A novel capacitative calcium entry channel expressed in excitable cells

Abstract

In addition to voltage-gated calcium influx, capacitative calcium entry (CCE) represents a major pathway for calcium entry into the cell. Here we report the structure, expression and functional properties of a novel CCE channel, TRP5. This channel is a member of a new subfamily of mammalian homologues of the Drosophila transient receptor potential (TRP) protein, now comprising TRP5 (also CCE2) and the structurally related CCE1 (also TRP4). Like TRP4, TRP5 forms ion channels mainly permeable for Ca2+ which are not active under resting conditions but can be activated by manoeuvres known to deplete intracellular calcium stores. Accordingly, dialysis of TRP5-expressing cells with inositol-(1,4,5)-trisphosphate evokes inward rectifying currents which reversed polarity at potentials more positive than +30 mV. Ca2+ store depletion with thapsigargin induced TRP5-mediated calcium entry dependent on the concentration of extracellular calcium, as seen by dual wavelength fura-2 fluorescence ratio measurements. TRP5 transcripts are expressed almost exclusively in brain, where they are present in mitral cells of the olfactory bulb, in lateral cerebellar nuclei and, together with TRP4 transcripts, in CA1 pyramidal neurons of the hippocampus, indicating the presence of CCE channels in excitable cells and their participation in neuronal calcium homeostasis.

Full text

The EMBO Journal Vol.17 No.15 pp.4274–4282, 1998
A novel capacitative calcium entry channel
expressed in excitable cells
Stephan Philipp
1
, Joerg Hambrecht,
Leonid Braslavski, Gregor Schroth,
Marc Freichel, Manabu Murakami,
Adolfo Cavalie
´
and Veit Flockerzi
Institut fu
¨
r Pharmakologie und Toxikologie der Universita
¨
t des
Saarlandes, D-66421 Homburg/Saar, Germany
1
Corresponding author
e-mail: stephan.philipp@med-rz.uni-sb.de
In addition to voltage-gated calcium influx, capacitative
calcium entry (CCE) represents a major pathway for
calciumentryintothe cell.Herewereportthe structure,
expression and functional properties of a novel CCE
channel, TRP5. This channel is a member of a new
subfamily of mammalian homologues of the Drosophila
transient receptor potential (TRP) protein, now com-
prising TRP5 (also CCE2) and the structurally related
CCE1 (also TRP4). Like TRP4, TRP5 forms ion chan-
nels mainly permeable for Ca
2F
which are not active
under resting conditions but can be activated by
manoeuvres known to deplete intracellular calcium
stores. Accordingly, dialysis of TRP5-expressing cells
with inositol-(1,4,5)-trisphosphate evokes inward recti-
fying currents which reversed polarity at potentials
more positive than F30 mV. Ca
2F
store depletion with
thapsigargin induced TRP5-mediated calcium entry
dependent on the concentration of extracellular cal-
cium, as seen by dual wavelength fura-2 fluorescence
ratio measurements. TRP5 transcripts are expressed
almost exclusively in brain, where they are present in
mitral cells of the olfactory bulb, in lateral cerebellar
nuclei and, together with TRP4 transcripts, in CA1
pyramidal neurons of the hippocampus, indicating the
presence of CCE channels in excitable cells and their
participation in neuronal calcium homeostasis.
Keywords: capacitative calcium entry/InsP
3
/store
depletion/store-operated calcium channel/TRP5
Introduction
Activation of the inositol-(1,4,5)-trisphosphate (InsP
3
) sig-
nalling cascade by neurotransmitters, hormones and
growth factors leads to the formation of InsP
3
which binds
to InsP
3
receptors and thereby initiates the release of Ca
21
from intracellular stores. Depletionof the storesis followed
by the influx of Ca
21
into the cell through store-operated
Ca
21
-selective plasma membrane channels, a mechanism
which has been described as capacitative calcium entry
(CCE; Putney, 1986). Ion currents associated with CCE
have been studied extensively in non-excitable tissues
(Hoth and Penner, 1992; Zweifach and Lewis, 1993;
Lu
¨
ckhoff and Clapham, 1994). However, neurons, like
4274
© Oxford University Press
virtually all cells, have InsP
3
- and ryanodine-sensitive
Ca
21
stores, and it has been envisaged for some time that
in excitable cells Ca
21
release from intracellular stores
and Ca
21
influx into the cell may also be coupled (Stu
¨
hmer
and Parekh, 1993; Gomez et al., 1995; Garaschuk et al.,
1997). Store-operated channels that have been described
so far differ in their biophysical properties, including
ion selectivity and single channel conductance, but their
molecular nature has not been clearly identified.
Two photoreceptor cell-specific gene products have
been isolated from Drosophila melanogaster, which have
been implicated to function as Ca
21
entry channels. The
transient receptor potential (trp) gene product is a protein
of 1275 amino acids with multiple transmembrane domains
(Montell and Rubin, 1989). Heterologous expression of
trp cDNA in eukaryotic cells led to the formation of ion
channels which could be activated by the depletion of
intracellular Ca
21
stores (Petersen et al., 1995) and which
were primarily permeable to Ca
21
(Vaca et al., 1994).
The transient receptor potential-like (trpl) gene product is
39% identical to TRP (Phillips et al., 1992), but in vitro
studies revealed that TRPL is a non-selective cation
channel which is not store operated but constitutively
active (Hu et al., 1994; Hu and Schilling, 1995; Zimmer
et al., 1997). Both proteins may assemble into hetero-
multimeric complexes leading to ion channels with
different ion selectivity compared with those of homo-
multimeric channels (Gillo et al., 1996; Xu et al., 1997). It
has also been proposed that TRP–TRPL heteromultimeric
complexes are associated with additional proteins such as
phospholipase C, protein kinase C and the PDZ-domain
protein inactivation-no-afterpotential D (INAD) (Huber
et al., 1996; Chevesich et al., 1997; Tsunoda et al., 1997).
Recently, the cDNAs of mammalian homologues of the
dipterian TRP/TRPL proteins have been cloned. One
group of cDNAs including htrp1, htrp3 and mtrp6 encodes
Ca
21
-permeable but non-selective cation channels when
expressed in eukaryotic cells (Zitt et al., 1996; Boulay
et al., 1997; Zhu et al., 1998). The recombinant channels
hTRP1 and hTRP3 are active under resting conditions
(Sinkins and Schilling, 1997; Hurst et al., 1998), which
may reflect the lack of auxiliary subunits that may regulate
channel activity. In addition, hTRP3 and mTRP6 appear
to be insensitive to Ca
21
store depletion (Boulay et al.,
1997; Zitt et al., 1997).
A bovine homologue of TRP/TRPL, bCCE (now
bCCE1, also bTRP4) represents the first member of
another group of cDNAs, which—when expressed in
human embryonic kidney (HEK) cells (Philipp et al.,
1996) or Chinese hamster ovary (CHO) cells (Warnat
et al., 1998)—confers a store-operated, highly Ca
21
-
permeable ion channel to these cells. Additional evidence
that TRP4 underlies CCE came from recent studies in
which cDNA fragments of the murine TRP4 in antisense

Molecular and functional analysis of TRP5 channels
orientation were transfected into mouse L cells resulting
in an inhibition of the endogenous CCE (Birnbaumer
et al., 1996).
We have now isolated cDNAs from rabbit and mouse
brain encoding a protein TRP5 which shows significant
homology to the dipterian TRP (41% amino acid identity)
and bovine TRP4 (69% amino acid identity). Expression
of TRP5 in HEK cells was sufficient to induce the
formation of a plasma membrane channel which was
mainly permeable for Ca
21
and was activated by InsP
3
or
thapsigargin via depletion of intracellular Ca
21
stores.
Accordingly, TRP5 together with TRP4 are the first
members of the subfamily of CCE channels structurally
related to TRP/TRPL from Drosophila. Due to the expres-
sion pattern of TRP5 transcripts in brain, the TRP5 channel
may be responsible for CCE in certain neurons.
Results
Primary structure of rabbit and mouse TRP5
We amplified a 133 bp cDNA fragment C (Figure 1A)
from rabbit brain using degenerate oligonucleotides
derived from TRP and TRPL. By screening brain cDNA
libraries, the full-length cDNA sequence of rabbit TRP5
(rTRP5) was obtained. The translation initiation site was
assigned to the first ATG triplet that appears downstream
of a nonsense codon found in-frame. The deduced amino
acid sequence of rTRP5 is composed of 974 residues,
with a calculated molecular mass of 111 533 Da (Figure
1B). Hydropathy analysis reveals a hydrophobic core in
the rTRP5 protein with six peaks likely to represent
membrane-spanning helices (Figure 1C; S1, S2, S3, S4,
S5 and S6) and a putative pore region between S5 and
S6. The hydrophobic core is flanked by long presumptive
cytoplasmic domains at the N- and C-termini. A similar
topology has been proposed for TRP, TRPL and their
known mammalian homologues including bTRP4. Con-
sistent with this topology, two potential cAMP-dependent
phosphorylation sites within rTRP5 (Ser122 and Thr167)
are located in the cytoplasm. As shown in Figure 1B,
rTRP5 shares 68.5% sequence identity with the bTRP4
protein. Their N-termini (residues 1–329 in rTRP5) and
the hydrophobic cores (residues 330–624 in rTRP5) share
considerable amino acid identity of 80.2 and 84.9%,
respectively, whereas the C-terminal regions of rTRP5
and bTRP4 reveal a lower level of identity (42.4%). In
contrast, amino acid sequence comparison of rTRP5
reveals 34.5 (bTRP2), 40.6 (dTRP), 41.1 (dTRPL), 41.7
Fig. 1. Primary structure of TRP5. (A) Cloning strategy: lines indicate
cDNAs with a translation start site at position 11, a bar represents the
corresponding protein. Stop codons in-frame are indicated by asterisks.
(B) The deduced amino acid sequence of TRP5 from rabbit brain
(rTRP5) is shown in alignment with the sequence from mouse brain
(mTRP5) and with the bovine TRP4 sequence (Philipp et al., 1996).
Amino acid residues are numbered on the right; numbering of mTRP5
is in accordance with that of rTRP5. Residues within mTRP5 and
bTRP4 identical to rTRP5 are indicated by blanks, gaps by dashes,
and the location of stop codons by asterisks. Limits of the mTRP5
sequence obtained are denoted by ,.. The putative transmembrane
segments (S1–S6) and the putative pore region are indicated.
(C) Hydropathy profile (Kyte and Doolittle, 1982) of rTRP5:
transmembrane segments S1–S6 were defined as regions with a
hydropathy index ù1.5 using a window of 19 amino acids.
4275
(mTRP6), 42.9 (hTRP3) and 47.3% (hTRP1) identity with
TRP and its homologues.
Using degenerate oligonucleotides corresponding to the
N- (amino acids 1–8) and C-termini (amino acids 959–
965) of rTRP5, the mouse TRP5 (mTRP5) cDNA was

S.Philipp
et al
.
amplified by RT–PCR from mouse brain mRNA as tem-
plate. The mTRP5 cDNA covers a sequence of 369 bp
recently amplified from mouse brain (Zhu et al., 1996).
The mTRP5 protein shares 97.5% amino acid sequence
identity with rTRP5, with few exchanges, mainly within
the C-terminus, indicating a high conservation throughout
evolution.
Expression of TRP5 in excitable cells of the
brain—co-expression with TRP4
By Northern analysis, we found that TRP5 mRNA is
expressed predominantly in the brain. The rTRP5 probe
hybridized with transcripts of 4.2, 8.0 and 10.5 kb in
rabbit brain and to a much lower degree in kidney (Figure
2A). No TRP5 transcripts were detected in liver, ureter,
ovary, lung, aorta, spleen and thymus, or in adrenal gland
and testis, where TRP4 transcripts primarily are expressed
(Philipp et al., 1996). The 4.2 kb mRNA corresponds to
the size of the rTRP5 cDNA (4549 bp) cloned from rabbit
brain (Figure 1A). The longer transcripts may result from
alternative mRNA processing. In fact, cDNA clones,
which differ in their 59- and 39-untranslated sequences,
were isolated from the cDNA libraries employed. In
human brain, the rTRP5 probe hybridized with transcripts
of 10.5 kb (Figure 2B), indicating a different pattern of
hybridization signals as compared with rabbit mRNA,
presumably due to species-specific TRP5 gene structure
and mRNA processing. The TRP5 mRNA in human brain
is present in the cerebellum and the occipital pole, and at
a lower level in the medulla and the frontal lobe. To
localize the transcripts in cerebellum, we used in situ
hybridization histochemistry. As shown in Figure 2C,
mTRP5 mRNA is present in neurons of the lateral cerebel-
lar nucleus. In addition, TRP5 transcripts were detected
in the entorhinal cortex (not shown). Thus, the main
expression of TRP5 seems to occur in excitable cells of
the brain, in contrast to the expression of other mammalian
TRP-related proteins which are expressed predominantly,
but not exclusively, in non-neuronal tissues (Wes et al.,
1995; Philipp et al., 1996; Zhu et al., 1996; Boulay et al.,
1997; Wissenbach et al., 1998).
At present, we do not know whether CCE channels
are heteromultimeric in nature and whether TRP5 co-
assembles with other TRP homologues, e.g. TRP4. In the
mouse brain, we found TRP5 transcripts throughout the
Ammon’s horn (CA) of the hippocampus (Figure 3A, left
panel). Using TRP4 cRNA as probe, a very similar
hybridization pattern was obtained (Figure 3A, right
panel), indicating that both channels might be expressed
in the same cells. In fact, TRP5 and TRP4 transcripts
appear to be co-expressed in CA1 hippocampal pyramidal
neurons (Figure 3B). TRP5 and TRP4 mRNAs were also
detected in cells within the olfactory bulb (Figure 3C and
D). TRP5 transcripts seem to be restricted to the mitral
cell layer and the glomerular layer (Figure 3C and D, left
panel), whereas TRP4 transcripts are expressed in the
internal granular layer (Figure 3C, right panel), but appear
not to be present in mitral cells (Figure 3D, right panel).
Apparently, depending on the tissue and the cell type, co-
expression of TRP5 and TRP4 transcripts may occur.
The TRP5 protein is a CCE channel
To examine the functional properties of TRP5, HEK cells
were transiently transfected with the cDNA of rTRP5. We
4276
Fig. 2. TRP5 is expressed predominatly in brain. Northern blot
analysis of rTRP5 expression in rabbit tissues (A) and in human
brain (B). Lower panels show signals after hybridization of human
GAPDH cDNA to the same filters. (C) In situ hybridization of mTRP5
antisense cRNA to a horizontal section of the mouse lateral cerebellar
nucleus (dark-field microscopy). A white rectangle indicates the
location of a magnification shown as an inset (bright-field
microscopy). Bars represent 100 and 10 µm (inset).
used a dicistronic expression vector (see Materials and
methods) which contains the rTRP5 cDNA upstream of
an internal ribosome entry site (IRES) followed by the
cDNA of the green fluorescent protein (GFP). The IRES
sequence allows the simultaneous translation of TRP5 and
GFP from one transcript. Thus, transfected cells can be
detected unequivocally by the development of green
fluorescence. In order to empty intracellular Ca
21
stores,
the transfected cells were dialysed with 10 µM InsP
3

Molecular and functional analysis of TRP5 channels
Fig. 3. TRP5 and TRP4 are co-expressed in hippocampal cells but not in the olfactory bulb. In situ hybridization of mTRP5 (left panels) and
mTRP4 (right panels) antisense cRNA to frontal sections through the hippocampus (A and B) and the olfactory bulb (C and D) of a mouse brain.
Insets show controls using sense cRNA probes. White rectangles indicate the locations of magnifications shown in (B) and (D) (bright-field
microscopy). Bars represent 100 µm (A, C and insets, dark-field microscopy) and 10 µm (B and D). CA, Ammon’s horn; PyC, CA1 pyramidal cells;
IGr, internal granular layer; Mi, mitral cell layer; EPl, external plexiform layer; Gl, glomerular layer; MiC, mitral cell. Note the expression signals in
pyramidal cells and their presence or absence in mitral cells (arrowheads).
via a patch–clamp pipette. Additionally, the dialysate
contained 10 mM EGTA to buffer the intracellular Ca
21
below 50 nM. Whole-cell currents were elicited with
voltage-clamp ramps from –100 to 160 mV every 5 s
(holding potential 0 mV) in the presence of 10 mM
external Ca
21
. As the dialysis of the cells with InsP
3
progressed, a clear increase of inward currents was
observed (Figure 4A). The inward currents develop 200 s
after the onset of InsP
3
dialysis whereas the outward
current remained stable throughout the experiment (Figure
4B). Analysis of leak-subtracted currents (Figure 4C)
showed that TRP5 mediated inwardly rectifying currents.
As expected for the dicistronic expression of TRP5 and
GFP, similar inwardly rectifying currents were detected
in eight out of nine green fluorescent cells. On average,
4277
the current density of TRP5-transfected cells was 6.71 6
2.81 pA/pF (n 5 8) at –80 mV. As previously reported
(Philipp et al., 1996), InsP
3
also activated inward currents
in non-transfected cells. The density of leak-subtracted
inward currents was 0.57 6 0.21 pA/pF (n 5 15) at
–80 mV in non-transfected HEK cells after dialysis with
10 µM InsP
3
. Similar results were also obtained in HEK
cells that were mock transfected with the same dicistronic
vector containing lacZ instead of TRP5 (0.41 6 0.13 pA/
pF at –80 mV, n 5 3). Thus, expression of TRP5 in HEK
cells increased the amplitude of inward currents activated
by InsP
3
~11-fold.
The results shown in Figure 5 demonstrated that recom-
binant TRP5 currents display the typical features of
currents associated with CCE, i.e. strong inward rectifica-

S.Philipp
et al
.
Fig. 4. Inward rectifying currents of TRP5-expressing HEK cells after
store depletion. (A) Representative whole-cell currents in TRP5-
transfected HEK cells during the dialysis with 10 µM InsP
3
in the
presence of 10 mM extracellular Ca
21
, obtained at the times indicated
and displayed without subtraction of leak currents. (B) Time course of
current amplitudes at –80 mV and 140 mV. (C) Leak-subtracted
current–voltage relationship of a TRP5-expressing cell 318 s after
InsP
3
dialysis.
tion and a reversal potential above 130 mV. Additionally,
these results indicate that recombinant TRP5 channels are
probably Ca
21
selective like those detected in TRP4-
transfected cells (Philipp et al., 1996; Warnat et al., 1998).
Therefore, it can be expected that the CCE of cells
expressing TRP5 is highly sensitive to changes in the
extracellular Ca
21
concentration. We tested this idea by
comparing the extracellular Ca
21
concentration ([Ca
21
]
o
)
dependence of CCE in control and TRP5-transfected cells.
For these experiments, we established a HEK cell line,
HekCCE2, that stably expresses the rTRP5 cDNA. As
illustrated in Figure 5A, Northern analysis revealed the
expression of 4.1 and 5.3 kb transcripts in HekCCE2 cells
whereas no hybridization signals were detected in non-
transfected HEK control cells. The length of the 5.3 kb
mRNA in the HekCCE2 cells corresponds to the expected
size for transcription of TRP5 in the dicistronic expression
vector, whereas the 4.1 kb mRNA may arise from alternate
RNA processing or cryptic transcription initiation
(Kaufman et al., 1991).
Measurements of the cytosolic free Ca
21
concentration
([Ca
21
]
i
) were conducted in steady state and during
transient changes in the [Ca
21
]
o
below physiological levels
to detect a possible Ca
21
-selective entry. In cells that were
equilibrated in a solution containing 500 µM [Ca
21
]
o
, the
steady-state [Ca
21
]
i
was 107.7 6 22.9 nM (n 5 14),
independent of TRP5 expression (Figure 5B). As has been
reported for various cell types (e.g. Montero et al., 1990),
the removal of extracellular Ca
21
with EGTA induced
store depletion in HEK cells (not shown). Therefore,
.10 min equilibration was usually required to attain
steady-state [Ca
21
]
i
levels. After prolonged equilibration
in a Ca
21
-free solution, the [Ca
21
]
i
levels dropped close
to 50 nM. Subsequent equilibration in 500 µMCa
21
restored the basal [Ca
21
]
i
both in controls and in TRP5-
expressing cells to 165.8 6 42.2 nM (n 5 8, Figure 5B).
These results suggest that expression of TRP5 does not
alter basal [Ca
21
]
i
levels in HEK cells. A further implica-
tion of these results is that the majority of recombinant
4278
channels formed by TRP5 are probably not spontaneously
active but only become active upon depletion of Ca
21
stores, as can be inferred from the experiment shown in
Figure 4. Therefore, we analysed [Ca
21
]
i
levels in cells
equilibrated in a nominal Ca
21
-free solution after treatment
with thapsigargin (Figure 5C). After readdition of 500 µM
[Ca
21
]
o
, [Ca
21
]
i
increased more rapidly and to higher
levels in HekCCE2 cells than in controls. When [Ca
21
]
o
in the solution used in the Ca
21
readdition step was
varied between 10 µM and 3 mM, the [Ca
21
]
o
–[Ca
21
]
i
relationship in HekCCE2 was much steeper than in control
cells (Figure 5D). Additionally, the [Ca
21
]
o
–[Ca
21
]
i
curve
of HekCCE2 cells was shifted to concentrations of external
Ca
21
~10 times lower when compared with the [Ca
21
]
o
–
[Ca
21
]
i
curve of controls. At 3 mM [Ca
21
]
o
, the [Ca
21
]
i
levels of HekCCE2 cells were 1.7 times higher than in
controls. As illustrated in Figure 5E, 3-fold higher [Ca
21
]
i
levels were also observed after readdition of 3 mM [Ca
21
]
o
to HekCCE2 cells that were incubated in thapsigargin plus
EGTA for 30 min (HekCCE2: 436.1 6 77.3 nM, n 5 8;
control cells: 142.8 6 52.8 nM, n 5 12).
Using the [Ca
21
]
i
–[Ca
21
]
o
curves of Figure 5D, the
EC
50
can be estimated to be ~70 µM [Ca
21
]
o
and1mM
[Ca
21
]
o
for HekCCE2 and control cells, respectively.
These EC
50
values are probably underestimates because
no saturation was observed below 3 mM [Ca
21
]
o
, but the
EC
50
of control cells lies within the range reported
previously (Takemura and Putney, 1989). The straightfor-
ward interpretation of the results of Figure 5D is that
expression of TRP5 increased the Ca
21
sensitivity of the
CCE naturally present in HEK cells in two ways: (i) CCE
occurs at lower [Ca
21
]
o
and (ii) the dependence of CCE
on [Ca
21
]
o
appears to be much steeper than in control
cells. Taken together, these results support the findings of
the patch–clamp experiments that TRP5forms capacitative
Ca
21
channels which are highly Ca
21
selective.
Discussion
In this study, we describe the structure of the CCE channel
TRP5, the functional properties of the recombinant channel
and the expression of its mRNA in excitable cells of the
brain. TRP5 shares 68.5% amino acid sequence identity
with TRP4 but has significantly lower sequence homology
(,48%) to other TRP-related proteins. Figure 6 shows
the evolutionary relationship among mammalian TRP
homologues. At least three subfamilies can be distin-
guished, with one subclass comprising TRP5 and TRP4
(Figure 6B). The sequences of the various TRP1 ortho-
logues are the most closely related to TRP5 and TRP4 in
terms of their evolutionary distance, whereas TRP3 and
TRP6 belong to an independent and less related subclass.
TRP2 might be a member of a fourth subclass; however,
it has not been functionally expressed so far. This evolu-
tionary classification apparently corresponds to functional
features of the channel proteins including their activation
mechanisms: TRP1, like TRP5 and TRP4, appears to be
activated by Ca
21
store depletion, but underlies non-
selective cation currents (Zhu et al., 1996; Zitt et al.,
1996), whereas the trp3 and trp6 cDNAs encode non-
selective cation channels stimulated by G protein-coupled
receptors rather than by depletion of intracellular Ca
21
stores (Zitt et al., 1997; Hurst et al., 1998; Zhu et al., 1998).

Molecular and functional analysis of TRP5 channels
Fig. 5. Store-operated calcium entry in stably TRP5-expressing HEK cells. (A) Northern blot analysis of TRP5 transcripts in clone HekCCE2 and
non-transfected HEK control cells (upper panel) as well as GAPDH mRNA (lower panel). (B) Measurements of steady-state [Ca
21
]
i
levels in
HekCCE2 cells (upper panel) and in control cells (lower panel) equilibrated in solutions containing either 500 µM [Ca
21
]
o
or no extracellular Ca
21
(500 µM EGTA). Broken lines represent 20 min equilibration periods. (C) Transient [Ca
21
]
i
changes after store depletion. Cells were kept in
nominal Ca
21
-free buffer before 2.5 µM thapsigargin (arrow) was added to deplete intracellular stores. Upon readdition of 500 µM calcium to the
bath, the [Ca
21
]
i
increased in HekCCE2 cells (upper panel) faster and to levels higher than in control cells (lower panel). Representative traces of
three independent cells are displayed. (D) [Ca
21
]
i
–[Ca
21
]
o
relationship. [Ca
21
]
i
was measured 300 s after readmission of various concentrations of
external Ca
21
in experiments as illustrated in (C) with HekCCE2 cells (d) and control cells (s). Values are given as means 6 SEM. (E) HekCCE2
and control cells were incubated for 30 min in nominal free extracellular Ca
21
in the presence of 5 µM thapsigargin and 2 mM EGTA. At the time
indicated, 3 mM Ca
21
was introduced into the bath. Representative traces of three independent cells are displayed.
So far, TRP4 and TRP5 are the only members of the
family of mammalian TRP homologues possessing the
basic functional features of CCE channels, i.e. they appear
to be store-operated and they are Ca
21
selective. To
emphasize these properties, TRP4 and TRP5 might also be
called CCE1 (Philipp et al., 1996) and CCE2, respectively.
In HEK cells, CCEmediated by TRP5/CCE2is activated
by Ca
21
store depletion either by cell dialysis with InsP
3
in the presence of 10 mM EGTA or by cell perfusion with
thapsigargin. This CCE depends on the [Ca
21
]
o
and is
most obvious at concentrations above 50 µMupto1mM.
At higher [Ca
21
]
o
, TRP5/CCE2-mediated Ca
21
entry
might be difficult to distinguish from the CCE endogenous
to HEK cells (e.g. at 3 mM [Ca
21
]
o
, Figure 5D). Accord-
ingly, Okada et al. (1998) reported that thapsigargin
4279
increased [Ca
21
]
i
in TRP5-transfected HEK293 cells to
levels similar to those incontrol cells at a [Ca
21
]
o
of 2 mM.
Although TRP5/CCE2 and TRP4/CCE1 are more
closely related to each other than to any other of the
known mammalian TRP homologues, their C-terminal
regions revealed only a few similarities. Nevertheless,
these regions account for the overall length of the two
proteins, which are significantly longer (974 and 981
amino acid residues) than any of the other mammalian
clones reported so far. This may be significant since the
C-terminal region seems to confer thapsigargin sensitivity
to TRP channels and might, therefore, be involved in
linking plasma membrane channels to store depletion
(Sinkins et al., 1996). Additionally, it has been shown
that in Drosophila photoreceptor cells, TRP interacts with

S.Philipp
et al
.
Fig. 6. Phylogenetic tree of mammalian TRP/TRPL homologues. Only
complete coding cDNA sequences of mammalian TRP/TRPL
homologues have been included. TRPC1 (Wes et al., 1995) and
TRPC1A (Zitt et al., 1996) are variants of hTRP1 apparently encoded
by the same gene. The DDBJ/EMBL/GenBank accession No. of
bTRP2 is AJ006304.
a PDZ-domain of the INAD protein via its C-terminus
(Shieh and Zhu, 1996), integrating the TRP channel into
a multi-signalling complex (Huber et al., 1996; Chevesich
et al., 1997; Tsunoda et al., 1997). Interestingly, the
C-terminal five amino acid residues VTTRL of TRP5
comprise the amino acid motif TXL resembling the known
PDZ-domain interaction site S/TXV of other ion channels
(Kornau et al., 1997). This sequence motif of TRP5 is
conserved in the TRP4 protein and may be responsible
for binding of TRP4 and TRP5 to mammalian INAD-like
proteins, as for example hINADL (Philipp and
Flockerzi, 1997).
Recent reports provided evidence for an interaction of
TRP and TRPL which might contribute channel subunits
to form heteromultimeric channels (Gillo et al., 1996; Xu
et al., 1997). TRP5 may be a subunit of a homo- or a
heteromultimeric complex composed of TRP5 and, for
example, another mammalian TRP-related protein. At
present, the actual subunit arrangement of CCE channels
is unknown, and we cannot rule out the possibility that
endogenous proteins may also contribute to the new
currents detected in TRP5-transfected cells. As shown in
Figure 3, formation of heteromultimeric TRP5–TRP4
channels may occur in CA1 pyramidal neurons of the
hippocampus. However, the specific expression of both
genes in different cells and tissues implies that TRP5 and
TRP4 function as store-operated Ca
21
channels independ-
ently of each other.
TRP5 transcripts were also detected in the olfactory
bulb and the lateral cerebellar nuclei. So far, CCE in
neurons has been implicated to be involved in the migration
of growth cones (Gomez et al., 1995) but, in contrast to
its well-established functional roles in non-excitable cells,
little is known about its contribution to neuronal Ca
21
homeostasis. In pyramidal neurons, refilling of ryanodine-
sensitive stores requires a transmembrane Ca
21
influx
pathway that is active at the resting membrane potential
(Garaschuk et al., 1997). To account for this Ca
21
influx,
4280
the existence of a Ca
21
entry pathway in the plasma
membrane has been hypothesized (Garaschuk et al., 1997)
which is activated by depletion of ryanodine-sensitive
Ca
21
stores. This mechanism would be analogous to the
CCE, activated by agonist-induced depletion of InsP
3
-
sensitive Ca
21
stores.
In conclusion, we have shown that TRP5 is activated
by Ca
21
store depletion and, therefore, is responsible for
CCE. The recombinant TRP5 currents display inward
going rectification and are reversed at positive potentials,
indicating that TRP5 channels are calcium selective.
Accordingly, expression of TRP5 increase the Ca
21
sensit-
ivity of CCE in the host cells. The cloning of TRP5 and
the characterization of its function and expression pattern
in the brain will facilitate efforts to identify the role of
CCE in excitable cells.
Materials and methods
Cloning of TRP5 from rabbit brain (rTRP5) and mouse brain
(mTRP5)
Using degenerate oligonucleotide primers corresponding to conserved
amino acidregionsin TRP and TRPL (W
644
GLLMFG and E
682
WKFART,
numbering according to TRPL), the 133 bp cDNA fragment C (nucleo-
tides 1799–1931, Figure 1A and B) was amplified from rabbit brain
poly(A)
1
RNA by RT–PCR. This fragment showed significant amino
acid sequence homology to TRP and TRPL and was used as a probe to
screen an oligo(dT)-primed cDNA library from rabbit brain. Three
independent clones were obtained, including N45 (nucleotides 1584–
3773, Figure 1A). The 59 nucleotide sequence was identified by screening
a specifically primed rabbit brain cDNA library using a synthetic primer
complementary to nucleotides 1886–1867 for reverse transcription and
a cDNA fragment covering nucleotides 1584–1718 as probe. Eight
independent clones were analysed, including clone M14 (Figure 1A).
All cDNA clones obtained revealed sequences identical within the
protein coding region. All cDNAs were sequenced on both strands using
a laser fluorescence DNA sequencer. To obtain the cDNA sequence of
mTRP5, two degenerate oligonucleotides corresponding to amino acids
M
1
AQLYYKK (59-ATG GCN CAR YTN TAY TAY AAR AA-39) and
M
960
HKWGDG (59-CCR TCN CCC CAY TTR TGC AT-39) of rTRP5,
respectively, were used to amplify a 2897 bp cDNA fragment which
was subcloned. Four independent cDNA clones were sequenced on both
strands. The nucleotide sequences of rTRP5 and mTRP5 cDNA have
been deposited in DDBJ/EMBL/GenBank under the accession Nos
AJ006203 and AJ006204, respectively.
Northern blot analysis
Northern blot analysis was performed as described (Philipp et al.,1996).
The probe was the –1 to 2571 nucleotide fragment spanning most of the
protein coding region of rTRP5 (Figure 1A) labelled by random priming
with [α-
32
P]dCTP. For analysis of rabbit tissues,10 µg of poly(A)
1
RNA
were applied. A human brain multiple tissue Northern blot (Clontech)
was hybridized under the same conditions but at 37°C. As a control for
the integrity and amount of the transferred RNA, all filters were
stripped and rehybridized with a 239 bp cDNA fragment of the human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Filters were
exposed to X-ray films for 9 days (TRP5 probe), 3 days (GAPDH probe,
rabbit tissues) or 2 h (GAPDH probe, human tissues).
In situ hybridization
Sections (8 µm thick) from freshly frozen adult mouse brains were
mounted on silane-coated glass slides and immersed in 4% paraform-
aldehyde (PFA) in phosphate-buffered saline (PBS; Sambrook et al.,
1989). After two washing steps in PBS for 5 min, the sections were
dehydrated through a series of 2 min incubations in 30, 60, 80, 95 and
100% ethanol, air dried and stored in desiccant at –80°C. For hybridiz-
ation, slides were rehydrated and treated with 1 µg/ml proteinase K in
100 mM Tris–HCl, 50 mM EDTA (pH 7.5) at 37°C for 30 min. After
refixation in 4% PFA/PBS, the specimens were immersed in 100 mM
triethanolamine-HCl (pH 8.0) and twice in 0.25% acetic anhydride/
0.1 M triethanolamine-HCl (pH 8.0), dehydrated and air dried. As a
probe, we subcloned a 1497 bp cDNA fragment corresponding to amino

Molecular and functional analysis of TRP5 channels
acids 467–965 of mTRP5 (Figure 1B) in pBluescript. As a probe for
detection of mouse TRP4, we used a 528 bp cDNA fragment amplified
with primers 59-ACA GTG ATC TGA ACC CAC GG-39 (Freichel et al.,
1998) and 59-ATT CTA TCT GCA TGG TCG GC-39 (Petersen et al.,
1995) covering part of the hydrophobic core region of mTRP4. Sense
and antisense RNAprobes were synthesized with SP6and T7 polymerase,
respectively, in the presence of [α-
33
P]UTPusing the linearized recombin-
ant plasmid. The cRNA probes were purified by gel chromatography,
and the probe length was adjusted to 200 bases by alkaline treatment.
The probes were precipitated and dissolved at a concentration of
60 ng/ml of hybridization solution containing 0.02% Ficoll, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin, 0.5 mg/ml yeast
tRNA, 10% dextran sulfate and buffer A [10 mM dithiothreitol (DTT),
50% formamide, 30 mM NaCl, 10 mM NaH
2
PO
4
/Na
2
HPO
4
,5mM
EDTA, 20 mM Tris–HCl, pH 6.8]. Hybridization was carried out with
8 µl of solution per section. Sections were covered with parafilm,
incubated in a moist chamber at 55°C overnight followed by sequentially
rinsing the slides twice in buffer A at 55°C for 60 min, twice in 0.5 M
NaCl, 10 mM Tris–HCl, 5 mM EDTA pH 7.5 (NTE) at 37°C for 15 min
and once in 20 µg/ml of RNase A in NTE at 37°C for 30 min. After a
further 15 min incubation in NTE at 37°C and 60 min in buffer A at
55°C, the slides were washed twice at room temperature in 23 SSC,
and once at 65°C and once at room temperature in 0.13 SSC for 15 min
each. Finally, the sections were dehydrated through a graded series of
ethanol containing 300 mM ammonium acetate followed by two washes
in 100% ethanol and air drying. After exposure to an X-ray film for
5 days, the slides were dipped in Kodak NTB-2 emulsion and exposed
for 3 weeks. Sections were stained with haematoxylin and eosin and
photographed using a Contax 167 MT camera adapted to a Zeiss
Axioskope microscope.
Construction of the dicistronic TRP5/GFP expression
plasmid, transient transfection and selection of stable
TRP5-expressing HEK cells
To obtain the recombinant dicistronic expression plasmid pdiCCE2
carrying the entire protein coding regions of rTRP5 and the GFP (Prasher
et al., 1992), the 59- and 39-untranslated sequences of the rTRP5 cDNA
were removed, the consensus sequence for initiation of translation in
vertebrates (Kozak, 1987) was introduced immediately 59 of the transla-
tion initiation codon and the resulting cDNA was subcloned in pcDNA3
downstream of the cytomegalovirus promoter. The IRES derived from
encephalomyocarditis virus (Mountford et al., 1994) followed by the
GFP cDNA containing a Ser65Thr mutation (Heim et al., 1995) was
then cloned 39 to the rTRP5 cDNA. When indicated, the same plasmid
containing the lacZ cDNA instead of the TRP5 cDNA was used as
control (pdiLacZ).
For transient expression of TRP5, HEK cells (ATCC CRL 1573) were
transfected with pdiCCE2 using lipofectamine (Lifetechnologies) as
described previously (Philipp et al., 1996). Electrophysiological record-
ings of cells showing green fluorescence were performed 48–72 h post-
transfection. Stable cell lines including HekCCE2 were selected by
addition of 400 µg/ml geneticin and by their green fluorescence.
Electrophysiological recordings
HEK cells transfected with pdiCCE2 were detected by development of
green fluorescence. As controls, either non-transfected HEK cells or
pdiLacZ mock-transfected cells were used. Single cells were voltage-
clamped in the whole-cell mode (Hamill et al., 1981) using an EPC-9
(HEKA) patch–clamp amplifier as described (Philipp et al., 1996). The
pipette solution contained 115 mM CsCl, 4 mM MgCl
2
, 10 mM EGTA,
10 mM HEPES (pH 7.4 adjusted with CsOH). The bath solution
contained 115 mM NaCl, 5 mM KCl, 2 mM MgCl
2
, 10 mM CaCl
2
,
10 mM HEPES (pH 7.4 adjusted with NaOH). Whole-cell currents were
elicited by 250 ms voltage-clamp ramps from –100 mV to
1 60 mV every 5 s (0.64V/s). For calculation of current densities, the
membrane capacitance (C
m
) was read from the settings provided by the
amplifier after automatic cancellation of transients occurring in the
whole-cell mode. Inward and outward currents were evaluated as the
mean current amplitude measured within a 10 mV window placed at
–80 mV and 140 mV, respectively. Data are given as means 6 SD.
Measurements of [Ca
2
F
]
i
in stable transformed HEK cells
Measurements of [Ca
21
]
i
in single HEK cells were performed with a
digital imaging system (T.I.L.L. Photonics). Cells grown on coverslips
for 3 days were loaded with 1 µM fura-2/AM (Molecular Probes) for
30 min at 37°C in minimal essential medium containing 10% fetal calf
serum. Cells were washed three times with 300 µl of a buffer containing
4281
115 mM NaCl, 2 mM MgCl
2
, 5 mM KCl, 10 mM HEPES (pH 7.4).
The extracellular Ca
21
concentration was varied between 10 µM and
3 mM. Nominal Ca
21
-free solutions contained ~2 µM [Ca
21
]
o
.In
some experiments, EGTA was added to the external solution at the
concentrations given in the text. [Ca
21
]
i
was calculated from fluorescence
ratios obtained at 340 and 380 nm excitation wavelengths essentially as
described (Garcia et al., 1994). Experiments were repeated three times,
with measurements ofthree cells in each experiment. A modified protocol
was applied (Zitt et al., 1996) in some experiments (Figure 5E) in which
eight cells were measured in three independent assays. Unless otherwise
stated, values are given as means 6 SD.
Miscellaneous methods
Sequences were analysed using the Heidelberg Unix Sequence Analysis
Resources (HUSAR) of the biocomputing unit at the German Cancer
Research Centre Heidelberg. The phylogenetic distances of proteins
were calculated with the clustal/clustree program (Saitou and Nei, 1987;
Thompson et al., 1994), and the similarity of protein sequences in pairs
with the bestfit program (Needleman and Wunsch, 1970). Photographs
were scanned and processed using Corel Photo-Paint/Corel Draw and
Adobe PhotoShop.
Acknowledgements
We thank Ute Soltek and Martin Simon-Thomas for excellent technical
assistance, Peter Wollenberg for helpful discussions, and Herbert
Schwegler for advice concerning the histology of the brain. This work
was supported in part by the Deutsche Forschunggemeinschaft and the
Fonds der Chemischen Industrie.
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Received April 7, 1998; revised June 3, 1998; accepted June 4, 1998