TRPC5 as a candidate for the nonselective cation channel
activated by muscarinic stimulation in murine stomach
Young Mee
Lee*,
Byung Joo
Kim*,
Hyun Jin
Kim,
Dong Ki
Yang,
Mei Hong
Zhu,
Kyu Pil
Lee,
Insuk
So, and
Ki Whan
Kim
Department of Physiology and Biophysics, Seoul National
University College of Medicine, Seoul 110 - 799, Korea
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ABSTRACT |
We investigated which transient
receptor potential (TRP) channel is responsible for the nonselective
cation channel (NSCC) activated by carbachol (CCh) in murine stomach
with RT-PCR and the electrophysiological method. All seven types of TRP
mRNA were detected in murine stomach with RT-PCR. When each TRP channel was expressed, the current-voltage relationship of mTRP5 was most similar to that recorded in murine gastric myocytes. mTRP5 showed a
conductance order of Cs+ > K+ > Na+, similar to that in the murine stomach. With 0.2 mM
GTP
S in the pipette solution, the current was activated transiently
in both NSCC in the murine stomach and the expressed mTRP5. Both NSCC
activated by CCh in murine stomach and mTRP5 were inhibited by
intracellularly applied anti-Gq/11 antibody, PLC inhibitor U-73122, IICR inhibitor 2-aminoethoxydiphenylborate, and nonspecific cation channel blockers La3+ and flufenamate. There
were two other unique properties. Both the native NSCC and mTRP5
were activated by 1-oleoyl-2-acetyl-sn-glycerol. Without the activation
of NSCC by CCh, the NSCC in murine stomach was constitutively active
like mTRP5. From the above results, we suggest that mTRP5 might be a
candidate for the NSCC activated by ACh or CCh in murine stomach.
transient receptor potential protein
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INTRODUCTION |
IT IS WELL KNOWN THAT
ACH and carbachol (CCh) induce depolarization and,
consequently, cause the contraction of mammalian gastrointestinal
smooth muscle. In mammalian gastrointestinal smooth muscle cells,
muscarinic agonists bind to M2 and M3 muscarinic receptors (27,
39) and then activate a nonselective, voltage-sensitive inward
current (3). On the other hand, muscarinic agonists inhibit the outward current in the toad stomach (42).
Since the first report by Benham et al. (3), the
characteristics of nonselective cation channel (NSCC) activated by ACh
(NSCCACh) or CCh (NSCCCCh) have been
reported. First, the channel is voltage dependent. Initial
reports of the voltage-dependent activation indicated a half-maximal
activation potential (V1/2) of
50 mV and steepness factor
(k) of
15 mV (14, 18, 42). However, the V1/2 value depended on the concentrations of agonists
and the type of extracellular cations used for current recordings (19). Second, the channel has the similar permeability to
Na+, K+, Cs+, and Li+
(15, 23, 45). It is also permeable to Ca2+
(21). Third, its activation depends on G protein activity
(13, 26). We have also showed that Go type
among GTP-binding proteins is responsible for activating the channel
(22). Fourth, the unitary conductance was ~25-30 pS
(3, 13, 18, 42). The open probability is modulated by
extracellular monovalent cation (18). Fifth, it is
regulated by intracellular Ca2+ concentration
([Ca2+]i) and calmodulin (22).
There is also a desensitization phenomenon depending on
[Ca2+]i and protein kinase C (1,
23).
In many tissues, mammalian homologs of the Drosophila
transient receptor potential (TRP) channel family (TRPC1-7) have
been implicated as molecular candidates for the receptor-operated
Ca2+ entry channels (ROCC) and store-operated
Ca2+ channels (SOCC). ROCCs are activated by G
protein-coupled receptor-PLC (GPCR-PLC) pathway and independently of
store depletion by various messengers of the signal transduction. SOCCs
are activated by depletion of intracellular Ca2+ stores
(8, 32). In contrast to the abundance of reports on the
TRP channels in heterologous expression systems, relatively little
information is available on their role in native tissues. In most
studies on the role of TRPCs in native tissues, these channels have
been implicated as a component of SOCC (2, 5, 6, 30, 33, 38,
46). However, a role for TRPCs in store-independent ROCCs has
also been proposed (16, 17, 28). It is suggested that
second messengers such as G proteins, inositol 1,4,5-trisphosphate (IP3), diacylglycerol (DAG), arachidonic acid, and
Ca2+ directly activate TRP channels.
Although smooth muscle cells are known to express voltage-activated
Ca2+ channels, the NSCCs stimulated by GPCR
(
1-adrenoceptor in vascular smooth muscle or muscarinic
receptor in visceral smooth muscle)-PLC activation form an additional
important Ca2+-entry pathway, i.e., ROCCs in smooth muscle
cells. The TRP6 was shown to be the molecular identity for
1-adrenoceptor-activated NSCC permeable to
Ca2+ (16). Likewise, TRPC6 was proposed to be
a molecular component of ROCCs in A7r5 smooth muscle cells
(17). For the NSCC activated by ACh, Schaefer et al.
(40) suggested that mouse (m) TRP4 and 5 might be
candidates. We tried to identify the molecular candidate for
the NSCC activated by CCh. To pursue the goal, we planned to record the
NSC current in the single cells isolated from the murine stomach and
then compare the properties of NSCCCCh in the murine
stomach with those of mTRP5.
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METHODS AND MATERIALS |
Molecular biological methods.
Ion-channel genes were transiently expressed in Chinese hamster ovary
(CHO) or human embryonic kidney (HEK) cells using the pFx-8 cationic
lipid transfection reagent (Invitrogen) according to the
manufacturer's instructions. As a marker of transient transfection in
CHO or HEK cells, plasmid DNA (pEGFP-N1) containing the cDNA for Green
Fluorescent Protein (Clontech) was cotransfected with the TRP cDNAs.
Single-cell dissociation of mouse stomach.
Gastric myocytes were isolated enzymatically from the antral region of
the Institute for Cancer Research (ICR) mouse. Mice of either
sex weighing 20-30 g were anaesthetized with carbon dioxide and
killed by cervical dislocation. The antral part of the stomach was cut,
and the mucous layer was dissected from the smooth muscle layer using
fine scissors and cut into small segments (~2-3 mm). The tissue
chunks were then incubated for ~20-25 min at 37°C in a
digestion medium that was a Ca2+-free Tyrode solution (see
Solutions and drugs) containing 0.1% collagenase (Wako or
Sigma type IA), 0.1% dithiothreitol, 0.1% trypsin inhibitor, and
0.2% bovine serum albumin. Single myocytes were dispersed by gentle
agitation of the digested segments with a wide-bored glass pipette.
Isolated myocytes were kept at 4°C until use. All experiments were
carried out within 10 h of harvesting cells and at room temperature.
Preparation of ICC cells and tissues.
ICR mice (0-15 days old) of either sex were anesthetized with
carbon dioxide and killed by cervical dislocation. Small intestines, from 1 cm below the pyloric ring to the cecum, were removed and opened
along the myenteric border. Luminal contents were washed away with
Krebs-Ringer bicarbonate solution. Tissues were pinned to the base of a
Sylgard dish, and the mucosa was removed by sharp dissection. Small
strips of intestinal muscle were equilibrated in Ca2+-free
Hanks' solution for 30 min, and cells were dispersed with an enzyme
solution containing (in mg/ml) 1.3 collagenase (Worthington Type II), 2 bovine serum albumin (Sigma, St Louis, MO), 2 trypsin inhibitor
(Sigma), and 0.27 ATP. Cells were plated onto sterile glass coverslips
coated with murine collagen (2.5 µg/ml, Falcon/BD) in 35-mm culture
dishes. The cells were cultured at 37°C in a 95% O2-5%
CO2 incubator in smooth muscle growth medium (Clonetics, San Diego, CA) supplemented with 2% antibiotic/antimycotic (GIBCO, Grand Island, NY) and murine stem cell factor (5 ng/ml, Sigma). Interstitial cells of Cajal (ICC) were identified immunologically with
a monoclonal antibody for Kit protein labeled with Alexa Fluor 488 (Molecular Probes, Eugene, OR).
Electrophysiological recordings.
Membrane currents were measured with an Axopatch 200A patch-clamp
amplifier (Axon Instruments) filtered at 5 kHz. Glass pipettes with a
resistance of ~2-4 M
were used to make a gigaseal. The pClamp
V.8.0 and Digidata-1200 (all from Axon Instrument) were used for the
acquisition of data and the application of command pulses. The data
were stored on a digital tape recorder (DTR 1204, Biologic, France) for
later analysis. Recorded data were played back and digitized using
Digidata 1200 at 1 or 5 kHz and low-pass filtered at 0.5 or 1 kHz for
illustration. With the use of pClamp 8.0 and Origin software (Microcal
Software), data were analyzed.
Solutions and drugs.
The physiological salt solution (Na+-Tyrode) contained (in
mM) 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 glucose, and 10 HEPES, and pH was adjusted to 7.35 with NaOH. In most
experiments that recorded CCh current (ICCh),
both Na+ and K+ were replaced with the same
concentration of Cs+; pH was adjusted to 7.35 with CsOH,
and CsCl was added to bring extracellular Cs+ concentration
([Cs+]o) to 140 mM. The pipette
solution consisted of (in mM) 3 MgATP, 0.2 Tris-GTP, 2 EGTA, and 10 HEPES; pH was adjusted to 7.3 with CsOH, and CsCl was added to bring
[Cs+]o to 140 mM. For different external
monovalent cationic solutions, Cs+ was replaced with 140 mM
Na+, K+, Li+, or
N-methyl-D-glucamate. In all experiments, a 3 M
KCl agar-bridge reference electrode was used, and corrections were made
for liquid junction potentials. For application of drugs, the
experimental chamber was superfused by gravity at a rate of ~2-3
ml/min. All drugs were purchased from Sigma. Anti-Gq/11
(SA-232, Biomol) and Go (SA-130, Biomol) antibody were
applied intracellularly with dilution (1:1,000). Experiments were
carried out at room temperature (20-23°C). Averaged results
throughout this paper are given as means ± SE. Student's
unpaired t-test was performed, and P values <0.05 were regarded as significant.
RNA Preparation and RT-PCR.
Total RNAs were extracted from antral smooth muscle tissues (without
mucosa layer) of murine stomach, ICC (1 × 106 cells),
and brain using a SNAP Total RNA Isolation kit (Invitrogen, Carlsbad,
CA) following the procedures of the manufacturer as previously
described (41). First-strand cDNA was synthesized from the
RNA preparations with a Superscript II RNase Transcriptase kit
(GIBCO-BRL, Gaithersburg, MD); RNA (1 pg) was reverse transcribed by
using random hexamers (50 µg/µl). To perform nested PCR, the following sets of primers were used: mtrp1 forward (nucleotides 1583-1600, 1601-1608) and reverse (nucleotides
2283-2300, 2301-2318, gene accession no. NM_011643); mtrp2
forward (nucleotides 2783-2800, 2801-2818) and reverse
(nucleotides 3483-3500, 3501-3518, AF111107); mtrp3 forward
(nucleotides 1030-1047, 1048-1065) and reverse (nucleotides 1749-1966, 1749-1966, AF190645); mtrp4 forward (nucleotides 1483-1500, 1501-1518) and reverse (nucleotides
2183-2200, 2201-2218, AF190646); mtrp5 forward (nucleotides
1749-1766, 1767-1784) and reverse (nucleotides
2449-2466, 2467-2484, AF060107); mtrp6 forward (nucleotides
603-620, 621-638) and reverse (nucleotides 1303-1320,
1321-1338, AF057748); and mtrp7 forward (nucleotides 2065-2082, 2083-2100) and reverse (nucleotides
2765-2782, 2783-2800, NM_012035). Complementary DNA (20% of
the first-strand reaction) was combined with first sense and antisense
primers (20 µM), 1 mM deoxynucleotide triphosphates, 60 mM
Tris · HCl (pH 8.5), 15 mM
(NH4)2SO4, 1.5 mM
MgCl2, 2.5 U of Taq (Bioneer), and RNase-free water to a final volume of 50 µl. The reaction occurred in a
PerkinElmer thermal cycler under the following conditions: an initial
denaturation at 94°C for 4 min, followed by 40 cycles at 94°C for
30 s, 42°C for 30 s, 72°C for 1 min, with a final
extension step at 72°C for 7 min. Five microliters of the
first-round PCR product were then added to a new reaction mixture
containing all of the components listed above except for second sense
and antisense primers (20 µM), and 40 additional cycles of PCR were
then performed. PCR products were separated by 2% agarose gel
electrophoresis. The sets of primers for mtrp1, mtrp2, mtrp3, mtrp4,
mtrp5, mtrp6, and mtrp7 were predicted to yield 700-, 700-, 700-, 718-, 700-, 700-, and 700-bp products, respectively. Two sets of negative control experiments were performed by including primers without cDNA or
by including primers with RNA that had not been reverse transcribed (no
RT added). To confirm murine TRP channels, PCR products of mtrp1-8
were digested with restriction enzymes. PCR product of mtrp1 was
digested into 395 and 305 bp by EcoR I. PCR product of mtrp2
was digested into 378 and 322 bp by MluI. PCR product of
mtrp3 was digested into 391 and 319 bp by SmaI. PCR product
of mtrp4 was digested into 400 and 318 bp by MluI. PCR product of mtrp5 was digested into 400 and 300 bp by BamHI.
PCR product of mtrp6 was digested into 400 and 300 bp by
SmaI. And PCR product of mtrp7 was digested into 400 and 300 bp by EcoRI as expected from the nucleotide sequences of
murine TRP channels. Primers were designed with the aid of the designer
program Primer3 at
http://www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi using the
corresponding murine mRNA sequences. The specificity of the primers for
the target gene was checked against the databases using Fasta3 at
http://www2.ebi.ac.uk/fasta3/, and primers were checked for hairpin
loops and palindromes using the Cybergene utility at
http://www.cybergene.se/primer.html. The oligonucleotides were
synthesized by Bionics (Seoul, South Korea).
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RESULTS |
RT-PCR and expression of TRP channels.
We used RT-PCR to see which types of TRP mRNA exist in the murine
stomach (Fig. 1). To test whether each
primer can detect each TRP mRNA, the murine brain was used. All seven
types of TRP mRNA were detected in the brain (Fig. 1A).
These primers were used to detect TRP mRNA in murine ICC and gastric
myocytes. In the stomach, all TRP mRNAs were detected (Fig.
1B), whereas TRP mRNAs except mtrp5 were detected in ICC
(Fig. 1C). To confirm the nucleotide sequences of the PCR
products, we digested the PCR products with restriction enzymes based
on the nucleotide sequence. We found that the PCR product of each TRP
subtype was digested into two fragments of the expected size from the
nucleotide sequences (Fig. 1D). Next, we expressed all types
of TRP genes except trp2, because TRPC2 is a pseudogene in
humans (47) and a potential pseudogene coding for an
NH2-terminal truncated protein in the bovine system
(48). First, no discernible currents were activated by 10 mM BAPTA or intracellular 0.2 mM GTP
S under the condition of
intracellular and extracellular 140 mM Cs+ in control cells
transfected with the empty vector (
115 ± 49 pA at
100 mV,
mean ± SE, n = 6). We recorded a current from
each TRP channel with a pipette containing intracellular 10 mM BAPTA or
intracellular 0.2 mM GTP
S under the condition of intracellular and
extracellular 140 mM Cs+ (Figs.
2 and 3).
The currents in human (h) TRP1 (n = 6), hTRP3 (n = 5), mTRP4 (n = 8), mTRP5
(n = 5), mTRP6 (n = 3), and mTRP7 (n = 3) were recorded under the condition of
intracellular 10 mM BAPTA and 140 mM Cs+ and extracellular
140 mM Cs+ by applying ramp pulses from 100 to
100 mV for
2 s from a holding potential of
60 mV (Fig. 2). In Fig.
2G, we obtained the ratios to compare current-voltage
(I-V) curves quantitatively: the current (under the
condition of intracellular 10 mM BAPTA and 140 mM Cs+ and
extracellular 140 mM Cs+) at 100 mV to the current at 25 mV
and the current at
100 mV to the current at 25 mV. When intracellular
0.2 mM GTP
S was used in hTRP1 (n = 3)-, hTRP3
(n = 3)-, mTRP4 (n = 5)-, mTRP5
(n = 3)-, mTRP6 (n = 4)-, and mTRP7
(n = 4)-expressing cells, I-V relationships were obtained, respectively (Fig. 3). In Fig. 3G, we
obtained the ratios to compare I-V curves quantitatively:
the current (under the condition of intracellular 0.2 mM GTP
S and
140 mM Cs+ and extracellular 140 mM Cs+) at 100 mV to the current at 25 mV and the current at
100 mV to the current
at 25 mV. In mouse stomach and mTRP5, current ratios of 100/25 and
100/25 mV are similar: 11.8 ± 1.1 and
7.1 ± 1.7 in
mouse and 10.8 ± 0.8 and
9.8 ± 2.6 in mTRP5, respectively (P > 0.05). However, there are differences
between the rectifying ratios (100/25 and
100/25 mV) of other TRPs
and those of murine stomach (P < 0.05). TRP6 was
suggested as the molecular identity for
1-adrenoceptor-activated NSCC (16). In our
study, the I-V shape of TRP6 has unique voltage
dependence. At 0-30 mV, there is a range in which little
current flows in the outward direction, whereas at more positive
potential (30 mV), a prominent outward rectification is seen. At
negative potential (
40 mV), there is a marked voltage-dependent
inhibition (Fig. 3E) as shown by Inoue et al.
(16) and Jung et al. (17). However, mTRP5
showed a slightly doubly rectifying appearance. In addition, the TRP6
maintained their activity when intracellular GTP
S was used for
recording the currents in our results (data was not shown) as well as
other reports (16), whereas the NSCC activated by
intracellular GTP
S in the murine stomach did not [see
Activation in murine stomach (see Fig. 11)]. Thus
we focused on TRP5 and compared the electrophysiological properties
with those recorded in the murine stomach.

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Fig. 1.
Tissue distribution of transient receptor potential (TRP) channels.
Detection of trp mRNA with RT-PCR in brain (A), stomach of
mouse (B), interstitial cells of Cajal (ICC) (C),
and digestion (D). Specificity of each primer was tested in
the brain. Each primer for each trp mRNA detected all trp mRNA in the
brain. D: confirmation of each PCR product with restriction
enzyme.
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Fig. 2.
The expression of each TRP channel in human embryonic
kidney (HEK) cells. A: human (h)TRP1. B: hTRP3.
C: mouse (m)TRP4. D: mTRP5. E: mTRP6.
F: mTRP7. All the pipette solution containing 140 mM
Cs+ and 10 mM BAPTA was used. The external solution
contained 140 mM Cs+. G:
I100mV/I25mV and
I 100mV/I25mV, where
I is the current (at 100 mV to the current at 25 mV and the
current at 100 mV to the current at 25 mV under the conditions of
intracellular 10 mM BAPTA and 140 mM Cs+ and extracellular
140 mM Cs), were calculated in antral myocytes of mouse stomach and
mTRP1-7.
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Fig. 3.
The expression of each TRP channel in HEK cells.
A: hTRP1. B: hTRP3. C: mTRP4.
D: mTRP5. E: mTRP6. F: mTRP7. Pipette
solution containing 140 mM Cs+ and 0.2 mM GTP S was used.
The external solution contained 140 mM Cs+. G:
I100mV/I25mV and
I 100mV/I25mV (the current at 100 mV to the current at 25 mV and the current at 100 mV to the current
at 25 mV under the conditions of intracellular 140 mM Cs+
and 0.2 mM GTP S and extracellular 140 mM Cs+) were
calculated in antral myocytes of mouse stomach and mTRP1-7.
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Electrophysiological properties of NSCCCCh in murine
stomach.
We isolated single cells from the murine stomach and used the same
protocol as that in the guinea pig to record the NSCCACh (18-20, 22). CCh, similar substance to ACh, induced
NSCC in isolated smooth muscle cells from the murine stomach (Fig.
4, A and B). The
current trace and I-V relationship recorded in murine
stomach were similar to those in mTRP5 expressed in HEK cells (Fig. 4, C and D). The currents displayed a reversal
potential of 0 mV. In both, the I-V relationship showed
slightly voltage-dependent inhibition at negative potential. This
voltage-dependent inhibition was also observed for mTRP5 in other
studies (35, 40). When the extracellular chloride ion was
replaced with aspartate, the current was activated by CCh. The reversal
potentials in the presence of aspartate ion (0.8 ± 1.3 mV,
n = 5) were not different from those in the presence of
Cl
(0.5 ± 1.2 mV, n = 6).

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Fig. 4.
The carbachol (CCh)-activated inward current and its
current-voltage (I-V) relationship in murine gastric
myocytes and mTRP5-expressing cells. Internal solution contained 140 mM
CsCl. Bath applied 50 µM CCh-induced inward current, which decayed
spontaneously during CCh treatment. Slow ramp depolarization from +100
to 100 mV was applied before (a) and during (b) the treatment with 50 µM CCh in murine stomach (A) and mTRP5 (C). The
I-V relationship was obtained from the difference in current
(b a) by digital subtraction in murine stomach (B)
and mTRP5 (D). The I-V relationship of murine
gastric myocytes and mTRP5 showed a voltage-dependent inhibition at
strongly negative potential. At more positive potential, outward
rectification was prominent.
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The channel in murine stomach has similar permeability to monovalent
cations such as those in mTRP5 (Fig. 5).
Relative permeability was calculated using the biionic equation
modified from the Goldman-Hodgkin-Katz equation. The reversal
potentials for Na+ and K+ in the murine stomach
were
4.0 ± 1.1 and 4.0 ± 1.0 mV (n = 4), respectively. Relative permeability of Cs+ to
Na+ to K+ was 1:0.80:1.19. The relative
permeability of Cs+ to Na+ to K+ in
mTRP5 was 1:0.98:1.1. On the other hand, the order of conductance was
Cs+ > K+ > Na+. The
current amplitude at
60 mV for Na+, K+, and
Cs+ solution in murine stomach was 143 ± 23 (n = 3), 210 ± 38 (n = 3), and
700 ± 24 pA (n = 10), respectively. The current
amplitude at
60 mV for Na+, K+, and
Cs+ solution in mTRP5 was 137 ± 101 (n = 4), 690 ± 122 (n = 4), and 1,400 ± 388 pA (n = 7), respectively. The
relative ratio of the current amplitude (Cs+ > K+ > Na+) and relative permeability ratio
(K+ > Cs+ > Na+) in
murine stomach are similar to those of the NSCCCCh in
mTRP5.

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Fig. 5.
The I-V relationships of nonselective cation
channels (NSCC) activated by CCh in murine stomach and mTRP5 recorded
under various ionic conditions. Ramp pulses were applied before and
during treatment with CCh. Traces were recorded from different cells
and in different ionic conditions, as shown above each I-V
relationship. Differences in currents were obtained by digital
subtraction and plotted against membrane potential.
A-C: murine stomach. D-F: mTRP5.
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Another property of NSCCCCh is the dependence upon
extracellular Ca2+ concentration (12, 18). The
NSCC was also modulated by extracellular calcium in murine stomach
(Fig. 6). After the activation of the currents, the external Ca2+ was changed to nominally free
solution and then 10 mM Ca2+. The current under the
Ca2+-free condition decreased, and then the current under
the 10 mM Ca2+ condition increased in both
NSCCCCh in the murine stomach and mTRP5. In our study, the
mTRP5 was shown to be increased when [Ca2+]o
was raised above physiological levels as in the other studies of
mTRP4/5 (35, 40).

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Fig. 6.
The effect of external calcium on NSCC activated by CCh
in murine stomach and mTRP5. As the external calcium increased, the
current increased. The current was activated under the condition of 1.5 mM Ca2+. During the activation of the current, the external
Ca2+ was changed first to nominally Ca2+-free
and then 10 mM Ca2+ and finally back to 1.5 mM
Ca2+. After a sudden jump of extracellular Ca2+
concentration ([Ca2+]o) from 0 to 10 mM, the
amplitude of both CCh current (ICCh) in murine
gastric myocytes (A) and mTRP5 (B) increased
immediately. The potentiating action of
[Ca2+]o on mTRP5 is essentially the same as
that in the murine gastric myocytes.
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We have used flufenamate to see whether it increases or decreases the
current, because Inoue et al. (16) recommended flufenamate could be used to distinguish TRP6 from other TRP channels. This compound has been shown to reversibly enhance the currents mediated by
mTRP6, whereas the currents mediated by mTRP3 and mTRP7 were inhibited
by the drug (16). Flufenamate (100 µM) inhibited
NSCCCCh by 87 ± 3% in the murine stomach
(n = 5) and 92 ± 3% in mTRP5 (n = 5; Fig. 7). To further confirm the
similarity between mTRP5- and carbachol-activated nonselective cation
channels in the murine stomach, we investigated the effects of a
nonspecific but frequently used cation channel blocker
La3+. Interestingly, in this study, La3+ also
inhibited the NSCCCCh in murine stomach. In Fig.
7E, the concentration-inhibition curves for La3+
block of NSCCCCh in mTRP5 and murine stomach gave similar
IC50 values (88 and 109 µM, n = 4-10, respectively) and similar Hill coefficients (0.48 and 0.50, respectively).

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Fig. 7.
The effect of La3+ and flufenamate on NSCC
activated by CCh in the murine stomach and mTRP5. A and
B: murine stomach. C and D: mTRP5.
Cation blockers 1 mM La3+ and 100 µM flufenamate
inhibited the currents. The effect was reversible. Sharp current
deflections are ramp pulses from 100 to 100 mV for 2 s from a
holding potential of 60 mV. E: the
concentration-inhibition curves for mTRP5 and murine stomach by
La3+ (n = 4-10). Curves are best
nonlinear fits to the Hill equation:
1/(1+{[La3+]/Ki}n),
where [La3+], Ki, and n
denote the concentration of La3+ applied, dissociation
constant, and Hill coefficient, respectively.
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Activation mechanism in murine stomach.
The main discrepancy between TRP and NSCC in visceral smooth muscle is
the G protein that activates channels. TRPCs are activated downstream
of G protein-coupled receptors, which induce PLC-mediated phosphoinositide breakdown. In the TRPCs shown to be activated by
store-independent pathways, Gq/11 is the mediator for the
activation of channels. On the other hand, in visceral smooth muscle,
pertussis toxin-sensitive G protein is responsible for the activation
of the channels. When ACh binds to the muscarinic receptor, information is transferred to the channel protein through a pertussis
toxin-sensitive GTP binding protein (13, 19). We performed
experiments to see whether Gq/11 is involved in the
activation of NSCC by muscarinic stimulation in the murine stomach.
Anti-Gq/11 antibody blocked the activation of NSCC in the
murine stomach (Fig. 8).
Anti-Gq/11 antibody decreased the current amplitude from
500 ± 50 (n = 5) and 600 ± 40 (n = 8) to 5.0 ± 1.5 (n = 4) and
15 ± 1.3 pA (n = 4) in murine stomach and
mTRP5-expressing cells, respectively. Anti-Go antibody did
not inhibit the currents in murine stomach and mTRP5; the peak
amplitude was 475 ± 140 (n = 4) and 575 ± 60 pA, respectively (n = 4). We have tested inactivated
anti-Gq/11 antibody in murine stomach and mTRP5.
Inactivated anti-Gq/11 antibody did not inhibit the
currents (n = 5). PLC inhibitor (500 µM neomycin sulphate and 1 µM U-73122, n = 4) blocked the
activation of NSCC in the murine stomach. U-73122 (1 µM) inhibited
NSCCCCh by 88 ± 2% in the murine stomach. The
inhibitors of IP3-induced Ca2+ release
[100 µM 2-aminoethoxydiphenylborate (2-APB) and 1 µM xestospongin C, n = 4] also blocked the activation of
NSCC in the murine stomach. 2-APB (100 µM) inhibited
NSCCCCh by 87 ± 7% in the murine stomach. 2-APB (100 µM; n = 5) and U-73122 (1 µM; n = 5) also blocked NSCCCCh by 91 ± 5 and 92 ± 3%
in mTRP5-expressing cells, respectively (Fig.
9). U-73343 (1 µM; inactive analog of U-73122), however, also inhibited the activation of NSCC in murine stomach and mTRP5 (n = 3). For DAG as activator, we
used 1-oleoyl-2-acetyl-sn-glycerol (OAG). OAG itself
activated NSCC a little in the murine stomach (40 ± 5 pA,
mean ± SE, n = 3; Fig.
10). Similarly, OAG can activate a
little inward current in mTRP5-expressing cells (147 ± 82 pA, n = 4; Fig. 10). OAG activated inward current
in murine stomach and mTRP5-expressing cells that has similar
I-V relationship to NSCCCCh. On the other hand,
OAG did not activate inward currents in control cells transfected with
the empty vector (n = 3). However, the current
activation by DAG analogs was reported as a characteristic feature of
the TRPC3/6/7 subfamily of TRP channels (10, 34), not the
TRPC4/5 subfamily (40). In addition, when we coexpressed the muscarinic receptor (M3) and mTRP5 in CHO cells, which have endogenous P2Y purinoceptor but lack endogenous muscarinic receptors, CCh activated currents. When we expressed mTRP5 only or coexpressed mTRP5 and M2 muscarinic receptors in CHO cells, CCh did not activate currents, whereas ATP did (data not shown). It seems that in murine stomach, NSCC is activated by a similar mechanism in the murine portal
vein, that is, the muscarinic receptor-Gq/11-PLC pathway.

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Fig. 8.
The effect of anti-G protein antibodies on NSCC activated by CCh in
murine stomach and mTRP5. A and B: murine
stomach. D and E: mTRP5. In the presence of
anti-Gq/11 antibody within the patch pipette, the current
was not activated by CCh (A). The same result was obtained
in mTRP5 (D). However, the anti-Go antibody did
not inhibit the activation of NSCC by CCh in murine stomach and mTRP5
(B and E). ICCh recorded
in the presence of anti-G protein antibodies was compared with the mean
value of control ICCh; averaged values are shown
(mean ± SE; C). The activation of NSCC by muscarinic
stimulation may be preferentially mediated by Gq/11 subtype
protein in the murine gastric myocytes and mTRP5-expressing cells.
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Fig. 9.
Effects of 2-aminoethoxydiphenylborate (2-APB) on
carbachol-induced current in murine stomach and mTRP5. 2-APB (100 µM;
inhibitor of inositol 1,4,5-trisphosphate-induced Ca2+
release) blocked the activation of NSCC in murine stomach
(A) and mTRP5 (B; n = 3).
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Fig. 10.
The activation of current by 1-oleoyl-2-acetyl-sn-glycerol (OAG)
in murine stomach and mTRP5-expressing cells. The holding potential was
60 mV. A and B: bath-applied membrane-permeable
analogs of diacylglycerol (DAG), OAG (20 µM)-activated NSCC in murine
stomach, which have similar I-V relationships to NSCC
activated by CCh, although the amplitude was very small compared with
that of NSCC activated by CCh and mTRP5. B and C:
OAG-activated inward current in mTRP5-expressing cells, which has a
similar I-V relationship to NSCC activated by CCh.
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|
NSCC in the murine stomach and mTRP5 was activated transiently by
intracellular 0.2 mM GTP
S (Fig. 11).
The activation was not maintained in the murine stomach and decayed to
the level before the activation (desensitization). The facilitation
induced by depolarizing the ramp pulse was observed. We tested whether the desensitization process depends on
[Ca2+]i or not. The desensitization process
was compared under the different concentrations of EGTA. The degree of
desensitization was estimated by calculating the relative values of the
current 5 min after peak to the current at peak
(I5min/Ipeak). The values for 0.5, 2, 2.5, 3, and 3.5 mM EGTA were 12.0 ± 2.2 (n = 25), 10.0 ± 1.1 (n = 4),
11.0 ± 1.7 (n = 3), 12.0 ± 3.5 (n = 3), and 10.0 ± 3.0% (n = 3), respectively. The nonselective cation current was not activated
under the condition of 5 mM EGTA. The desensitization process was not
dependant on [Ca2+]i, but PLC
1 seems to be
involved in the desensitization process, because the desensitization
remarkably slowed in PLC
1 knockout mice (data not shown;
n = 3).

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Fig. 11.
The transient activation of current by intracellularly applied
GTP S. The arrow indicates the rupture of the cell membrane. After
the rupture, the current was activated as the 0.2 mM GTP S diffused
into the cell. The activation was not maintained in murine stomach but
decayed to the control level (A). During the decay, the
application of depolarizing pulses increased the current amplitude
transiently, so-called facilitation. B: mTRP5.
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|
One unique property of mTRP5 is the constitutive activity
(40). We investigated whether NSCC in the murine stomach
has such an activity (Fig. 12). In
control HEK cells transfected with the empty vector, there was no
constitutive activity in normal tyrode and 140 Cs+
(n = 4). The I-V relationship recorded under
the condition of 140 mM Cs+ in control HEK cells was not
similar to that of NSCCCCh and that activated by mTRP5.
When we superfused external Cs+ solution without the
muscarinic stimulation, there was an increase in the conductance,
suggesting that there is a constitutive activity in NSCCCCh
in the murine stomach.

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Fig. 12.
The basal activity of NSCC activated by CCh in murine
stomach and mTRP5. A: murine stomach. B: mTRP5.
Without the stimulation by CCh, the change of external monovalent
cation from Na+ to Cs+ increased the current
amplitude. The I-V relationship recorded under the condition
of 140 mM Cs+ and normal Tyrode in murine stomach was
similar to that of mTRP5-expressing cells. In addition, the
I-V relationships recorded under the condition of 140 mM
Cs+ were similar to those of NSCC activated by CCh and
mTRP5. C: in control HEK cells transfected with the empty
vector, the I-V relationship recorded under the condition of
140 mM Cs+ was not similar to that of NSCC activated by CCh
(n = 4).
|
|
 |
DISCUSSION |
In this study, we investigated the molecular identity of the NSCC
activated by muscarinic stimulation (ACh or CCh) in the murine stomach.
Similarity between mTRP5 and NSCCCCh in murine gastric
myocytes, i.e., unique voltage dependence, permeability ratio
(K+ > Cs+ > Na+) and
conductance order (Cs+ > K+ > Na+), dependence on [Ca2+]o,
block by flufenamic acid and La3+, activation pathway
(muscarinic-Gq/11-PLC pathway), transient activation by
intracellular GTP
S, activation by OAG, and the constitutive
activity, suggests that the mTRP5 protein is a molecular component of
NSCCCCh in murine gastric myocytes.
Our results support this conclusion. First, CCh-induced nonselective
cation currents in murine gastric myocytes were found to display a
characteristic doubly rectifying I-V relationship that
resembles the I-V relationship for hTRPC3/6 (10,
24), mTRPC4/5 (40). In this study, the I-V shape of
mTRP6 has unique voltage dependence as in Inoue et al.
(16) and Jung et al. (17). On the other hand,
mTRP5 showed a slightly doubly rectifying appearance (Figs. 2 and 3).
Second, the channel in both the murine stomach and mTRP5-expressing
cells has a similar permeability to monovalent cation
(K+ > Cs+ > Na+) and
conductance order (Cs+ > K+ > Na+; Fig. 4). Third, NSCC was modulated by extracellular
calcium, as in murine stomach and mTRP5-expressing cells (Fig. 5). The mTRP6 exhibits a dual dependence on [Ca2+]o.
The current is partially inhibited by [Ca2+]o
in the physiological range, and the amplitude increases when [Ca2+]o is decreased. Nevertheless, the
complete removal of external Ca2+ did not further
potentiate the currents but rather led to a decrease in the amplitude
of inward currents (16, 17). A similar complex dependence
of agonist-evoked cation currents on [Ca2+]o
has been reported for norepinephrine-evoked currents in rabbit portal
vein smooth muscle cells (9, 16). A potentiating effect of
decreasing [Ca2+]o has been described for
hTRPC1 (29, 43), hTRPC3 (29), and mTRPC7
(34). In our study, however, the mTRP5 was shown to
increase when [Ca2+]o was raised above
physiological levels as in the other study of mTRPC4/5 (35,
40). Fourth, the pharmacological properties of the murine
stomach are similar to mTRP5 in our study (Fig. 6). Flufenamate
increased mTRPC6 currents but inhibited currents mediated by the
TRPC3/7 subfamily (16). Flufenamate decreased NSCCCCh in the murine stomach. La3+ also
inhibited the NSCCCCh in murine stomach. The
IC50 value of murine stomach is similar to that of mTRP5.
Fifth, both mTRP5 and NSCC activated by CCh in the murine stomach were
activated through the M3-Gq/11-PLC pathway (Fig. 7). TRPCs
are activated downstream of G protein-coupled receptors, which induce
PLC-mediated phosphoinositide breakdown. However, the downstream
signaling pathways that finally activate TRPCs remain highly
controversial. For nearly all of the functionally expressed TRPCs,
there is at least one report proposing a store-operated mechanism of
activation (4, 25, 31, 36, 37). On the other hand, there
is growing evidence for the involvement of store-independent pathways
in the regulation of TRPC3 (10, 11, 49), TRPC5
(35), TRPC6 (4, 10), and TRPC7
(34). In the TRPCs shown to be activated by
store-independent pathways, Gq/11 is the mediator for the
activation of channels. In the murine stomach, the NSCC is activated by
similar mechanism in the murine portal vein, that is, muscarinic
receptor-Gq/11-PLC pathway. Finally, both mTRP5 and NSCC
activated by CCh in the murine stomach were activated transiently by
intracellular GTP
S (Fig. 10). The mTRP6 maintained their activity
when intracellular GTP
S was used for recording the currents
(16), whereas NSCC activated by intracellular GTP
S in
the murine stomach did not.
When mTRP5 was expressed in HEK in our laboratory, two properties were
different from a previous report by Schaefer et al. (40).
First, OAG could activate the nonselective cation current in our
experiment, although the current amplitude is small compared with that
activated by CCh or GTP
S. Application of OAG stimulated the current
independently of protein kinase C, a characteristic property of the
TRPC3/6/7 subfamily (10, 34) not shared with the TRPC4/5
subfamily (40). Schaefer et al. (40) recorded the intracellular calcium change and found that OAG did not increase the [Ca2+]i. From our results, we assume that
the calcium influx through the activation by OAG is not enough to
record the change in the [Ca2+]i. Second,
La3+ blocked the mTRP5 in our results, whereas Schaefer et
al. (40) showed that La3+ increased the
[Ca2+]i using the Mn2+ quenching
experiment. One characteristic biophysical feature of
ICRAC is a specific block by low micromolar
concentrations of La3+. The TRPC3/6/7 subfamily (7,
34) was also blocked by La3+. La3+ (100 µM) did not inhibit, but, similar to 10 mM Ca2+, even
potentiated GTP
S-induced currents through mTRPC4/5 without changing
the reversal potential. The TRPC4/5 subfamily was potentiated by
La3+ in two studies (40, 44) but not in
another (35). Okada et al. (35) showed that
La3+ decreased the [Ca2+]i when
mTRP5 was expressed. Inoue et al. (16) also showed that La3+ blocked the current by phenylephrine when mTRP6 was
expressed in HEK cells.
In conclusion, we suggest that mTRP5 is a candidate for
NSCCACh in the murine stomach.
 |
ACKNOWLEDGEMENTS |
We thank Drs. A. Tobin and T. Bonner for providing m3
and m2 receptor genes, respectively. We thank Drs. M. Zhu,
Y. Mori, and G. Schultz for providing htrp1 and
3, mtrp6 and 8, and mtrp4 and 5 genes, respectively.
 |
FOOTNOTES |
*
Y. M. Lee and B. J. Kim contributed equally to
this work.
This study was supported by a grant from the Ministry of Health and
Welfare (01-PJ1-PG3-21400-0015), by the Advanced Backbone IT
Technology Development Project from the Ministry of Information and
Communication (IMT-2000-C3-5), and by the year 2001 BK21 project for medicine, dentistry, and pharmacy.
Address for reprint requests and other correspondence: I. So, Dept. of
Physiology and Biophysics, Seoul National Univ. College of Medicine, 28 Yongon-Dong, Chongno-Gu, Seoul 110-799, Korea (E-mail:
insuk{at}plaza.snu.ac.kr).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00069.2002
Received 20 February 2002; accepted in final form 1 November 2002.
 |
REFERENCES |
1.
Ahn, SC,
Kim SJ,
So I,
and
Kim KW.
Inhibitory effect of phorbol 12,13 dibutyrate on carbachol-activated nonselective cationic current in guinea-pig gastric myocytes.
Pflügers Arch
434:
505-507,
1997[ISI][Medline].
2.
Balzer, M,
Lintschinger B,
and
Groschner K.
Evidence for a role of Trp proteins in the oxidative stress-induced membrane conductances of porcine aortic endothelial cells.
Cardiovasc Res
42:
543-549,
1999[ISI][Medline].
3.
Benham, CD,
Bolton TB,
and
Lang RJ.
Acetylcholine activates an inward current in single mammalian smooth muscle cells.
Nature
316:
345-347,
1985[ISI][Medline].
4.
Boulay, G,
Brown DM,
Qin N,
Jiang M,
Dietrich A,
Zhu MX,
Chen Z,
Birnbaumer M,
Mikoshiba K,
and
Birnbaumer L.
Modulation of Ca2+ entry by polypeptides of the inositol 1,4,5-trisphosphate receptor (IP3R) that bind transient receptor potential (TRP): evidence for roles of TRP and IP3R in store depletion-activated Ca2+ entry.
Proc Natl Acad Sci USA
96:
14955-14960,
1999[Abstract/Free Full Text].
5.
Golovina, VA,
Platoshyn O,
Bailey CL,
Wang J,
Limsuwan A,
Sweeney M,
Rubin LJ,
and
Yuan JX.
Upregulated TRP and enhanced capacitative Ca2+ entry in human pulmonary artery myocytes during proliferation.
Am J Physiol Heart Circ Physiol
280:
H746-H755,
2001[Abstract/Free Full Text].
6.
Groschner, K,
Hingel S,
Lintschinger B,
Balzer M,
Romanin C,
Zhu X,
and
Schreibmayer W.
Trp proteins form store-operated channels in human vascular endothelial cells.
FEBS Lett
437:
101-106,
1998[ISI][Medline].
7.
Halaszovich, CR,
Zitt C,
Jüngling E,
and
Lückhoff A.
Inhibition of TRP3 channels by lanthanides. Block from the cytosolic side of the plasma membrane.
J Biol Chem
275:
37423-37428,
2000[Abstract/Free Full Text].
8.
Hartneck, C,
Plant TD,
and
Schultz G.
From worm to man: three subfamilies of TRP channels.
Trends Neurosci
23:
159-166,
2000[ISI][Medline].
9.
Helliwell, RM,
and
Large WA.
Dual effect of external Ca2+ on noradrenaline-activated cation current in rabbit portal vein smooth muscle cells.
J Physiol
492:
75-88,
1996[Abstract].
10.
Hofmann, T,
Obukhov AG,
Schaefer M,
Harteneck C,
Gudermann T,
and
Schultz G.
Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol.
Nature
397:
259-263,
1999[ISI][Medline].
11.
Hurst, RS,
Zhu X,
Boulay G,
Birnbaumer L,
and
Stefani E.
Ionic currents underlying HTRP3 mediated agonist-dependent Ca2+ influx in stably transfected HEK293 cells.
FEBS Lett
422:
333-338,
1998[ISI][Medline].
12.
Inoue, R.
Effect of external Cd2+ and other divalent cations on carbachol-activated non-selective cation channels in guinea-pig ileum.
J Physiol
442:
447-463,
1991[Abstract].
13.
Inoue, R,
and
Isenberg G.
Acetylcholine activates nonselective cation channels in guinea-pig ileum through a G protein.
Am J Physiol Cell Physiol
258:
C1173-C1178,
1990[Abstract/Free Full Text].
14.
Inoue, R,
and
Isenberg G.
Effect of membrane potential on acetylcholine-induced inward current in guinea-pig ileum.
J Physiol
424:
57-71,
1990[Abstract].
15.
Inoue, R,
Kitamura K,
and
Kuriyama H.
Acetylcholine activates single sodium channels in smooth muscles.
Pflügers Arch
410:
69-74,
1987[ISI][Medline].
16.
Inoue, R,
Okada T,
Onoue H,
Hara Y,
Shimizu S,
Naitoh S,
Ito Y,
and
Mori Y.
The transient receptor potential protein homologue TRP6 is the essential component of vascular
1-adrenoceptor-activated Ca2+-permeable cation channel.
Circ Res
88:
325-332,
2001[Abstract/Free Full Text].
17.
Jung, S,
Strotmann R,
Schultz G,
and
Plant TD.
TRPC6 is a candidate channel involved in receptor-stimulated cation currents in A7r5 smooth muscle cells.
Am J Physiol Cell Physiol
282:
C347-C359,
2002[Abstract/Free Full Text].
18.
Kang, TM,
Kim YC,
Sim JH,
Rhee JC,
Kim SJ,
Uhm DY,
So I,
and
Kim KW.
The properties of carbachol-activated nonselective cation channels at single channel level in guinea pig gastric myocytes.
Jpn J Pharmacol
85:
291-298,
2001[ISI][Medline].
19.
Kim, SJ,
Ahn SC,
So I,
and
Kim KW.
Quinidine blockade of the carbachol activated nonselective cation current in guinea-pig gastric myocytes.
Br J Pharmacol
115:
1407-1414,
1995[Abstract].
20.
Kim, SJ,
Ahn SC,
So I,
and
Kim KW.
Role of calmodulin in the activation of carbachol activation of carbachol activated cationic current in guinea-pig gastric antral myocytes.
Pflügers Arch
430:
757-762,
1995[ISI][Medline].
21.
Kim, SJ,
Koh EM,
Kang TM,
Kim YC,
So I,
and
Kim KW.
Ca2+ influx through carbachol activated nonselective cation channels in guinea-pig gastric myocytes.
J Physiol
513:
749-760,
1998[Abstract/Free Full Text].
22.
Kim, YC,
Kim SJ,
Sin JH,
Cho CH,
Juhnn YS,
Suh SH,
So I,
and
Kim KW.
Suppression of the carbachol activated nonselective cationic current by antibody against alpha subunit of Go protein in guinea-pig gastric myocytes.
Pflügers Arch
436:
494-496,
1998[ISI][Medline].
23.
Kim, YC,
Kim SJ,
Sin JH,
Jun JY,
Kang TM,
Suh SH,
So I,
and
Kim KW.
Protein kinase C mediates the desensitization of CCh-activated nonselective cationic current in guinea-pig gastric myocytes.
Pflügers Arch
436:
1-8,
1998[ISI][Medline].
24.
Kiselyov, K,
Shin DM,
Wang Y,
Pessah IN,
Allen PD,
and
Muallem S.
Gating of store-operated channels by conformational coupling to ryanodine receptors.
Mol Cell
6:
421-431,
2000[ISI][Medline].
25.
Kiselyov, K,
Xu X,
Mozhayeva G,
Kuo T,
Pessah I,
Mignery G,
Zhu X,
Birnbaumer L,
and
Muallem S.
Functional interaction between Ins P3 receptors and store-operated Htrp3 channels.
Nature
396:
478-482,
1998[ISI][Medline].
26.
Komori, S,
and
Bolton TB.
Role of G-proteins in muscarinic receptor inward and outward currents in rabbit jejunal smooth muscle.
J Physiol
427:
395-419,
1990[Abstract].
27.
Komori, S,
Unno T,
Nakayama T,
and
Ohashi H.
M2 and M3 muscarinic receptors couple, respectively, with activation of nonselective cationic channels and potassium channels in intestinal smooth muscle cells.
Jpn J Pharmacol
76:
213-218,
1998[ISI][Medline].
28.
Li, HS,
Xu XZ,
and
Montell C.
Activation of a TRPC3-dependent cation current through the neurotrophin BDNF.
Neuron
24:
261-273,
1999[ISI][Medline].
29.
Lintschinger, B,
Balzer-Geldsetzer M,
Baskaran T,
Graier WF,
Romanin C,
Zhu MX,
and
Groschner K.
Coassembly of Trp1 and Trp3 proteins generates diacylglycerol- and Ca2+-sensitive cation channels.
J Biol Chem
275:
27799-27805,
2000[Abstract/Free Full Text].
30.
Liu, X,
Wang W,
Singh BB,
Lockwich T,
Jadlowiec J,
O'Connell B,
Wellner R,
Zhu MX,
and
Ambudkar IS.
Trp1, a candidate protein for the store-operated Ca2+ influx mechanism in salivary gland cells.
J Biol Chem
275:
3403-3411,
2000[Abstract/Free Full Text].
31.
Mizuno, N,
Kitayama S,
Saishin Y,
Shimada S,
Morita K,
Mitsuhata C,
Kurihara H,
and
Dohi K.
Molecular cloning and characterization of rat trp homologues from brain.
Mol Brain Res
64:
41-51,
1999[ISI][Medline].
32.
Montell, C,
and
Rubin GM.
Molecular characterization of the Drosophilia trp locus: a putative integral membrane protein required for phototransduction.
Neuron
2:
1313-1323,
1989[ISI][Medline].
33.
Moore, TM,
Brough GH,
Babal P,
Kelly JJ,
Li M,
and
Stevens T.
Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1.
Am J Physiol Lung Cell Mol Physiol
275:
L574-L582,
1998[Abstract/Free Full Text].
34.
Okada, T,
Inoue R,
Yamazaki K,
Maeda A,
Kurosaki T,
Yamakuni T,
Tanaka I,
Shimizu S,
Ikenaka K,
Imoto K,
and
Mori Y.
Molecular and functional characterization of a novel mouse transient receptor potential protein homologue TRP7. Ca2+-permeable cation channel that is constitutively activated and enhanced by stimulation of G protein-coupled receptor.
J Biol Chem
274:
27359-27370,
1999[Abstract/Free Full Text].
35.
Okada, T,
Shimizu S,
Wakamori M,
Maeda A,
Kurosaki T,
Takada N,
Imoto K,
and
Mori Y.
Molecular cloning and functional characterization of a novel receptor-activated TRP Ca2+ channel from mouse brain.
J Biol Chem
273:
10279-10287,
1998[Abstract/Free Full Text].
36.
Philipp, S,
Cavalié A,
Freichel M,
Wissenbach U,
Zimmer S,
Trost C,
Marquardt A,
Murakami M,
and
Flockerzi V.
A mammalian capacitative calcium entry channel homologous to Drosophila TRP and TRPL.
EMBO J
15:
6166-6171,
1996[Abstract].
37.
Philipp, S,
Hambrecht J,
Braslavski L,
Schroth G,
Freichel M,
Murakami M,
Cavalié A,
and
Flockerzi V.
A novel capacitative calcium entry channel expressed in excitable cells.
EMBO J
17:
4274-4282,
1998[Abstract/Free Full Text].
38.
Philipp, S,
Trost C,
Warnat J,
Rautmann J,
Himmerkus N,
Schroth G,
Kretz O,
Nastainczyk W,
Cavalié A,
Hoth M,
and
Flockerzi V.
TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.
J Biol Chem
275:
23965-23972,
2000[Abstract/Free Full Text].
39.
Rhee, JC,
Rhee PL,
Park MK,
So I,
Uhm DY,
Kim KW,
and
Kang TM.
Muscarinic receptors controlling the carbachol-activated nonselective cationic current in guinea pig gastric smooth muscle cells.
Jpn J Pharmacol
82:
331-337,
2000[ISI][Medline].
40.
Schaefer, M,
Plant TD,
Obukhov AG,
Hofmann T,
Gudermann T,
and
Schultz G.
Receptor-mediated regulation of the nonselective cation channels TRPC4 and TRPC5.
J Biol Chem
275:
17517-17526,
2000[Abstract/Free Full Text].
41.
Sim, JH,
Yang DK,
Kim YC,
Park SJ,
Kang TM,
So I,
and
Kim KW.
ATP-sensitive K+ channels composed of Kir6.1 and SUR2B subunits in guinea pig gastric myocytes.
Am J Physiol Gastrointest Liver Physiol
282:
G137-G144,
2002[Abstract/Free Full Text].
42.
Sim, SM,
Singer JJ,
and
Walsh JV.
Cholinergic agonists suppress a potassium current in freshly dissociated smooth muscle cells of the toad.
J Physiol
367:
503-529,
1985[Abstract].
43.
Sinkins, WG,
Estacion M,
and
Schilling WP.
Functional expression of TrpC1: a human homologue of the Drosophila Trp channel.
Biochem J
331:
331-339,
1998[ISI][Medline].
44.
Strübing, C,
Krapivinsky G,
Krapivinsky L,
and
Clapham DE.
TRPC1 and TRPC5 form a novel cation channel in mammalian brain.
Neuron
29:
645-655,
2001[ISI][Medline].
45.
Vogalis, F,
and
Sanders KM.
Cholinergic stimulation activates a non-selective cation current in canine pyloric circular muscle cells.
J Physiol
429:
223-236,
1990[Abstract].
46.
Warnat, J,
Philipp S,
Zimmer S,
Flockerzi V,
and
Cavalié A.
Phenotype of a recombinant store-operated channel: highly selective permeation of Ca2+.
J Physiol
518:
631-638,
1999[Abstract/Free Full Text].
47.
Wes, P,
Chevesich J,
Jeromin A,
Rosenberg C,
Stetten G,
and
Montell C.
TRPC1, a human homolog of a Drosophilia store-operated channel.
Proc Natl Acad Sci USA
92:
9652-9656,
1995[Abstract].
48.
Wissenbach, U,
Schroth G,
Philipp S,
and
Flockerzi V.
Structure and mRNA expression of a bovine TRP homologue related to mammalian TRP2 transripts.
FEBS Lett
429:
61-66,
1998[ISI][Medline].
49.
Zhu, X,
Jiang M,
and
Birnbaumer L.
Receptor-activated Ca2+ influx via human Trp3 stably expressed in human embryonic kidney (HEK)293 Cells.
J Biol Chem
273:
133-142,
1998[Abstract/Free Full Text].
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