the bath, followed by the combined application of ionomycin (1 µM) and Ca2+ (1.2 mM). Again, ionomycin had
no effect in the absence of Ca2+ (n = 7), whereas large
currents were evoked after readdition of extracellular
Ca2+ (mean increase from baseline by 247 ± 193 pA; n = 7;
no such currents occurred in controls, n = 5). Interestingly, there was a delay of 15-45 s before these currents
started to develop. This delay was not completely explained by technical problems with the bath exchange. For
example, the block of the currents by NMDG in Fig. 5 occurred much faster than the stimulation by Ca2+. Taken
together, our findings demonstrate that ionomycin requires extracellular Ca2+ to stimulate TRPC3, consistent
with the requirement for Ca2+ influx for the action of the
ionophore.
If Ca2+ influx activates TRPC3, a similar effect should
be observed if the cells were dialyzed with a pipette solution containing an elevated Ca2+ concentration. Fig. 6 A
shows such an experiment in which the pipette solution
contained 10 µM Ca2+. Again, spontaneous cation currents were present right after obtaining the whole-cell configuration, but under these conditions, a considerable current increase developed over the next 20 s, followed by a
much slower decline than that after stimulation with ionomycin. Similar results were obtained in three experiments. When the pipette solution contained 10 mM EGTA without CaCl2, currents were initially present but decreased
rapidly (Fig. 6 B; n = 8). No Ca2+-induced currents were
evoked in cells not expressing TRPC3 (n = 4).
To estimate the single-channel conductance of TRPC3,
we performed a simple noise analysis (Neher and Stevens,
1977
In cell-attached patches, channel activity (expressed as
NPo) was constitutively present and considerably increased by ionomycin (1 µM) added to the bath solution
(Fig. 9, n = 2 out of 4). As in the whole-cell measurements,
the effect of ionomycin required extracellular Ca2+ because it was not observed in the absence of Ca2+ in the
bath solution (n = 5, not shown). Thus, TRPC3 channels were activated in a Ca2+-dependent manner. Experiments
in which the cytosolic side of inside-out patches was exposed to either 0, 1, 10, or 30 µM Ca2+, however, failed to
reveal any obvious stimulation of the open probability by
Ca2+ (Fig. 10 A). This situation was not changed when
calmodulin (0.2 µM) was added along with Ca2+ (1 µM,
Fig. 10 B). No obvious activation of channel activity was observed in 11 out of 14 patches. It should be noted that
the remaining three patches showed moderate increases in
activity in response to Ca2+ and calmodulin (increase in
NPo by a factor of ~3). Thus, the single-channel experiments confirmed the whole-cell data to the extent that
TRPC3 is activated by Ca2+, but no evidence was obtained
for a direct interaction of Ca2+ with the TRPC3 channel.
This study was designed to functionally characterize the
gene product of TRPC3 (or Htrp3), a member of the
growing gene family of mammalian homologues of the trp
gene from Drosophila. In particular, we tested whether
TRPC3, previously shown to support receptor-mediated Ca2+ entry (Zhu et al., 1996 After expression of TRPC3 in CHO cells with intranuclear injection of the corresponding cDNA, currents were
constitutively active, without requirement for previous depletion of calcium stores. No stimulation of TRPC3 was
evoked with either InsP3 or thapsigargin, both the classical
substances to induce store depletion and store-operated
currents in the presence of high intracellular concentrations of the calcium chelator EGTA. Only the calcium ionophore ionomycin led to a profound increase in TRPC3
currents. Ionomycin induces Ca2+ flux through all kinds of
biological membranes. Therefore, it can be used to mobilize calcium from intracellular stores, to an extent exceeding that of InsP3 (Morgan and Jacob, 1994 Our study extends that of Zhu et al. (1996) The activation of TRPC3 by Ca2+ may represent either
a direct interaction of Ca2+ with the channel or may be the
result of a more complex process involving a Ca2+-dependent step. Since Ca2+-depleted cells showed no immediate
but rather delayed responses to ionomycin applied along
with extracellular Ca2+, and since these responses occurred only in a part of the cells, the hypothesis of a direct
interaction of TRPC3 with Ca2+ already appears unlikely.
To test the hypothesis more rigorously, we studied TRPC3
in inside-out patches and exposed the cytosolic side of the
patches to various Ca2+ concentrations ranging from 0 to
30 µM. We did not find significant changes in channel activity. Thus, Ca2+ alone is not sufficient for a regulation of
TRPC3. The fact that Ca2+-mediated activation was occasionally observed in the presence of calmodulin cannot be
interpreted to indicate a calmodulin action on TRPC3,
since the effect occurred inconsistently. Therefore, the exact cascade of events eventually leading to the activation of TRPC3 remains elusive; it may be speculated that not
only Ca2+ but also calmodulin has a role. At any rate, the
sustained channel activity in inside-out patches again argues against a role of store depletion for channel stimulation.
The expression of a Ca2+-activated, Ca2+-permeable
channel may be considered hazardous for a cell because it
may result in an uncontrolled Ca2+ influx leading to Ca2+
overload and cell death. Indeed, we noted that 2 d after injection of TRPC3 cDNA, the majority of cells were dead
and that the surviving cells were those with little expression of TRPC3. We suggest that the spontaneous activity
of TRPC3 channels leads to a lethal Ca2+ overload, enhanced by the Ca2+-triggered activation of the channels.
After one day of TRPC3 expression, however, Ca2+ homeostasis appeared well compensated since basal [Ca2+]i
levels were not higher than in control cells. The Na+ permeability of TRPC3 may help to keep Ca2+ influx at compensable levels since it is expected to lead to depolarization and therefore to a reduction of the driving force for
Ca2+ influx. The finding that overexpression of TRPC3 is
lethal is another striking difference to our experience with
overexpression of TRPC1A, which resulted in fairly small
currents routinely measured 2 d after microinjection. On
the other hand, our results in an overexpression model do
not exclude that TRPC3 may well have a role in normal
cells expressing it in much lower numbers, with potent
counterregulatory mechanisms available to keep net Ca2+
influx at bay. Ca2+-dependent Ca2+ entry may be used as
convenient way to amplify an initial [Ca2+]i signal. Indeed,
Ca2+-activated nonselective cation channels were among
the first described ion channels demonstrated to carry
receptor-mediated Ca2+ influx in nonexcitable cells (von
Tscharner et al., 1986 With a growing number of members of the trp family
cloned and functionally characterized, it appears that its
members obey very distinct regulatory principles. While
some, like the parent member trp in Drosophila, can be activated by calcium store depletion (Vaca et al., 1994 In summary, we have demonstrated that TRPC3 codes
for a Ca2+-permeable nonselective cation channel. Since
TRPC3 supports receptor-mediated Ca2+ influx, it may
contribute to the refilling of intracellular calcium stores.
However, depletion of calcium stores is not the mechanism by which activation of TRPC3 is initiated. In the opposite, TRPC3 is activated by a Ca2+-dependent process.
) or
by second messengers such as InsP3 (Kuno and Gardner,
1987
; Restrepo et al., 1990
; Mozhayeva et al., 1991
), inositol-1,3,4,5-tetrakisphosphate (Lückhoff and Clapham,
1992
), or cGMP (Bahnson et al., 1993
), but these mechanisms appear to be confined to a few specialized cells. One
other Ca2+ entry mechanism, however, appears to be of
particular importance in a broad range of different cell
types that is named "capacitative" or "store-operated"
(Putney, 1990
; Berridge, 1995
; Clapham, 1995
). The term
describes the phenomenon that depletion of intracellular calcium stores generates an unknown signal that is communicated to Ca2+-permeable channels in the plasma
membrane and activates these channels. Recently, several
cDNAs have been found in different species, including
human (Petersen et al., 1995
; Wes et al., 1995
; Zhu et al.,
1996
; Philipp et al., 1996
), which are homologous to the trp
gene in Drosophila (Montell and Rubin, 1989
). Like trp,
all functionally expressed members of the new trp gene
family code for Ca2+-permeable cation channels. We have
reported that expression of one of these trp homologues
named TRPC1A, a shortened variant of the previously
cloned cDNAs TRPC1 (Wes et al., 1995
) or Htrp1 (Zhu et
al., 1995
), results in currents with the typical characteristics of store-operated Ca2+ entry pathways (Zitt et al., 1996
).
Specifically, these currents were stimulated by store-depletion with InsP3 or thapsigargin, but were inhibited by
Ca2+. In terms of ion permeability and predicted single-channel conductance, however, TRPC1A showed properties sharply distinct from previously described store-operated Ca2+ currents (Hoth and Penner, 1992
; Zweifach and
Lewis, 1993
; Parekh et al., 1993
; Lückhoff and Clapham,
1994
). Therefore, other members of the trp family may exist that are store-operated as well, but may more closely
resemble the other known pathways. Evidence for this
possibility has been provided by the study of Zhu et al. (1996)
, which demonstrates that capacitative Ca2+ entry in
murine Ltk
cells was virtually abolished by expression of
a mixture of antisense RNAs directed against six murine
members of the trp family. In the same study, Zhu et al.
expressed the cDNA Htrp3 (a full-length cDNA of the
PCR fragment TRPC3 described by Wes et al., 1995
) and
found that Htrp3 induced a significant and sustained enhancement of receptor-mediated increases in [Ca2+]i.
Since expression of Htrp1 resulted in less pronounced increases in Ca2+ influx than that of Htrp3 (Zhu et al., 1996
),
one might consider Htrp3 (or TRPC3) a promising candidate as a channel responsible for store-operated Ca2+ influx (Birnbaumer et al., 1996
) and an important tool to
study this elusive mechanism in greater detail.
and Zhu et al. (1996)
,
we have cloned TRPC3 and report here its functional
characterization. With fluorometric methods, we confirmed that expression of TRPC3 in CHO cells enhanced
receptor-activated Ca2+ influx. More detailed studies with
the patch-clamp method revealed that TRPC3 did not exhibit any of the properties expected for store-operated
channels. In contrast, TRPC3 currents were stimulated by
Ca2+. This finding can explain the effects of TRPC3 expression on [Ca2+]i, which may be interpreted as Ca2+-induced Ca2+ entry. Thus, TRPC3 represents the prototype of a Ca2+ channel activated by a Ca2+-dependent
mechanism.
Materials and Methods
). The standard bath solution contained (mM): NaCl 138, KCl 6, MgCl2 1, CaCl2 1, glucose 5.5, Hepes 20, pH 7.4.
). The standard intracellular solution contained (mM): CsCl 140, MgCl2 2, EGTA 1 or 10, ATP 0.3, GTP 0.03, Hepes 10, pH 7.2. Thapsigargin (3 µM) or InsP3 (10 µM) was added as indicated. In some experiments, the Ca2+ concentration in the pipette solution was set to a calculated value (Schubert, 1996
) of 10 µM with a mixture of EGTA (10 mM) and CaCl2 (9.85 mM). The standard bath solution
contained (mM): NaCl 140, MgCl2 1.2, CaCl2 1.2, glucose 10, Hepes 11.5, pH 7.4. In some experiments, EGTA (5 or 10 mM) was added, or NaCl
was substituted with N-methyl-D-glucamine (NMDG)/HCl, or CaCl2 was changed to 0 or to 10 mM. The standard holding potential was
70 mV.
Currents were normally filtered with 1 kHz. The procedure for noise analysis has been described (Zitt et al., 1996
).
). If calmodulin (0.2 µM) was added to the bath, it contained also ATP (0.3 mM, taken into account as Ca2+ chelator for the calculation of [Ca2+]i).
The pipette solution contained (mM): CsCl 140, MgCl2 1, CaCl2 1.8 (or
sometimes EGTA 1), glucose 10, Hepes 10, pH 7.4. Data were sampled at
50 kHz and filtered at 5 kHz. Analysis was performed with the Pclamp 6 software (Axon Instruments, Foster City, CA). Channel activity is expressed as NPo, the product of the number (N) of channels in the patch
and the open probability. All experiments were performed at room temperature (21-26°C). Data are presented as mean ± SEM.
Results
, we found different
bases at five positions within the coding region (data not
shown), one of them resulting in a change in the amino acid sequence (A 820 E). The cDNA of TRPC3 was
cloned into the eukaryotic expression plasmid pcDNA3
and injected into the nucleus of CHO cells for transient
expression of TRPC3.
), cation currents were observed in 73% of cells injected with cDNA coding for
TRPC1A. In the present study with TRPC3, however,
only 3 out of more than 50 cells exhibited cation currents markedly larger than control cells (injected with pcDNA3
alone). Addition of Ca2+ to the bath (1.2 or 10 mM) failed
to elicit Ca2+-selective currents in any cell (data not
shown).
). Only cells exhibiting marked GFP fluorescence
were measured 1 d after injection. Under the new conditions, most of the cells showed inward currents at a holding potential of
70 mV that were considerably larger
than in control cells (intranuclear injection of GFP and
pcDNA3 only). Specifically, 10 out of 16 cells exceeded the range of currents in control cells (Fig. 1 A). The median of the peak cation current density in cells expressing
TRPC3 was about 15 times higher than in control cells
(29.5 vs. 2.05 pA/pF). It should be noted that the peak inward current density in control cells was usually smaller
than 6 pA/pF (20 out of 22 cells), and currents below this
limit were considered for analysis without further test for
their ionic nature. In all cells revealing higher current densities (2 control cells, 13 TRPC3-injected cells), the relevant cation currents are given as the difference between the peak inward current and the current remaining after
application of NMDG, which discriminates cation currents
from chloride and leak currents.
Fig. 1.
Cation currents expressed in CHO cells after
intranuclear injection of
TRPC3. (A) Comparison of
current densities (peak inward currents at 70 mV divided by the cell capacity) in
TRPC3-expressing cells and
control cells (injected with
pcDNA3). All cells additionally expressed GFP and were
identified by their marked
fluorescence. (B) Current-
voltage relation of TRPC3
currents obtained during voltage ramps from
90 to +60 mV in three different bath solutions with the following cation concentrations
(mM): Na+ 140, Ca2+ 1.2 (1); Na+ 131, Ca2+ 10 (2); and NMDG 140 (3). The insert shows a magnified view of the currents at potentials
around 0 mV.
[View Larger Version of this Image (13K GIF file)]
20 ± 8 nM (n = 12; P < 0.01, rank sum test). Thus, expression of TRPC3
markedly enhances Ca2+ influx in response to receptor
stimulation. Therefore, we used electrophysiological techniques to study the mechanisms of TRPC3 activation in
detail. Specifically, we tested whether TRPC3 is store-operated or activated by some other mechanisms.
Fig. 2.
Effects of TRPC3
on [Ca2+]i in CHO cells.
[Ca2+]i was measured with
the fura-2 method in cells
coinjected with cDNAs for
GFP, angiotensin II receptor
AT1A, and either TRPC3 or
pcDNA3 (control). Angiotensin II (1 µM) was added
10 s after the beginning of
the measurements (arrow).
Each trace represents the
mean value of five cells obtained during one experiment; two further experiments showed similar results.
[View Larger Version of this Image (17K GIF file)]
Fig. 3.
Stimulation of TRPC3 currents by angiotensin II. Shown is the tracing of the whole-cell current in a cell expressing TRPC3, GFP,
and the angiotensin II receptor (A) and in a control cell (expressing GFP and the angiotensin II
receptor, B). The whole-cell configuration was
obtained at the time indicated (wc). Angiotensin II (1 µM) was applied at the time point indicated
by the arrow. At the end of the experiment, the
bath was exchanged (arrow) to a solution containing NMDG instead of Na+ and Ca2+. The
holding potential was 70 mV.
[View Larger Version of this Image (20K GIF file)]
Fig. 4.
Stimulation of TRPC3 currents by ionomycin in the presence of extracellular Ca2+. To
a cell expressing TRPC3 and the angiotensin II
receptor, angiotensin II and ionomycin at increasing concentrations were cumulatively
added. See also the legend to Fig. 3.
[View Larger Version of this Image (11K GIF file)]
Fig. 5.
Requirement for extracellular Ca2+ for ionomycin effects on TRPC3 currents. A cell expressing TRPC3 was kept first
in a Ca2+-free solution (5 mM EGTA) and then in a solution with
1.2 mM Ca2+, as indicated by the bars. Extracellular cations were
changed to NMDG in the end. The cell was stimulated with ionomycin (1 µM) present in the bath over the indicated time. The insert shows current traces during voltage ramps from 90 to +60
mV which were obtained at the time points indicated (1, 2, and 3)
in the main trace.
[View Larger Version of this Image (21K GIF file)]
Fig. 6.
Stimulation of TRPC3 currents by intracellular Ca2+.
(A) For whole-cell experiments, a pipette solution with a Ca2+
concentration of 10 µM was used. The bath was changed to an
NMDG solution during the times indicated by the bars. (B) An
experiment in which the pipette solution contained 10 mM
EGTA but no CaCl2. The tracings appear noisier than in the
other figures because they were obtained with a filtering of 5 kHz. The holding potential was 60 mV.
[View Larger Version of this Image (14K GIF file)]
) by correlating the mean current (I) within several
200-ms intervals with the current variance (s) during these
intervals in experiments (n = 3) in which the initial
TRPC3 current declined rapidly. From the linear regression analysis of the s/I plot, we calculated a single-channel
conductance of 45, 49, and 52 pS (at
70 mV; data not
shown). Channels with a comparable size were found in single-channel measurements in the cell-attached and inside-out mode on patches from cells coexpressing GFP
and TRPC3 (n = 92 out of 110), but not in any patches
(n = 34) from control cells (Fig. 7 A). The calculated slope
conductance in inside-out patches was 66 pS (Fig. 7, B and
C). The mean open time was shorter than 0.2 ms, as assessed from the dwell-time distribution of channel events
recorded at 50 kHz and filtered at 5 kHz (Fig. 7 D). The channels were permeant to Na+ as well as to Cs+, but essentially not to NMDG or Cl
, as shown in Fig. 8. In this
experiment, an inside-out patch was exposed first to a bath
(facing the cytosolic side of the channel) containing
NMDG as main cation and then to a bath with 140 mM
Na-gluconate. The pipette contained the standard solution (140 mM CsCl). In NMDG, channel events were observed
at a transmembrane potential of
60 mV, but not at +60
mV. After substitution of NMDG with Na+, channel
events became visible at +60 mV, thus demonstrating that
the currents were carried by Na+ and Cs+, and ruling out
that they were carried by Cl
.
Fig. 7.
Characteristics of
TRPC3 in single-channel
analysis of inside-out patches
from cells expressing
TRPC3 (and GFP). (A) Sample tracings at a membrane
potential of 60 mV (corresponding to a holding potential of +60 mV). (B) Amplitude histogram of channel events at
60 mV, indicating
a mean amplitude of
4.5
pA. (C) Amplitude-voltage
relation of channel openings.
Each point derives from amplitude histograms from 4-25
patches. The regression line yields a single-channel conductance of 66 pS. (D) Open-time distribution of TRPC3
channels. The dwell times
were logarithmically binned, and the amplitudes of the
bins were fitted to a monoexponential function (Sigworth
and Sine, 1987
). Note that
the filtering (5 kHz) precludes recording of short
events, such that the mean
open time can be smaller
than the calculated 0.13 ms.
[View Larger Version of this Image (17K GIF file)]
Fig. 8.
Cation selectivity of TRPC3 channels. (A) A continuous single-channel recording from an inside-out patch when the
bath solution (facing the cytosolic side of the patch) initially contained NMDG as main cation (no Na+, no Ca2+) and was then
changed to a solution with Na-gluconate (140 mM), as indicated
by the bar. The pipette solution facing the extracellular side of
the patch contained Cs+ (140 mM). The membrane potential was
either +60 or 60 mV, as indicated. (B) Time-expanded recordings from the same patch, from times indicated (1, 2, and 3) in A. 1 was taken in NMDG solution at +60 mV, showing the absence
of currents under these conditions. 2 was also taken in NMDG,
but at
60 mV, showing inward currents carried by Cs+. 3 was
taken in Na+ at +60 mV, showing outward currents carried by Na+.
[View Larger Version of this Image (18K GIF file)]
Fig. 9.
Stimulation of TRPC3 channels by ionomycin in a cell-attached patch. The channel activity (expressed as NPo) of
TRPC3 channels in a cell-attached patch was recorded over time.
Ionomycin (0.5 µM) was added to the cell at the time indicated
by the arrow. Note that the bath contained 1.8 mM Ca2+. The inserts show sample channel recordings at two different times. The
holding potential was +60 mV.
[View Larger Version of this Image (12K GIF file)]
Fig. 10.
Absence of stimulation by Ca2+ and calmodulin of TRPC3 channels in
inside-out patches. (A)
Channel activity (expressed
as NPo) when the Ca2+ concentration in the cytosolic solution was changed from 0 to 30 µM. (B) Channel activity
in another patch when the
cytosolic side was initially exposed to a solution with 0 Ca2+ and then to a solution
with 1 µM Ca2+ plus 0.2 µM
calmodulin. Representative
tracings are shown on the
right side. The membrane
potential was 60 mV.
[View Larger Version of this Image (24K GIF file)]
Discussion
), constitutes a Ca2+-permeable channel that enables currents similar to store-operated Ca2+ currents like ICRAC in mast cells (Hoth and Penner, 1992
) and lymphocytes (Zweifach and Lewis, 1993
).
Indeed, expression of TRPC3 yielded whole-cell currents
partly carried by Ca2+. In our hands, however, these
currents were not activated by any measures known to
deplete intracellular calcium stores. Therefore, TRPC3
currents should not be considered store-operated or responsible for capacitative calcium entry.
). In the present
study, however, the effects of ionomycin were strictly dependent on the presence of extracellular Ca2+ and must
therefore be attributed to Ca2+ entry through the plasma
membrane. Thus, we conclude that TRPC3 currents are
activated by Ca2+. This conclusion is supported by our
finding that dialysis of the cells with a pipette solution containing 10 µM Ca2+ led to an increase of the currents considerably above the level of spontaneous activity. On the
other hand, release of Ca2+ from intracellular stores,
evoked by application of ionomycin or dialysis of the cells
with InsP3 or thapsigargin, was obviously not sufficient in
the presence of intracellular EGTA. This might indicate
the requirement for fairly high levels of Ca2+ in the vicinity of the channels, i.e., the submembraneous space. The
steady decline of the currents in the course of the whole-cell experiments are consistent with the decline of [Ca2+]i
by dialysis of the cell interior with the pipette solution, although other mechanisms of current inactivation probably
play a role since the currents decreased also in the presence of ionomycin and elevated intracellular Ca2+ concentrations.
, who showed
that increases in [Ca2+]i induced by stimulation of membrane receptors were prolonged in cells expressing Htrp3
(TRPC3). Our measurements of [Ca2+]i revealed very similar results. We believe, however, that this kind of experiment does not unequivocally reveal the detailed mechanisms of how TRPC3 is activated. Led by the evidence
from our electrophysiological measurements, we propose
that any initial increase in [Ca2+]i by any mechanism will
be enhanced in the presence of TRPC3 because the Ca2+-dependent activation of this Ca2+-permeable channel
provides a positive feedback mechanism leading to Ca2+-induced Ca2+ entry. In general, it may be difficult to discriminate Ca2+-induced from store-operated Ca2+ entry
with the fura-2 method as long as only changes in [Ca2+]i
are measured. A more direct demonstration of cation entry may be required, such as quenching of fura-2 by entry
of manganese, combined with probing the state of intracellular calcium stores. Using this technique, Jacob (1990)
has shown that cation entry into endothelial cells takes
place in the absence of an agonist when calcium stores are
depleted, but not when the stores have been allowed to refill. Alternatively, cation influx may be quantified with the
patch clamp technique that furthermore allows the intracellular application of InsP3 or other substances for store
depletion. In this way, the store-operated regulation of
ICRAC (Hoth and Penner, 1992
), as well as of the human trp
homologues TRPC1A (Zitt et al., 1996
) and bCCE (Philipp et al., 1996
), has been demonstrated. These currents
develop slowly during exchange of the cytosol with the pipette solution, in sharp contrast to the TRPC3 currents.
).
), it
has been shown that this is not true for others. For example, trpl may be directly controlled by G proteins of the
Gq/11 family (Obukhov et al., 1996
) and also stimulated by
Ca2+-calmodulin-dependent processes (Lan et al., 1996
).
Our present study demonstrates a Ca2+-dependent mechanism by which receptor stimulation can lead to the activation of a human trp homologue, with the same mechanism already demonstrated in mammalian calcium channels of
unknown molecular structure (von Tscharner et al., 1986
).
Thus, the trp family may provide a broad spectrum of various Ca2+-permeable channels. Alternatively, proteins of
the trp family may be subunits of heteromultimeric channels on which multiple regulatory pathways may act in
concert, possibly in a cell-specific manner (Birnbaumer et
al., 1996
).
Received for publication 9 May 1997 and in revised form 4 July 1997.
Address all correspondence to Günter Schultz, Institut für Pharmakologie, Thielallee 69-73, Freie Universität Berlin, D-14195 Berlin, Germany. Tel.: 49-30-8386360. Fax: 49-30-8315954.We thank Sabina Naranjo Kuchta and Evelyn Glaß for excellent technical assistance. This work was supported by the Bundesministerium für Forschung und Technologie, Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie.
GFP, green fluorescent protein; InsP3, inositol-1,4,5-trisphosphate; NMDG, N-methyl-D-glucamine.
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