Department of Physiology and Cell Biology, University of Nevada, School of Medicine, Reno, Nevada 89557
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Interstitial cells of Cajal (ICC) are the
pacemaker cells responsible for the generation and propagation of
electrical slow waves in phasic muscles of the gastrointestinal (GI)
tract. The pacemaker current that initiates each slow wave derives from
a calcium-inhibited, voltage-independent, nonselective cation channel. This channel in ICC displays properties similar to that reported for
the transient receptor potential (TRP) family of nonselective cation
channels, particularly those seen for TRPC3 and TRPC4. We have
identified transcripts for TRPC4 in individually isolated ICC and have
cloned the two alternatively spliced forms of TRPC4, TRPC4 and
TRPC4
, from GI muscles. TRPC4
is missing an 84-amino acid segment
from the carboxy terminus. Expression of either form using the whole
cell patch-clamp technique led to calcium-inhibited, nonselective
cation channels as determined by
N-methyl-D-glucamine replacement experiments and
BAPTA dialysis. Expression of TRPC4
channels recorded at the whole
cell level had characteristics similar to the nonselective cation
current in ICC. The single-channel conductance of TRPC4
was
determined to be 17.5 pS. Application of calmidazolium to cells
expressing TRPC4
led to a significant increase in the inward current
of these cells at both the whole cell and single-channel level, and
currents were sensitive to block by 10 µM lanthanum, niflumic acid,
and DIDS. Comparison of the properties reported for the nonselective
cation current in ICC and those identified here for TRPC4
led us to
conclude that a TRPC4-like current encodes the plasmalemmal pacemaker
current in murine small intestine.
cation channel; gastrointestinal; smooth muscle; calmodulin
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
INTERSTITIAL CELLS OF CAJAL (ICC) are the pacemaking cells in gastrointestinal (GI) muscles that generate the rhythmic oscillations in membrane potential known as slow waves (7, 16). Slow waves propagate within ICC networks, conduct into smooth muscle cells via gap junctions, and initiate phasic contractions via activation of Ca2+ entry through L-type Ca2+ channels. Ablation of ICC networks by genetic means (25) or through inactivation of Kit receptors with neutralizing antibodies (21) results in elimination of slow wave activity and alterations in GI motility. The pacemaker mechanism has been shown to involve rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) in a compartment near the plasma membrane that controls the open probability of channels responsible for pacemaker currents. This mechanism involves Ca2+ release from D-myo-inositol 1,4,5-trisphosphate (IP3) receptor-operated stores and uptake of Ca2+ by mitochondria (26). Mitochondrial Ca2+ uptake activates voltage-independent, Ca2+-inhibited, nonselective cation channels with a unitary conductance of 13 pS (10). The molecular species responsible for the pacemaker conductance has not been identified.
Transient receptor potential (TRP) channels were first cloned from
Drosophila (TRP and TRPL) and constitute a superfamily of
proteins encoding a diverse group of Ca2+-permeable cation
channels (14). One subfamily, the classic or canonical
TRPs, has seven members (TRPC1-7) and participates in functions as
diverse as store-operated Ca2+ entry (4),
vasorelaxation (2), and egg fertilization
(8). Some of the characteristics of the
Ca2+-inhibited, nonselective cation currents in ICC
(10) are similar to those of TRPC3 and TRPC4 currents.
Transcripts for both TRPC4 and TRPC6 have been identified in freshly
dispersed ICC (1), but transcripts for TRPC3 have not been
identified in this cell type (23). TRPC4 exists as two
alternatively spliced transcripts that are expressed in several tissue
and cell types (15, 23). TRPC4 represents the complete
form of the protein, whereas TRPC4
is missing an 84-amino acid
segment from the carboxy terminus (13, 17). TRPC4
,
originally cloned from bovine retina, was shown to activate upon
application of GTP
s or depletion of intracellular calcium stores
(15). Recently, Schaefer et al. (17) reported the expression of TRPC4
and TRPC4
, demonstrating the
receptor-dependent, but store-independent, activation of these channels
in HEK-293 cells. In addition, two independent studies by Tang et al.
(19) and Trost et al. (22) examined the
calmodulin-binding sites of TRPC4 and reported the
Ca2+-dependent binding of calmodulin at two locations in
the cytosolic COOH terminus of TRPC4
and one in TRPC4
. We have
previously reported the cloning of TRPC4
and TRPC4
from murine
colonic smooth muscle tissues (23).
In the present study, we examined the properties of TRPC4 splice
variants (TRPC4 and TRPC4
) to determine whether either isoform
could be responsible for the Ca2+-inhibited,
nonselective cation (pacemaker) conductance in ICC. We expressed
TRPC4
and TRPC4
in HEK-293 cells and used the patch-clamp technique to examine the properties of TRPC4 whole cell and unitary currents. The effects of calmidazolium (CMZ), a calmodulin inhibitor, were tested on whole cell and single-channel currents. The properties of TRPC4
were remarkably like the pacemaker conductance and led us
to conclude that slow wave activity in murine GI muscles may be due to
a TRPC4-like conductance.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular techniques:RT-PCR.
Total RNA was prepared from cell cultures by using the Trizol reagent
(Invitrogen, San Diego, CA) as per manufacturer's instruction. First
strand cDNA was prepared from the RNA preparations by using the
Superscript II reverse transcriptase kit (GIBCO BRL, Gaithersberg, MD), and 500 µg/µl of oligo dT primers were used to reverse
transcribe the RNA sample. The cDNA reverse transcription product was
amplified with specific primers by PCR as previously described
(23). The following PCR primers were used (the first
number represents the sense-bordering nucleotide positions, the second
number represents the antisense-bordering nucleotide positions, and the
number in parenthesis is the GenBank accession number): TRPC4 primers
(AF019663) nt 1667-1686 and 1742-1760, amplicon = 93 bp,
will amplify both and
forms of TRPC4; and TRPC4 wild-type
primers (AF019663) nt 2518-2538 and 2599-2620, amplicon = 102 bp, will amplify only the
form of TRPC4 and not the
alternatively spliced
form, because the primers were designed to
hybridize to the deleted region in the alternatively spliced transcript.
Adenoviral constructs.
Constructs used for expression in HEK-293 cells were developed in the
pcDNA3.1 mammalian expression vector (Invitrogen, San Diego, CA).
Plasmid DNA was prepared from overnight cultures by using the Qiagen
Miniprep kit and sequenced by the ABI Prism 310 genetic analyzer
(Applied Biosytems, Foster City, CA) to confirm the correct plasmid
construct. Recombinant adenoviruses for TRPC4 and TRPC4
were then
produced, purified, and amplified by using the AdEasy adenoviral vector
system (Stratagene, La Jolla, CA) and were used to infect HEK-293
cells. The virus without any recombinant insert (GFP) was used as a
control. Viral production and infection of cells are aided by the
presence of green fluorescent protein (GFP) encoded by a gene
incorporated into the viral backbone. For infection, HEK-293 cells were
plated 24 h before viral infection. TRP viral constructs were
added to the cells with fresh growth medium. Infected cells can be
monitored by observing how many cells are green under fluorescent
microscopy at 24 and 48 h and were subsequently used in
electrophysiological recordings and for molecular analysis.
Cell cultures. HEK-293 cells (ATCC, Manassas, VA) were obtained and cultured in low-glucose DMEM (Invitrogen, San Diego, CA) with 10% FBS. Cells were passaged weekly, and fresh medium was applied every 2-3 days.
Creation of TRPC4 stable lines.
TRPC4 and TRPC4
were cloned from murine colonic smooth muscle as
previously described (23) and ligated into pcDNA3.1
mammalian vector (Invitrogen). Clones in this vector were used to
generate stable lines in HEK-293 cells by use of the calcium phosphate method and G418 selection (Invitrogen). PCR analysis and whole cell
patch-clamp recordings confirmed stable lines.
Voltage-clamp experiments in HEK-293 cells.
The patch-clamp technique for whole cell recording was utilized in
these experiments. The patch pipettes were made from borosilicate glass
capillaries pulled with micropipette puller (P-80/PC, Sutter, CA) and
heat polished with a microforge (MF-83, Narishige, Japan). The pipette
resistances were 1-3 M for whole cell recordings and 5-8
M
for single-channel recordings. The average cell capacitance was
17 ± 1 pA. Currents were amplified with a List EPC-7
amplifier and/or Axopatch-1A amplifier and digitized with a 12-bit
analog-to-digital converter (Digidata 1322A; Axon instrument, Foster
City, CA). The data were stored directly and digitized online by using
pCLAMP software (version 8.0; Axon instrument). Data were sampled at 2 kHz for whole cell and 5 kHz for single-channel recordings and filtered
at 1 kHz using an 8-pole Bessel filter. Data were analyzed using pCLAMP
(version 8.0; Axon Instrument), GraphPad Prizm (version 3.0; San Diego,
CA), and Origin software (MicroCal Software, version 5.0; Northampton, MA).
Solutions.
The bath solution, Ca2+-phosphate-buffered saline (PSS),
contained (in mM) 5 KCl, 135 NaCl, 2 CaCl2, 10 glucose, 1.2 MgCl2, and 10 HEPES, pH 7.4, with Tris. The pipette
solutions contained (in mM) 110 Cs aspartate, 30 TEA-Cl, 5 MgCl2, 2.7 K2ATP, 0.1 Na2GTP, 2.5 creatine phosphate disodium salt, 10 BAPTA, and 5 HEPES, pH 7.2, with
Tris. BAPTA (10 mM) was replaced with EGTA (0.1 mM) in some experiments
to raise intracellular free Ca2+. Na+
replacement was performed by replacing equimolar amounts of
N-methyl-D-glucamine (NMDG+). For
single-channel recordings, pipette solutions for on-cell and excised
patches were Ca2+-PSS. The bath solution for on-cell
patches contained (in mM) 140 KCl, 1 EGTA, 0.61 CaCl2, and
10 HEPES adjusted to pH 7.4 with Tris. To test the Ca2+
sensitivity of channels in the excised patches, the bath solution contained (in mM) 110 potassium gluconate, 30 KCl, 1 EGTA, and 10 HEPES
adjusted to pH 7.4 with Tris. Ca2+ was added to a bath
solution buffered by 1 mM EGTA to create Ca2+ activities
from 107 to 10
6 M. Activities were
calculated with a program developed by C. M. Hai (University of
Virginia, Charlottesville, VA). LaCl3 (La3+)
(Sigma, St Louis, MO) was dissolved into water. CMZ, niflumic acid, and
DIDS were dissolved into DMSO to prepare stock solutions (10
1 M) and were added to the bath solution in some
experiments. All experiments were performed at room temperature.
Statistical analysis. Data are expressed as means ± SE. The "n values" in the text indicate the number of cells used in whole cell patch-clamp experiments or the number of membrane patches used in single-channel analysis. P values <0.05 were taken as a statistically significant difference.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular features of TRP channels.
Full-length cDNAs of TRPC4 and TRPC4
were amplified from
murine colonic smooth muscle RNA by using gene-specific primers (23). These cDNAs were used to produce a stable line of
HEK-293 cells expressing TRPC4
, as well as TRPC4
and TRPC4
adenoviruses for use in transient infections of cells (see
MATERIALS AND METHODS). Calmodulin-binding
domains (CaMBD) are present in all TRP genes within the carboxy
terminus (22). TRPC4
contains two CaMBD, but in
TRPC4
the second CaMBD is deleted due to alternative splicing. These
cDNAs, isolated from mouse colonic smooth muscle RNA, share complete
amino acid identity in the CaMBD to those previously reported in mouse
brain (22).
Native inward current in HEK-293 cells.
We investigated native currents in nontransfected HEK-293 cells and in
HEK-293 cells infected with a virus expressing only GFP to determine
the extent of endogenous TRP currents in this expression system. We
detected transcripts for TRPC4 and TRPC4
in HEK cells (data not
shown) similar to other investigators (27).
|
|
Expression of TRPC4 in HEK-293 cells.
Currents were recorded from HEK cells stably transfected with TRPC4
and TRPC4
or infected with similarly constructed adenovirus. To
determine the calcium regulation of these TRP channels, cells were
dialyzed with BAPTA (10 mM), which activated an inward current in cells
expressing TRPC4
averaging
618 ± 239 pA at
60 mV; n = 6, P < 0.05 (Fig. 1B).
Summarized data are shown in Fig. 1D. Inward currents were
of smaller magnitude in TRPC4
cells dialyzed with 0.1 mM EGTA
(
125 ± 23 pA; n = 9, P < 0.01)
than in cells dialyzed with BAPTA at a holding potential of
60 mV
(compare control in Fig. 2, C and D, to Fig. 1,
B and D). After full development of current after
dialysis with EGTA (0.1 mM), CMZ (10 µM) increased the inward current
to
482 ± 27 pA at
60 mV (n = 9).
La3+ blocked the inward current to control levels (Fig. 2,
C and D). Summarized data are shown in Fig.
2D.
Expression of TRPC4 in HEK-293 cells.
Dialysis of cells expressing TRPC4
with BAPTA (10 mM) induced inward
currents within 5-10 min after establishing the whole cell
configuration (Fig. 1C). The inward current activated by BAPTA was significantly increased (
691 ± 134 pA at
60 mV;
n = 16, P < 0.05) compared with
control, untransfected cells, or cells infected with a virus expressing
only GFP. Other cells were dialyzed with 0.1 mM EGTA. At a holding
potential of
60 mV, currents in these cells were smaller (e.g.,
233 ± 53 pA; n = 10, P < 0.05, control in Fig. 2, E and F) than in BAPTA
dialyzed cells. These data demonstrate that cells expressing TRPC4
and TRPC4
have a current available that is inhibited by
Ca2+.
Cation selectivity of TRPC4 currents in HEK-293 cells.
Inward current generated in TRPC4
cells by BAPTA dialysis were also
blocked by 10 µM La3+ (
691 ± 134 to
108 ± 22 pA; n = 5, Fig. 3,
A and B), as observed with currents activated by 0.1 mM EGTA dialysis. By using a ramp protocol (from
80 to +80 mV), the inward currents were activated by
BAPTA without a change in reversal potential throughout development (Fig. 3C). Application of La3+ inhibited the
BAPTA-induced current and also did not result in a change in the
reversal potential (Fig. 3B). The current activated by BAPTA
dialysis was reduced by replacement of external Na+ with
NMDG. This reduced the inward current from
471.4 ± 74 to
142 ± 25 pA (
60 mV; n = 4, P < 0.05, Fig. 3, E and F) and shifted the
reversal potential from
1 ± 0.3 to
14 ± 0.8 mV
(P < 0.01, Fig. 3, E and F; see
inset of 3F).
|
Effects of niflumic acid and DIDS on TRPC4 currents.
Niflumic acid and DIDS have been shown to block the
Ca2+-inhibited, nonselective cation currents in ICC
(10). Therefore, we tested the effects of niflumic acid on
HEK-293 cells stably expressing TRPC4
. In these experiments,
currents were evoked by application of 10
5 M CMZ.
Niflumic acid (30 µM) reduced the amplitude of the inward current
from
1,438 ± 273 to
247 ± 141 pA, (n = 6, P < 0.05). Use of a ramp protocol demonstrated that
application of niflumic acid did not cause a change in the reversal
potential, although the current at all potentials was
decreased. A similar pharmacological effect was noted with
application of 100 µM DIDS reducing the amplitude of the inward
current from
809 ± 129 to
274 ± 97 pA, (n = 4, P < 0.05).
Unitary currents in HEK-293 cells expressing TRPC4.
We further investigated the properties of TRPC4
currents at the
single-channel level. Unitary currents were recorded in on-cell patches
in TRPC4
expressing cells as the membrane potential of the patch was
stepped to various potentials. Recordings at potentials positive to +20
mV were too noisy to clearly discern single-channel currents, so
current-voltage curves were generated for potentials between
60 and
+20 mV (Fig. 4A). The averaged
data from 5 cells revealed a single-channel conductance of 17.5 ± 0.5 pS (Fig. 4B). A whole cell current was activated and
shown to reverse at 0 mV when cells were dialyzed with 10 mM BAPTA.
These observations suggested to us that reduced cytoplasmic
Ca2+ may activate these channels. To test the properties of
the channels more directly, patches were excised, allowing the interior
of the patch to be exposed to bathing solutions containing
10
6 M Ca2+ (pipette solution contained
Ca2+-PSS; EK and
ECl were
87 and
30 mV, respectively). We
examined the reverse calcium dependence of the unitary current using a cell-excised patch exposed to 10
6 M calcium (Fig.
4C). The open probability of the channel was greatly
increased upon exposure to 10
7 M calcium (Fig.
4C).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the present study, we have evaluated currents expressed in
HEK-293 cells after transfection of the cells with TRPC4 splice variants. We found that buffering Ca2+ to low levels within
the cells results in the activation of persistent inward currents that
reverse at ~0 mV and are reduced in amplitude by replacing
Na+ with NMDG+. TRPC4 currents could also be
induced by treating cells with CMZ, a calmodulin inhibitor. Previous
studies have shown that Ca2+/calmodulin binding to TRPC3
and TRPC4 channels causes inhibition of the intrinsically high open
probability of these channels (19, 30). Work by Zhang et
al. (30) demonstrated that TRPC3 bound Ca2+/calmodulin at a site that overlapped with the
IP3 receptor (IP3R) binding domain and could be
activated by CMZ. Tang et al. (19) went further to
show that other TRP proteins, including TRPC4, could also interact with
IP3R and calmodulin, but that one such site present in
TRPC4 was absent in TRPC4
. Given this observation, we addressed
this difference by examining the functional properties of the two TRPC4
splice variants at a whole cell and single-channel level. Some
properties of TRPC4
and TRPC4
were different between our study
and that of Schaefer et al. (17), including the
double rectification observed at very negative and positive potentials and the single-channel conductance (30 pS). This may be due to differences in recording conditions or solutions used during recording.
The single-channel conductance of expressed TRPC4 channels was 17.5 pS, and these channels were inhibited by Ca2+. Channel
openings with the same characteristics were activated in on-cell
patches and in excised patches by CMZ. These properties are similar to
the properties of the conductance in native ICC that is
responsible for the pacemaker current (see Table
1 for a comparison of the currents). ICC
express TRPC4 isoforms at the transcriptional level, and a
recent study showed immunochemical evidence suggesting TRPC4
expression, particularly in caveolae, in ICC (20).
Immunochemical staining of TRPC4 was also detected within putative
smooth muscle cells in these same preparations; however, the TRPC4
expression was clearly stronger in the colabeled ICC than in the other
cell types (20). This observation suggests that a
decreased translation of the TRPC4 protein within smooth muscle cells
could explain the lack of a Ca2+-inhibited current within
these cells. Taken together, these data suggest that TRPC4 or
TRPC4-like channels might encode the pacemaker conductance in ICC.
|
Transcripts of TRPC6 have also been identified in ICC (1).
Experiments with BAPTA dialysis in HEK-293 cells stably transfected with TRPC6, however, failed to generate a current similar to that seen
in TRPC4-transfected cells or in ICC (24). A recent
study has shown that TRPC4 is predominantly expressed in ICC, whereas smooth muscle cells in the same preparation predominantly express TRPC6
(20). TRP channels can form heterotetramers with slightly different properties to the homotetrameric channels (12,
29). A recent study demonstrates that heterotetramers can form
between TRPCs 1, 4, and 5 or TRPCs 3, 6, and 7, but not between the two channel subgroups (3, 6, 18). Thus, as TRPC1 and TRPC5 have not been identified in ICC, it is reasonable to assume that TRPC4
forms a homotetramer in ICC, although channels consisting of TRPC4
and TRPC4
cannot be ruled out.
Although ICC have Ca2+-inhibited, nonselective cation
channels (10), a similar conductance was not detected in
intestinal smooth muscle cells in spite of the fact that transcripts
for TRPC4 have been found in vascular and visceral smooth muscle cells (1, 23). Both ICC and smooth muscle cells contain
transcripts for each splice variant, with TRPC4 dominating in
quantitative studies involving tissue from different regions of the GI
tract, including tissue obtained from mouse intestine
(23). In addition, murine and canine fundus smooth
muscles, electrically quiescent tissues, expressed the TRPC4
isoform
predominantly as opposed to TRPC4
in isolated smooth muscle cells
(23). Interestingly, neither BAPTA dialysis nor
application of CMZ on freshly dispersed smooth muscle cells from mouse
ileum (n = 45; unpublished observations) yielded a
current comparable to that seen in cultured ICC or HEK cells expressing
TRPC4
or TRPC4
. A recent study from Schaefer et al.
(17) suggested that TRPC4
is capable of forming
heterotetramers with TRPC4
and works in a dominant negative fashion
to inhibit the currents generated from expression of TRPC4
homotetramers. It is possible that the function of TRPC4
in smooth
muscle cells is altered by the presence of other TRP isoforms or is
possibly inhibited by the expression of TRPC4
and that cells
predominantly expressing TRPC4
display little current from the
heterotetrameric channels.
Electrical slow waves are a fundamental property of phasic GI muscles,
and these events activate periodic Ca2+ entry and regulate
contractions in electrically coupled smooth muscle cells. A
Ca2+-inhibited, nonselective cation conductance contributes
to the pacemaker current that initiates slow wave activity (10,
11). In the present study, we identified a molecular entity,
TRPC4, that shares many similarities with native pacemaker channels. We
have demonstrated that a splice variant of TRPC4 (TRPC4) that eliminates a calmodulin-binding site in the carboxy terminus of channels generates robust currents when intracellular Ca2+
is decreased or calmodulin inhibitors are applied. The preponderance of
circumstantial evidence leads us to suggest that TRPC4
or a
TRPC4
-like channel encodes the pacemaker conductance in ICC. These
experiments have not ruled out the possibility that an as yet
unidentified TRP channel may be responsible for encoding the plasmalemmal pacemaker current in murine small intestine. However, we
have demonstrated that if an unknown conductance exists and is involved
in pacemaking, it must have properties very similar to TRPC4. Thus we
would conclude that the pacemaker current in ICC is either a TRPC4
conductance or a TRPC4-like conductance.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank L. Miller and H. Beck for excellent technical assistance.
![]() |
FOOTNOTES |
---|
* R. L. Walker and S. D. Koh contributed equally to this work.
The National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-41315 supported this work. R. L. Walker is a predoctoral fellow of the American Heart Association-Western States Affiliate.
Address for reprint requests and other correspondence: B. Horowitz, Dept. of Physiology and Cell Biology, Univ. of Nevada, School of Medicine, Reno, NV 89557 (E-mail: burt{at}physio.unr.edu).
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.
August 14, 2002;10.1152/ajpcell.00266.2002
Received 7 June 2002; accepted in final form 9 August 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Epperson, A,
Hatton WJ,
Callaghan B,
Doherty P,
Walker RL,
Sanders KM,
Ward SM,
and
Horowitz B.
Molecular markers expressed in cultured and freshly isolated interstitial cells of Cajal.
Am J Physiol Cell Physiol
279:
C529-C539,
2000
2.
Freichel, M,
Suh SH,
Pfeifer A,
Schweig U,
Trost C,
Weissgerber P,
Biel M,
Philipp S,
Freise D,
Droogmans G,
Hofmann F,
Flockerzi V,
and
Nilius B.
Lack of an endothelial store-operated Ca2+ current impairs agonist-dependent vasorelaxation in TRP4 /
mice.
Nat Cell Biol
3:
121-127,
2001[ISI][Medline].
3.
Goel, M,
and
Schilling WP.
Association of immunophilins with mammalian TRP channels (Abstract).
Biophysical Journal
82:
637a,
2002.
4.
Groschner, K,
Hingel S,
Lintschinger B,
Balzer M,
Romanin C,
Zhu X,
and
Schreibmayer W.
Trp proteins form store-operated cation channels in human vascular endothelial cells.
FEBS Lett
437:
101-106,
1998[ISI][Medline].
5.
Halaszovich, CR,
Zitt C,
Jungling E,
and
Luckhoff A.
Inhibition of TRP3 channels by lanthanides. Block from the cytosolic side of the plasma membrane.
J Biol Chem
275:
37423-37428,
2000
6.
Hofmann, T,
Schaefer M,
Schultz G,
and
Gudermann T.
Subunit composition of mammalian transient receptor potential channels in living cells.
Proc Natl Acad Sci USA
99:
7461-7466,
2002
7.
Huizinga, JD,
Robinson TL,
and
Thomsen L.
The search for the origin of rhythmicity in intestinal contraction; from tissue to single cells.
Neurogastroenterol Motil
12:
3-9,
2000[ISI][Medline].
8.
Jungnickel, MK,
Marrero H,
Birnbaumer L,
Lemos JR,
and
Florman HM.
Trp2 regulates entry of Ca2+ into mouse sperm triggered by egg ZP3.
Nat Cell Biol
3:
499-502,
2001[ISI][Medline].
10.
Koh, SD,
Jun JY,
Kim TW,
and
Sanders KM.
A Ca2+-inhibited nonselective cation conductance contributes to pacemaker currents in mouse interstitial cell of Cajal.
J Physiol
540:
803-814,
2002
11.
Koh, SD,
Sanders KM,
and
Ward SM.
Spontaneous electrical rhythmicity in cultured interstitial cells of Cajal from the murine small intestine.
J Physiol
513:
203-213,
1998
12.
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
13.
Mery, L,
Magnino F,
Schmidt K,
Krause KH,
and
Dufour JF.
Alternative splice variants of hTrp4 differentially interact with the C-terminal portion of the inositol 1,4,5-trisphosphate receptors.
FEBS Lett
487:
377-383,
2001[ISI][Medline].
14.
Montell, C.
Physiology, phylogeny, and functions of the TRP superfamily of cation channels.
Sci STKE
2001:
RE1,
2001.
15.
Philipp, S,
Cavalie A,
Freichel M,
Wissenbach U,
Zimmer S,
Trost C,
Marquart 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].
16.
Sanders, KM.
A case for interstitial cells of Cajal as pacemakers and mediators of neurotransmission in the gastrointestinal tract.
Gastroenterology
111:
492-515,
1996[ISI][Medline].
17.
Schaefer, M,
Plant TD,
Stresow N,
Albrecht N,
and
Schultz G.
Functional differences between TRPC4 splice variants.
J Biol Chem
277:
3752-3759,
2002
18.
Strubing, 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].
19.
Tang, J,
Lin Y,
Zhang Z,
Tikunova S,
Birnbaumer L,
and
Zhu MX.
Identification of common binding sites for calmodulin and inositol 1,4,5-trisphosphate receptors on the carboxyl termini of trp channels.
J Biol Chem
276:
21303-21310,
2001
20.
Torihashi, S,
Fujimoto T,
Trost C,
and
Nakayama S.
Calcium oscillation linked to pacemaking of interstitial cells of Cajal: requirement of calcium influx and localization of TRP4 in caveolae.
J Biol Chem
277:
19191-19197,
2002
21.
Torihashi, S,
Nishi K,
Tokutomi Y,
Nishi T,
Ward S,
and
Sanders KM.
Blockade of kit signaling induces transdifferentiation of interstitial cells of cajal to a smooth muscle phenotype.
Gastroenterology
117:
140-148,
1999[ISI][Medline].
22.
Trost, C,
Bergs C,
Himmerkus N,
and
Flockerzi V.
The transient receptor potential, TRP4, cation channel is a novel member of the family of calmodulin binding proteins.
Biochem J
355:
663-670,
2001[ISI][Medline].
23.
Walker, RL,
Hume JR,
and
Horowitz B.
Differential expression and alternative splicing of TRP channel genes in smooth muscles.
Am J Physiol Cell Physiol
280:
C1184-C1192,
2001
24.
Walker, RL,
Koh SD,
Greenwood IA,
Sergeant GP,
and
Horowitz B.
Functional characterization of smooth muscle TRP channels using an adenoviral expression system (Abstract).
Biophysical Journal
82:
422a,
2002.
25.
Ward, SM,
Burns AJ,
Torihashi S,
and
Sanders KM.
Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine.
J Physiol
480:
91-97,
1994[Abstract].
26.
Ward, SM,
Ordog T,
Koh SD,
Baker SA,
Jun JY,
Amberg G,
Monaghan K,
and
Sanders KM.
Pacemaking in interstitial cells of Cajal depends upon calcium handling by endoplasmic reticulum and mitochondria.
J Physiol
525:
355-361,
2000
27.
Wu, X,
Babnigg G,
and
Villereal ML.
Functional significance of human TRP1 and TRP3 in store-operated Ca2+ entry in HEK-293 cells.
Am J Physiol Cell Physiol
278:
C526-C536,
2000
28.
Wu, X,
Babnigg G,
Zagranichnaya T,
and
Villereal ML.
The role of endogenous human trp4 in regulating carbachol-induced calcium oscillations in HEK-293 cells.
J Biol Chem
277:
13597-13608,
2002
29.
Xu, XZ,
Li HS,
Guggino WB,
and
Montell C.
Coassembly of TRP and TRPL produces a distinct store-operated conductance.
Cell
89:
1155-1164,
1997[ISI][Medline].
30.
Zhang, Z,
Tang J,
Tikunova S,
Johnson JD,
Chen Z,
Qin N,
Dietrich A,
Stefani E,
Birnbaumer L,
and
Zhu MX.
Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain.
Proc Natl Acad Sci USA
98:
3168-3173,
2001