Statistical analysis

39 Pages·2000·2.34 MB·English

JBC Papers in Press. Published on May 17, 2000 as Manuscript M003408200
TRP4 (CCE1) protein is part of native CRAC-like channels in
adrenal cells
Stephan Philipp, Claudia Trost, Jan Warnat, Julia Rautmann, Nina Himmerkus,
Gregor Schroth, Oliver Kretz1, Wolfgang Nastainczyk2 Adolfo Cavalié, Markus Hoth3
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and Veit Flockerzi n
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Institut für Pharmakologie und Toxikologie, Institut für Anatomie1, ttp
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Institut für Medizinische Biochemie2 und Institut für Physiologie3 .jb
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Universität des Saarlandes, D-66421 Homburg, Germany. b/
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Correspondence should be addressed to Stephan Philipp
Tel.: [phone]
Fax.: [phone]
Email: [email protected]
Running title: TRP4 is part of CRAC-like channels
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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Summary
Mammalian TRP proteins have been implicated to function as ion channel subunits
responsible for agonist-induced Ca2+ entry. To date, TRP proteins have been extensively
studied by heterologous expression giving rise to diverse channels properties and activation
mechanisms including store-operated mechanisms. However, the molecular structure and the
functional properties of native TRP channels still remain elusive. Here we analyze the
properties of TRP4 (CCE1) channels in their native environment and characterize TRP
expression patterns and store-operated calcium currents that are endogenous to bovine adrenal
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cells. We show by Northern blot analysis, immunoblots and immunohistochemistry that TRP4 n
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transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. fro
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Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only other TRP ttp://w
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transcript found at considerable levels was TRP1, whereas TRP2, TRP3, TRP5(CCE2) and .jb
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TRP6 were not detectable. Depletion of calcium stores with InsP3 or thapsigargin activates by g/
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store-operated ion channels in adrenal cells. These channels closely resemble calcium release- t o
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activated Ca2+ (CRAC) channels. Expression of cce1(trp4) cDNA in antisense orientation ril 1
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significantly reduces both, the endogenous CRAC-like currents and the amount of native
TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of
native CRAC-like channels in adrenal cells.
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Introduction
Receptor operated Ca2+ signaling has been described in a variety of non-excitable cells and
has been attributed to basic and specific cell functions like cell growth, differentiation and
hormone release (reviewed by (1-3)). The receptor dependent and phospholipase C mediated
formation of inositol 1,4,5-trisphosphate (InsP ) leads to the depletion of intracellular Ca2+
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stores which in turn activates store-operated Ca2+ (SOC) channels in the plasma membrane.
The mechanisms linking store depletion to channel activation are still unknown but might
include direct coupling of store proteins to the plasma membrane channel (1;4-7), diffusible
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messengers (8-10) or exocytotic insertion of channels preformed in vesicles (11-13). The n
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current picture and implications of these activation mechanism have been reviewed recently fro
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(14;15). The best studied SOC channels in terms of function and biophysical properties are ttp://w
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calcium release-activated Ca2+ (CRAC) channels which have been originally described in .jb
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mast cells and T-lymphocytes (16;17). This prototype of a SOC channel is characterized by by/
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its inward rectifying currents and by its high selectivity for Ca2+. The molecular structure of t o
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CRAC channels, however, remains elusive. ril 1
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Mammalian homologues of the Drosophila TRP and TRPL ion channels are candidate
proteins underlying CRAC channels but also other store-operated and/or receptor operated
Ca2+ permeable ion channels. So far, the cDNAs of seven mammalian TRP genes have been
cloned and characterized as functional cation channels after heterologous expression in
various cell systems including HEK 293 cells, L-cells, CHO cells, SF9 insect cells and
Xenopus oocytes (reviewed in (18)). The gating mechanisms for the recombinant mammalian
TRP channels are still controversial. The channels formed by heterologously expressed TRP1,
TRP4 and TRP5 appear to be activated by maneuvers known to deplete Ca2+ stores and
represent SOCs (19-23) but store independent activation of cation channels has also been
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reported after heterologous expression of TRP4 and TRP5 (24). In addition, TRP3 appears to
be gated by Ca2+ store depletion via InsP (5;25;26) and it has been connected to InsP
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dependent cation influx in pontine neurones (27). However, direct activation by
diacylglycerol of TRP3 and TRP6 cation channels has also been demonstrated (28). TRP1,
TRP3 and TRP6 encode Ca2+ permeable but non-selective cation channels when expressed in
eucaryotic cells (19;29-32), whereas TRP4 and TRP5 form ion channels mainly permeable
for Ca2+ (20-23;33). Obviously, some recombinant mammalian TRP channels are sensitive to
store depletion and form ion channels mainly permeable to Ca2+ whereas others appear to be
non-selective cation channels insensitive to store depletion. Probably, depletion-insensitive
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TRP proteins may form store-operated channels through multimerization with a store n
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sensitive subunit. TRP4 and TRP5 are appropriate candidates to play the role of a dominant fro
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subunit of these channels in terms of Ca2+ selectivity. ttp://w
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To analyze the properties of TRP proteins, their cDNAs have been usually overexpressed in .jb
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cells which already may contain various endogenous TRP proteins as well as store-operated by/
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Ca2+ entry channels. HEK 293 cells, for example, show a significant amount of CRAC t o
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currents (34) and do express TRP transcripts to some extent (25;26;35;36). Expression of ril 1
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TRP cDNAs in HEK 293 cells might lead to heterooligomeric channels by multimerization of
recombinant and endogenous TRP proteins. In addition, homomultimeric recombinant
channels may be formed due to overexpression of the recombinant TRP cDNA in the
heterologous system. Consequently, the activity of recombinant TRP channels expressed in a
foreign cellular environment does not necessarily correspond to the activity of native store-
operated Ca2+ channels.
Further evidence linking TRP proteins to agonist induced Ca2+ entry was obtained by
expression of mammalian TRP4 cDNA fragments in antisense orientation in mouse
fibroblasts (37) and salivary gland cells (38). In clones of salivary gland cells stably
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expressing TRP1 antisense cDNA, Ca2+ influx in the presence of thapsigargin is decreased,
suggesting that TRP1 is a candidate protein for store-operated calcium Ca2+ entry
mechanisms (38). Using fura-2 Ca2+ measurements, Birnbaumer and colleagues (37) reported
that clonal lines of murine L(tk-) cells stably transfected with the muscarinic M5 receptor
cDNA together with a mouse TRP4 antisense cDNA fragments showed a lack of M5 receptor
stimulated calcium entry. This entry could be due either to the activation of receptor-operated
or store-operated channels, which might be selective for Ca2+. It remains to be shown which
TRP proteins are expressed in L(tk-) cells.
The TRP4 cDNA was originally isolated from bovine adrenal gland mRNA and we show
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now, that TRP4 transcripts and proteins are abundantly expressed in the cortex but not in the n
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medulla of the adrenal gland. To study TRP4 function in its native environment we used fro
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bovine adrenal cortex cells which were found to express significant amounts of TRP4. ttp://w
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Analyzing store-operated currents, we show that these cells display endogenous CRAC-like .jb
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currents after InsP3- or thapsigargin-induced Ca2+ store depletion. In this study we used an by g/
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antisense strategy to dissect the relation between the TRP4 protein expression and native t o
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store-operated Ca2+ entry channels. Transfection of bovine adrenal cortex cells with TRP4 ril 1
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antisense cDNA reduces both, the endogenous CRAC-like current as well as the amount of
endogenously expressed TRP4 protein strongly indicating that TRP4 is part of native CRAC-
like channels in adrenal cells.
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Experimental procedures
Construction of eukaryotic expression plasmids
The pCAGGS expression vector (39) and the pCAGGS-EGFP plasmid containing the cDNA
of the green fluorescent protein EGFP-N1 (Clontech) were kindly provided by Jun-ichi
Miyazaki. To create additional cloning sites the synthetic cDNA 5´CTC GAG CGG CCG
CTA GCC CGG GTA CCA TCG ATA TCT GAT CAG AGC TCG AG3´ was subcloned in
the XhoI-cleaved pCAGGS in both orientations yielding pCAGGS1 and pCAGGS2. The
NotI/AccI cDNA fragment of pEBFP (Clontech) encoding the blue fluorescent protein and
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the NotI/BspDI pCAGGS2 fragment were ligated to obtain the reporter plasmid pCAGGS2- n
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EBFP. The complete coding cDNAs of bovine TRP4 (20)(previously CCE or CCE1) and fro
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bovine TRP2 (40) were subcloned into pCAGGS2 in antisense orientation downstream of the ttp://w
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ß-actin promoter to obtain pCAGGS2-TRP4ANTI and pCAGGS2-TRP2ANTI. .jbc
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Culture and Transfection of SBAC cells and HEK 293 cells t o
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Bovine adrenocortical cells (SBAC, ATCC CRL 1796) were obtained seven passages after ril 1
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primary culture from the American Tissue and Cell Collection as frozen stocks. On thawing,
cells were grown in HAM´s F12 medium supplemented with 10% foetal calf serum (Life
technologies Lot no. 40F4566J), 50 U/ml penicillin, 50 µg/ml streptomycin at 37°C in a 5 %
CO humidified atmosphere. Cells were subcultured every third day and grown to ∼90 %
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confluence. Cells were detached from plates in the presence of 0.5 g/l trypsin (Life
technologies) and thereafter, trypsin was neutralized by addition of 10 volumes of serum
supplemented medium. Stocks were stored in 5% dimethyl sulfoxide in liquid nitrogen until
required for experiments.
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Transfection of SBAC cells (grown in 35 mm dishes to ∼80 % confluence) was carried out
essentially as described by the manufacturer of Superfect reagent (Quiagen) using 3µg
pCAGGS-EGFP together with 1µg pCAGGS2-TRP4 or 1 µg pCAGGS2-EBFP or
ANTI
pCAGGS2-TRP2 . Electroporation was carried out as follows: 107 cells were suspended in
ANTI
300 µl of fresh medium and cotransfected with 15 µg pCAGGS-EGFP and 5µg pCAGGS2-
TRP4 or 5µg pCAGGS2-EBFP, respectively, using a gene pulser transfection apparatus
ANTI
(Bio-Rad Laboratories) at a setting of 0.23 kV and 960 µF. Transfection of HEK 293 cells
with pCCE were carried out as described (20).
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Cloning of bovine TRP5 and TRP6 and human TRP6 cDNA n
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A 365 bp bovine TRP5 cDNA fragment was amplified from 5 µg DNaseI treated bovine brain fro
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total RNA using degenerated oligonucleotides corresponding to amino acids E467EWEMWH ttp://w
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(5´GAR GAR TGG GAR ATG TGG CA3´) and M HKWGDG (5`CC RTC NCC CCA .jb
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YTT RTG CAT3´) and nested primers corresponding to amino acids L DILKFL (5´CTT by/
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GAT ATA CTC AAA TTT CTC3´) and L630IADHAD (5´TC AGC ATG ATC GGC AAT G3´) t on A
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of mouse TRP5 (21). The alignment with the corresponding mouse and rabbit TRP5 ril 11
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sequences revealed 91% and 100% nucleotide and amino acid sequence identity, respectively.
Bovine TRP6 cDNA fragments were amplified from DNase I treated bovine brain total RNA
using primers corresponding to D IFKFMV (5´GAT ATC TTC AAA TTC ATG GTC3´)
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and S SFQEIE (5´CTC AAT TTC CTG GAA TGA AC3´) of mouse TRP6(30). The
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alignment with the corresponding mouse and human TRP6 sequences revealed 89,9 % and
94.7 % nucleotide and 100 % amino acid sequence identity. The complete coding sequence of
human TRP6 (2796 bp, Genbank acc. AJ006276) was amplified from human placenta poly
A+ RNA. We found two alternatively spliced transcripts encoding hTRP6 variants now called
hTRP6∆316-431 and hTRP6∆377−431 both lacking the first out of seven hydrophobic regions
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predicted to span the plasma membrane. The bTRP5, bTRP6, hTRP6, hTRP6∆ and
316-431
hTRP6∆377−431 sequences have been deposited to the GenBank database under
GenBankAccession Number AJ271070, AJ271069, AJ271066, AJ271067 and AJ271068,
respectively.
Analysis of the TRP expression pattern by RT-PCR
5 µg SBAC total RNA was treated with DNaseI for 30 min and reverse transcribed using
random primers and Supercript™ II reverse transcriptase (Life technologies). 1/7 of the
resulting single strand cDNA or of negative control reactions performed without reverse
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transcriptase, were used for PCR in the presence of 0.2 µM primers specific for each bovine n
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TRP sequence. PCR conditions were the same as described above for nested PCR, except that fro
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the annealing temperature was 56 °C. The following primers were used: bTRP1 (GenBank ://w
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acc. AF012900): 5´GAC AGA TGT CAG GTT ACC3´ and 5´ATT CTT TCA AGG GCT .jb
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b/
GGC3´; bTRP2 (GenBank acc. AJ006304): 5´CAC TTC TGC TGC AAA ACC3´ and 5´AGC y
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TCT GTC AGT CTG TTG3´; bTRP3 (GenBank acc. AJ006781): 5´CAA TCT GGT ACG t on
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AGA ACC3´ and 5´CAG TCC AAG TGA ACT GTG3´; bTRP4 (GenBank acc. X99792): ril 11
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5´TGG CGT CTC GCT GGT AC3´ and 5´AGG ACC CAC GGT AAT ATC3´; bTRP5
(bCCE2, this manuscript): 5´GTA CTG CTG GCT TTT GCC3´ and 5´TTC AGC AGC ACT
ACC AGG3´; bTRP6 (this manuscript): 5´TCA TCA TGG TGT TTG TGG C3´ and 5´GCA
AAA CAA TGA CCA TTG TAA3´. As positive controls we used PCR reactions with 1 amol
of recombinant plasmids containing the corresponding TRP cDNA as template. 1/10 of the
PCR products were analyzed by electrophoresis in 2 % agarose gels. The identity of the
amplified cDNA fragments was confirmed by restriction analysis in 5 % polyacrylamide gels
and by sequence analysis.
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Northern blot analysis
Northern blots were performed using 10 µg poly A+ RNA as described (20). For the analysis
of TRP transcripts in adrenal gland, cortex and medulla were separated macroscopically. The
following probes were labeled by random priming using [α32P]dCTP (6000 Ci/mmol): TRP1:
a 2289 bp cDNA fragment corresponding to the complete coding region of human TRP1 (19);
TRP3: a 395 bp fragment of bovine TRP3 (32) and a 2544 bp cDNA fragment spanning the
human TRP3 coding region (25); TRP4: a 2965 bp fragment corresponding to the coding
region of bovine TRP4 (20); TRP5: a 365 bp cDNA fragment of bovine TRP5 (see above), a
2895 bp cDNA fragment encoding mouse TRP5 and a 1630 bp cDNA fragment of rabbit
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TRP5 (21); TRP6: a 2796 bp fragment encoding hTRP6 (see above). The filters were exposed n
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to X-ray films with intensifying screens at -80°C for the times indicated. The filters used for fro
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analysis of TRP3 and TRP5 expression in SBAC cells were reused to analyse of TRP1 and ttp://w
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TRP6 expression. For that purpose filters were stripped, exposed to X-ray films to control .jb
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residual signals, hybridized with the second probe and, finally, with a 239 bp cDNA fragment by/
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of the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH). t on
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In situ hybridisation
8 µm thick sections of freshly frozen adult bovine adrenal glands as well as SBAC cells
grown overnight on silane-coated glass slides were hybridized to bovine TRP4 cRNA as
described (21). Sense and antisense cRNA probes corresponding to the complete coding
region of bovine TRP4 were labeled by in vitro transcription in the presence of [α33P]UTP
using a linearised recombinant plasmid.
Construction and purification of glutathione-S-transferase (GST)-TRP4 fusion proteins
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A EcoRV cDNA fragment corresponding to amino acids 843 to 981 of bovine TRP4 was
fused in frame 3´ of the GST cDNA into pGEX4T-2 (Pharmacia) to yield pGST-bTRP4c. E.
coli BL21 (DE3) cells transformed with pGST-bTRP4c or pGEX4T-2, were grown in LB
medium containing 2.5 mM betaine and 440 mM sorbitol. Protein expression was induced by
addition of 0.5 mM IPTG and then the cultures were incubated for 3 h at 29°C. The following
steps were carried out at 4°C: cells were harvested, resuspended in TBS/PMSF buffer (50
mM Tris-HCl, 150 mM NaCl, pH 7.5 containing 1 mM phenylmethylsulfonyl fluoride),
sonicated 3 times for 30 s and incubated for 1 h in TBS/PMSF buffer containing 1 % Triton
X-100. The clear supernatant obtained after centrifugation for 15 min at 12000 g, was
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incubated with glutathione-sepharose for 1 h and washed 5-7 times with 10 volumes n
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TBS/PMSF buffer. GST and GST-bTRP4c fusion proteins were eluted with 50 mM Tris-HCl fro
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pH 8.0 containing 10 mM reduced glutathione. Proteins were analyzed by SDS- ttp://w
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polyacrylamide gelelectrophoresis (SDS-PAGE) and stained with Coomassie blue. .jb
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Preparation of a TRP4 antibody t o
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The synthetic peptide D TITHEDYVTTRL of bovine TRP4 was coupled to keyhole ril 1
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limpet hemocyanine activated with N-Succinimidyl-3-maleinimido-propionate (Fluka) via an
introduced amino-terminal cysteine residue and injected into rabbit 236 together with
complete and incomplete Antibody-Multiplier-Adjuvans (Linaris). Serum was affinity-
purified using the synthetic peptide, which was coupled to epoxy-activated Sepharose
(Pharmacia).
Preparation of bovine adrenal cortex microsomes
All steps were performed at 4°C. Bovine adrenal glands were obtained from the local
slaughter house and the cortex was homogenized in TBSP buffer (TBS, 1 µg/ml antipain, 1
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