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J. Biol. Chem., Vol. 281, Issue 6, 3227-3236, February 10, 2006

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TRP Channels Are Inhibited by cAMP and Contribute to Pacemaking in Neurosecretory Insect Neurons^*

Dieter Wicher¹, Hans-Jürgen Agricola, Roland Schönherr^¶, Stefan H. Heinemann^¶, and Christian Derst^||

From the Department of Neurohormones, Saxon Academy of Sciences, 07743 Jena, Germany, the Institute of Zoology, Friedrich Schiller University, Jena, Germany, the ^¶Institute of Molecular Cell Biology, Friedrich Schiller University, Jena, Germany, and the ^||Center for Anatomy, Charité, Berlin, Germany

Received for publication, October 31, 2005 , and in revised form, November 22, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
From a neuronal cDNA library of the cockroach Periplaneta americana we isolated a 3585-bp cDNA sequence encoding Periplaneta transient receptor potential (pTRP), a protein of 1194 amino acids showing 65% identity to the orthologous Drosophila channel protein dTRP. Heterologous expression of pTRP in HEK293 cells produced a constitutively active, non-selective cation channel with a Ca²⁺:Na⁺ permeability ratio of 2. In contrast to dTRP-mediated currents, pTRP currents were partially inhibited by 8-bromo-cAMP, and this effect was not mediated by protein kinase A (PKA) activation. pTRPb, a truncated pTRP splice variant missing most of the C terminus, was insensitive to 8-bromo-cAMP. Thus, the critical cAMP-binding site seems to be located in the C-terminal part of pTRP, although there is no common cAMP-binding consensus sequence. While dTRP is only expressed in the photoreceptors, pTRP is expressed throughout the nervous system. In particular it is expressed in dorsal unpaired median (DUM) neurons. In these octopamine-releasing, neurosecretory cells a Ca²⁺ background current contributing to pacemaker activity was found to be up-regulated by the reduction of cAMP level. In addition, the Ca²⁺ background current was inhibited by LOE-908, 2-APB, and La³⁺, which similarly affected the pTRP current. We thus propose that the pTRP protein is involved in forming the channel passing the Ca²⁺ pakemaking background current in DUM neurons.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Proteins of the TRP² (transient receptor potential) family form cation-selective and Ca²⁺-permeable ion channels with multiple functions (1). The first TRP channel was described in Drosophila photoreceptors where mutants lacking this channel showed a transient receptor potential upon continuous light stimulation (2). In the fly photoreceptors two further TRP proteins, TRPL and TRP (3), contribute to visual transduction (4). TRP proteins have six transmembrane segments, and most of them probably assemble to homoor heterotetramers to form ion channels.

The superfamily of TRP channels has been grouped into several subfamilies (1, 5). Members of the mammalian TRPC (canonical TRP) family show the highest homology with the Drosophila TRP/TRPL/TRP variants. These channels appear to be receptor-operated in that they are activated by a variety of stimuli that lead to activation of phospholipase C (PLC).

The modulation of pacemaker conductances is a basic principle to adjust the neuronal activity to the physiological requirements. Certain peptide hormones affect ion channels involved in the regulation of pacemaking. Previous investigations on identified insect neurosecretory neurons, namely efferent dorsal unpaired median (DUM) neurons in the cockroach Periplaneta americana, have shown that a voltage-independent Ca²⁺ background current providing for non-capacitative Ca²⁺ entry (NCCE) changes the spike frequency of these cells. For example, up-regulation of this current by the Periplaneta adipokinetic hormone AKH I (pAKH I: pQ-V-N-F-S-P-N-W·NH₂, also named neurohormone D (6)), accelerates spiking (7, 8). A detailed analysis of the signal transduction mechanism, by which AKH I binding to the receptor leads to potentiation of Ca²⁺ background current, revealed a complex pattern of events including activation of PLC but no apparent contribution from intracellular Ca²⁺ stores (8). The final event of the signal transduction cascade was the down-regulation of the cAMP level, which directly enhanced the background current. Up-regulation of the cAMP level in DUM neurons attenuates the Ca²⁺ background current and thus the spike frequency (9).

We were interested in identifying the molecular basis for the Ca²⁺ background current in DUM neurons and thus looked for a channel that (i) may conduct a voltage-independent Ca²⁺ current and (ii) that is sensitive to cAMP. Since TRP channels are known to fulfill the first criterion, we screened a Periplaneta cDNA library for putative members of the TRPC family. We succeded in finding a Periplaneta homolog to the Drosophila TRP channel. In the present study, we report an unusual regulation of this TRP channel. We demonstrate that the Periplaneta but not the Drosophila TRP channel is downregulated by cAMP. Based on an analysis of the pharmacological profile of the Periplaneta TRP channel we propose that it is involved in forming the channel that conducts the Ca²⁺ background current in DUM neurons.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Degenerated RT-PCR and RACE Protocols—RNA from isolated Periplaneta thoracic and abdominal ganglia were isolated using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and reverse-transcribed using Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). According to sequence homologies between the three Drosophila melanogaster TRP channels dTRP (GenBank^TM accession number M34394 [GenBank] ), dTRPL (GenBank^TM accession number M88185 [GenBank] ), and dTRP (GenBank^TM accession number AJ277968 [GenBank] ) several degenerated primers were designed. Among these primers one pair (5'-tggtaygarggattgcc-3' coding for WYEGLP; 5'-gghcccagrtgcggatt-3' coding for NPHLGP) amplified a 650-bp fragment from the ganglial cDNA with strong homology to dTRP using AmpliTaq Gold polymerase (Applied Biosystems, Darmstadt, Germany). To obtain the full-length pTRP sequence we performed nested touch-down 5'- and 3'-RACE experiments as described previously (10) using pTRP-specific primers and a Periplaneta americana mixed tissue Marathon RACE library (RACE-F1, 5'-tgtacgtggcaaccgtggcact-3'; RACE-F2, 5'-gccgccaaggtgcaaacgtc-3'; RACE-B1, 5'-cgaaccactcgccgatcacaga-3'; RACE-B2, 5'-tgcgacgccaggatcagcag-3'). PCR and RACE products were either sequenced directly or cloned into pGEM-T (Promega, Mannheim, Germany) for sequencing.

RT-PCR Analysis of Periplaneta Tissues—A nested RT-PCR approach was developed for testing ganglion-specific expression and single-cell analysis of isolated DUM neurons. The following intron-spanning PCR primers were designed: Sc-F1, 5'-cagaaagaacgtcggctcat-3'; Sc-F2, 5'-caactgcccactgtagcaga-3'; Sc-B1, 5'-gggactgttgctgtggctat-3'; Sc-B2, 5'-cagagtcttccgacctgctc-3'. For single-cell RT-PCR analysis of pTRP expression the cytosol of single DUM neurons was harvested with patch pipettes and directly transferred to the RT reaction mixture. RT reaction was performed with either Superscript II reverse transcriptase (Invitrogen) and ganglion RNA or with Sensiscript reverse transcriptase (Qiagen) and isolated cytosol of single DUM neurons as described by the manufacturer, respectively. The two nested PCR reactions were performed with AmpliTaq Gold polymerase (Applied Biosystems) with initial 6 min at 94 °C enzyme activation followed by 40 cycles 30 s at 94 °C; 30 s at 55 °C; 1 min at 72 °C. PCR products of the second PCR reaction were visualized on a 2% agarose gel. As a positive control we amplified part of a ubiquitously expressed actin gene from P. americana.

Construction of a pTRP pIRES2-EGFP Expression Vector (HEK293 Expression)—Full-length pTRP was amplified using Advantage TaqII DNA polymerase mixture (Clontech, Palo Alto, CA) and ganglion cDNA using the following three primers: FL-for 1, 5'-aaaggcctcgagatgagtgacaattatgacgtg-3'; FL-for 2, 5'-acagcatcctcgagatgatggaggaggagaacgtc-3'; and FL-rev, 5'-tctgcgctcgagttatagccagcctgctgacat-3'. PCR was performed for 35 cycles with 30 s at 94 °C, 1 min at 64 °C (or 58 °C during initial two cycles), and 12 min at 68 °C. Finally, PCR products were cloned into the pIRES2-EGFP vector using XhoI sites incorporated into primer sequences (shown in italics). Among these clones a novel splice variant pTRPb was found, harboring a novel exon and introducing a stop codon resulting in a shorter C-terminaly truncated splice variant.

Production and Specificity of pTRP Antibodies—Two pTRP antisera were raised in rabbits against the synthetic peptides (1) QPVSGHNMSAGW and (2) QNKSRNG representing two different C-terminal motifs. The peptides were cross-linked to keyhole limpet hemocyanin (KLH) by means of glutaraldehyde. The peptides, the peptide-KLH conjugates, and the antiserum (1) were produced by Sigma Genosys (Cambridge, UK). The specificity of antiserum (1) was tested by Western blot (Fig. 2B). For immunocytochemical control of antiserum (2) we used the preimmune serum (Fig. 3, B and E), and we performed experiments without the primary antibody (data not shown). In both control experiments there was no staining.

Western Blot—Freshly isolated P. americana thoracic and abdominal ganglia from five animals were homogenized in 100 µl of lysis buffer (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% Triton X-100, 1 tablet Complete protease inhibitor mixture (Roche Diagnostics) per 50 ml). After electrophoretical separation of 20 µg protein per lane (SDS-PAGE) and blotting on polyvinylidene difluoride transfer membranes (Millipore), standard wash and detection procedures were used. Anti-pTRP antibody (1) was 1:2000 diluted, and peroxidase-coupled secondary antibodies (anti-rabbit IgG or anti-mouse IgG) were applied in 1:10,000 dilution, followed by chemoluminescence detection with the ECL Plus kit (Amersham Biosciences). For control we used pTRP-transfected and mock-transfected HEK293 cells. 2 days after transfection about 1 x 10⁵ cells were harvested and immediately lysed in 200 ml of lysis buffer, and 10 µl were used for SDS-PAGE. To exclude unspecific bands due to antibodies directed against KLH, control blots were performed with antiserum (5 µl) that was preincubated with 100 µg of KLH protein in 100 µl of phosphate-buffered saline, pH 7.4). This procedure prevented the Western blot detection of KLH protein by the serum but did not affect the 129 kDa signal in Periplanea tissues or in transfected HEK293 cells.

Immunocytochemistry—Adult P. americana L. were taken from laboratory colonies at the University of Jena (maintained at 27 °C under a 12 h light/12 h dark regime). Animals were anesthetized by cooling under crushed ice and decapitated. Brains and the sixth abdominal ganglia were dissected from animals of both sexes under ice-chilled Tris-HCl saline and transferred into freshly prepared fixative: 4% formaldehyde in 0.1 M Millonigs buffer (pH = 7.3–7.4) overnight at room temperature. The preparations were washed in Tris-HCl buffer (146 mM NaCl, 50 mM Tris-OH, pH = 7.4, for 12 h at 4 °C on a shaker. 40-µm-thick sections of agar-embedded tissues were cut on a vibratome (Technical Products, St. Louis, MO). The washed free-floating vibratome sections were then incubated in rabbit anti-TRP polyclonal antibody (cf. above) diluted 1:1000 in a solution of Tris-HCl buffer containing 2% normal goat serum, 0.25% Triton-X, 3% skim milk powder, and 0.25% bovine serum albumin (MPB). Subsequently, the tissue was washed in MPB overnight and incubated in the Cy3-tagged secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 3 h at adilution of 1:800 in MPB. The vibratome sections were mounted and embedded in Mowiol. For the photographic documentation we used the bright-field optics on a Zeiss Axiophot microscope, a Hamamatsu digital camera C4742-95, and the software OpenLab (Improvision Ltd., Coventry, England). HEK293 cells growing on coverslips were treated as the vibratome sections.

Electrophysiology—HEK293 cells were cultured at a density of 2 x 10⁴ per 35-mm dish and transfected with 1 µg of pTRP/pIRES2-EGFP using SuperFect (Qiagen). For some experiments we transfected cells with pTRPb/pIRES2-EGFP or with dTRP/pcDNA3 (a generous gift of Drs. C. Montell and L. Salkoff) and 0.8 µg pEGFP-C1 (Clontech).

Ion currents in HEK293 cells and in DUM neurons (isolated according to Ref. 7) were measured at room temperature using whole-cell patch clamp with appropriate compensation of series resistance and of capacitive and leakage currents. Some experiments were performed in the inside-out configuration. Pipettes having resistances of 2–4 M (HEK293 cells) or 0.5–0.8 M (DUM neurons) were pulled from borosilicate capillaries. Current measurements and data acquisition were performed using an EPC9 patch clamp amplifier controlled by PULSE software (HEKA Elektronik, Lambrecht, Germany).

HEK293 Cells—The pipette solution contained (in mM) 140 CsCl, 1 Mg-ATP, 10 EGTA, 10 HEPES (pH = 7.3), and the bath solution contained 140 CsCl, 15 glucose, 15 HEPES (pH = 7.4). For inside-out measurements the pipette contained the bath solution and the bath solution was exchanged by the pipette solution after establishing the seal. To evaluate the permeability of pTRP channel to Na⁺ and Ca²⁺ the bath solution contained 140 NaCl, 15 glucose, 15 HEPES, and 140 N-methyl-D-glucamine-Cl, 5 CaCl₂, 10 glucose, 15 HEPES, respectively. The permeability ratios were calculated according to Equations 1 and 2.

(Eq. 1)

(Eq. 2)

where P_ion are the permeabilities, [ion]_i and [ion]_o are the internal and external concentration of the respective ion, and V_rev is the reversal potential (11).

To rule out an effect on endogenous HEK293 channels (12), 8-Br-cAMP and all other substances used below were first tested in mocktransfected cells (n = 5–8). In no case we found any significant effect indicating that all reported effects of the agents relied on the expressed TRP channels. On the other hand, it cannot be excluded that heterologous expression of TRP proteins in HEK293 cells may lead to the formation of new TRP heteromultimers including endogenous TRPCs (13) or to up-regulation of TRPC expression. As outlined under "Discussion," this is rather unlikely in the case of the TRP proteins used in this study.

DUM Neurons—The pipette solution contained (in mM) 100 choline methyl sulfate, 60 CsOH, 8 CsCl, 30 tetraethylammonium-Br, 2 Mg-ATP, 1 CaCl₂, 3 EGTA, 10 HEPES, pH = 7.25. The bath solution contained 190 choline methyl sulfate, 2 CaCl₂, 3 SrCl₂, 10 HEPES, 0.5 µM tetrodotoxin, pH = 7.4. Background currents were measured 40 ms after stepping from holding potential of –50 mV, which corresponds to the resting potential of cells to –90 mV.

Data Analysis—Results were given as means ± S.E. of mean (S.E.), n = number of cells. The evaluation of statistical significance of differences was performed with Student's t test. For data analysis the software IgorPro (WaveMetrics, Lake Oswego, OR) and Prism 4 (Graph Pad Software, San Diego, CA) were used.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sequence Analysis of P. americana TRP—Using a combined degenerated PCR and RACE protocol, we were able to isolate the full-length sequence of the P. americana pTRP channel. The 3585-bp cDNA sequence (GenBank^TM accession number AY387857 [GenBank] ) is coding for a protein of 1154 amino acids showing all typical features of a TRP-like channel: four ankyrin-like repeats at the N terminus, a coiled-coil domain, six transmembrane regions, a TRP-pore sequence typical for a nonspecific cation channel, and a long C terminus harboring several conserved TRP-specific sequences. The pTRP sequence is 66% identical and 75% homologous to the orthologous D. melanogaster sequence (dTRP), whereas sequence similarity to paralogous Drosophila sequences (dTRP and dTRPL) is significantly lower and spans only the very conserved core region of 900 amino acids of the protein, with 50% identity and 65% similarity. An alignment of pTRP and dTRP indicating several conserved protein features is shown in Fig. 1. The C terminus is less conserved between Periplaneta and Drosophila TRP as seen in various gaps in the alignment and in a C-terminal extension of pTRP. A comparison with the Drosophila genomic sequence (3) indicated that alternative splicing only in one case might be responsible for these variations, i.e. the pTRP sequence lacked one dTRP exon (indicated in Fig. 1). In addition, when amplifying the full-length pTRP sequence, a short alternatively spliced variant, pTRPb, was discovered. This splice variant harbored the homologous dTRP exon missing in the full-length form, but it introduced a stop codon thereafter, resulting in a truncated pTRP version with only 791 amino acids, i.e. lacking most of the C-terminal sequence. In addition, a short variable region was identified in the C terminus, resulting in either deletion of a single amino acid (alanine 827) or in an expansion by four residues (GNTS) preceding alanine 827. The mechanism of this sequence variation is unclear but may represent alternative usage of exon-intron splice sites or polymorphisms.

Expression Analysis of pTRP—The expression of pTRP channels in neuronal tissue was tested by a nested RT-PCR approach. An intron-spanning 355-bp PCR fragment was amplified from Periplaneta brain as well as from all thoracic and abdominal ganglia indicating a widespread expression in cockroach neuronal tissue. In addition, in four out of ten investigated DUM neurons a pTRP signal was found on single-cell level as well (Fig. 2A). A Western blot analysis of neuronal tissues showed a distinct band with the expected molecular mass of 129 kDa in abdominal and thoracic ganglia with very low background staining (Fig. 2B). A protein of the same size was detected by the same antiserum (1) in transiently transfected HEK293 cells expressing pTRP but not in mock-transfected cells.

Distribution of pTRP in the Nervous System—Control immunostainings were performed with both pTRP antibodies in HEK293 cells transfected with pTRP DNA. 24 h after transfection, HEK293 cells showed significant immunofluorescence (Fig. 2, C and E), which was missing in non-transfected cells (Fig. 2, D and F). Immunostainings within the Periplaneta nervous system revealed a frequent but distinct expression of pTRP both in brain (Fig. 3, A–H) and abdominal ganglia (Fig. 3, J–L). Both antibodies, which were designed to recognize different C-terminal pTRP motifs, were seen to stain the same structures (Fig. 3, A, C, D, and F). Generally less intense immunofluorescence was seen in neuronal somata ranging from virtually absent to clearly pronounced staining as shown in Fig. 3, J and K, for the DUM neurons. Within the brain, immunostaining was found in the protocerebrum, in the deutocerebrum, and in the optic lobes. In the protocerebrum longitudinal and commissural fibers are stained. Within the central complex there is strong immunoreactivity in fibers around the upper (Fig. 3, D and F) and the lower (Fig. 3D) part of central body. Interestingly, the arborization areas within the central body do not show any immunoreactivity. This might indicate that pTRP is expressed in fibers exept for their terminal branches including synaptic areas (Fig. 3, D and F). Similarly, there was clear staining around the olfactory glomeruli in the antennal lobe but not at all within the glomeruli where olfactory signals are processed via manifold synaptic contacts (Fig. 3G). The antennocerebral tract containing the projection neuron fibers was extensively stained (Fig. 3H). Besides the regions of olfactory information processing there is prominent staining in the optic lobes, particularly within the medulla (Fig. 3I). In the abdominal ganglia the somata of DUM neurons (Fig. 3, J and K) and those of a few smaller, not identified neurons (data not shown) appear stained. As in the brain, some fiber tracts are prominently stained while others such as the giant axons lack any staining (Fig. 3, J and L).

cAMP Down-regulates pTRP but Not dTRP Currents—Expression of pTRP in HEK293 cells produced a constitutively active non-selective cation conductance, which was not found in mock-transfected cells. The pTRP current showed outward rectification, even with symmetrical ion concentrations (Fig. 4, A and B). The ratio of ion permeabilities was estimated from the dependence of the reversal potential on the composition of bath versus pipette solutions. With 5 mM Ca²⁺ and 140 mM Na⁺ in the bath versus 140 mM Cs⁺ in the pipette the reversal potential was –16.0 ± 0.9 mV (n = 5) and –0.4 ± 1.1 mV (n = 6), respectively. This yields, according to Equations 1 and 2, a permeability ratio of P_Na:P_Cs:P_Ca = 1:1.01:2.05, which is very similar to the corresponding ratio reported for dTRP (3). HEK293 cells expressing pTRP were thus exposed to sustained influx of ions including Ca²⁺ and showed considerably shortened survival time in culture compared with mock-transfected cells. 24 h after transfection the ratio of green fluorescing cells to non-fluorescing cells was 0.19 ± 0.08 (n = 200). Among the cells expressing pTRP the morphology of 26 ± 7(n = 38) cells was typical for HEK293 cells; the remaining cells appeared spherical. 48 h after transfection only 9 ± 3% of cells (n = 200) showed green fluorescence, and all these cells were of spherical shape.

View larger version (55K): [in this window] [in a new window]   FIGURE 1. Sequence alignment of Periplaneta and Drosophila TRP. Ankyrin-like repeats are underlined and in bold; the coiled-coil domain is shown in italics; transmembrane regions are shown in bold and are labeled with a +. The dTRP exon missing in pTRP is indicated by *; the C-terminal sequence of the truncated pTRPb splice variant is shown in bold italics. A second variable sequence stretch is found near alanine 787 (PGT-A787-GAG, shown in bold italics and underlined), where sequences can vary to PGT-GAG (deletion) or PGT-GNTSA-GAG (insertion).

View larger version (36K): [in this window] [in a new window]   FIGURE 2. A, RT-PCR expression analysis of pTRP in different tissues. An RT-PCR analysis of the expression in Periplaneta neuronal tissue is shown in the upper panel, and a singlecell RT-PCR analysis of isolated DUM neurons is shown in the lower panel. pBR322/HaeIII digest was used as DNA marker (M). B, Western blot analysis of pTRP expression. A band around the expected 129 kDa was recognized by antiserum 1 in HEK293 cells expressing pTRP, in abdominal and thoracic ganglia. This band is missing in mock-transfected HEK293 cells. Protein standard markers are indicated. C–F, anti-pTRP immunofluorescence in HEK293 cells transfected with pTRP DNA (C, E) and non-transfected HEK293 cells (D, F) using antiserum 1 (C, D) or antiserum 2 (E, F). Under identical illumination the immunostaining visible in pTRP-transfected cells does not appear in non-transfected cells. Highest fluorescence density in the cytoplasm of pTRP-transfected cells is seen with antiserum 1 (C). This serum provides some unspecific staining (D). In pTRP-transfected cells antiserum 2 recognizes a protein (E), which is missing in non-transfected cells (F). pTRP-transfected cells, in contrast to non-transfected cells, form spherical clusters (C, E). Bar,10 µm.

To our knowledge, no TRP channel has been found to be downregulated by cAMP. Therefore, we were interested to see whether Drosophila TRP (dTRP), the only other presently known TRP channel, was also sensitive to cAMP. When expressing dTRP in HEK293 cells we obtained, similarly to pTRP, an outwardly rectifying current that was constitutively active. But this current was resistant to cAMP (Fig. 5, A and B). Application of 8-Br-cAMP (2 µM) only yielded a current reduction by 4% (n = 8). For comparison, 2-APB (100 µM) strongly reduced the dTRP current (Fig. 5, A and B).

To characterize the selectivity of the putative cyclic nucleotide-binding site in the pTRP channel, we applied the membrane-permeant cGMP analog 8-Br-cGMP (2 µM). However, cGMP failed to affect the pTRP and dTRP currents (Fig. 5C). Thus, the down-regulation of TRP channels is specific for cAMP.

The depressing action of cAMP on the NCCE channel in DUM neurons was a direct cAMP effect and not mediated by channel phosphorylation via PKA (8). To confirm that the down-regulation of pTRP current directly relies on cAMP, we had to rule out a possible contribution of pTRP channel phosphorylation by endogenous PKA. We thus tested the effect of 8-Br-cAMP in presence of the PKA inhibitors KT5720, Rp-cAMPS, and myristoylated PKA-inhibiting peptide 14–22 amide (PKI). If the regulation of pTRP was PKA-dependent, the presence of an inhibitor should abolish the effect of cAMP. There was, however, neither a direct effect of PKA inhibitors on pTRP current (Fig. 6A) nor a significant change in the reducing effect of 8-Br-cAMP (Fig. 6B).

Searching for a putative constituent of a neuronal NCCE channel we thus found the pTRP channel, which displayed some of the expected properties including the unusual sensitivity to cAMP. For comparing the pharmacological profiles of TRP channels from Periplaneta and Drosophila we tested, besides typical TRP channel blockers such as 2-APB and La³⁺ (14) and SKF96365, which inhibits a variety of channels providing Ca²⁺ influx (15, 16), and LOE-908, which blocks NCCE in various preparations including DUM neurons (17, 2). The pTRP current was reduced by LOE-908 (10 µM)by26 ± 5% (n = 5), while dTRP was insensitive (Fig. 7). 2-APB (100 µM) reduced the pTRP current by 71 ± 6% (n = 9) and the dTRP current by 49 ± 6% (n = 7). SKF96365 (10 µM) did not affect either of the currents, and La³⁺ (1 mM) reduced the pTRP current by 50 ± 9% (n = 5) and the dTRP current by 23 ± 9% (n = 8).

View larger version (128K): [in this window] [in a new window]   FIGURE 3. Anti-pTRP immunofluorescence in the nervous system of adult Periplaneta. A–I, brain; J–L, sixth abdominal ganglion. A, protocerebrum and part of deutocerebrum (transversal slice, antiserum 2). B, control (parallel section, preimmune serum). C, protocerebrum and part of deutocerebrum (transversal slice, antiserum 1). The protocerebral structures indicated in A–C are the central complex with lobes (BL) and central body (CB) and the mushroom bodies formed by pedunculus (P) and calyces (C). The antennal lobe (AL) with the olfactory glomeruli (G) is part of the deutocerebrum. D, central complex (transversal slice, antiserum 2). E, control (parallel section, preimmune serum). F, central complex (transversal slice, antiserum 1). The structures indicated in (D–F) represent lobes (BL), upper central body (CBU), lower central body (CBL), and protocerebral bridge (PB). Note that there is no staining within the central body (CB), while the surrounding fibers show extensive staining. G, part of antennal lobe (transversal slice, antiserum 2). Note that there is no staining within the glomeruli (G), while surrounding fibers appear stained. H, deutocerebrum (transversal slice, antiserum 2). The antennocerebral tracts (ACT), which project from the antennal lobe (AL) to cerebral structures including the calyces show pronounced, dot-like staining. I, optic lobes (transversal slice, antiserum 2). The indicated structures are lamina (Lam), medulla (Med), first (1.OC) and second (2.OC) optic chiasma. Note the slight staining in the lamina and the prominent staining in the medulla. J, sixth abdominal ganglion (transversal slice, antibody 1). The somata of DUM neurons (*) appear stained. No staining is seen in giant axons (arrows). K, part of transversal slice around DUM neurons (*) (antiserum 1). There is also dot-like staining of fibers. L, horizontal slice, overview (antiserum 2). Bars, 100 µm (exept for K, 50 µm).

View larger version (25K): [in this window] [in a new window]   FIGURE 4. pTRP current is attenuated by cAMP. pTRP was expressed in HEK293 cells, and currents were measured in the whole-cell mode using symmetric CsCl solutions. A, families of currents obtained by 100-ms lasting voltage jumps from –80 to +100 mV from a holding potential of –50 mV. The currents measured before (Control) and 5 min after application of 2 µM 8-Br-cAMP are shown. Further application of 100 µM 2-APB caused nearly completely current block after 5 min (2-APB). B, time course of the current reduction by 8-Br-cAMP. Currents were measured at +100 mV and normalized to the current IControl that was obtained before application of 8-Br-cAMP (arrow). Data represent means ± S.E. of n = 5 cells. C, pTRP currents obtained by voltage ramps from –100 to +100 mV (in 400 ms). The pTRP current shows outward rectification, the reversal potential is near 0 mV. 8-Br-cAMP (2 µM, 5 min) caused a reduction of both inward and outward current. In the presence of 2-APB (5 min) only a residual current remained. D, pTRP currents obtained as described for C in the inside-out configuration. cAMP (1 mM, 5 min) reduced the inward and outward current. E, concentration dependence of the cAMP-effect at +100 mV. Filled squares represent means of 8–10 experiments. For comparison, the open square represents data obtained with 8-Br-cAMP (2 µM) in the whole-cell configuration (n =9). The dose-response curve is described by an IC50 of 74 nM and a Hill coefficient of 0.83. The saturating current reduction amounts to 48%.

View larger version (21K): [in this window] [in a new window]   FIGURE 5. dTRP current is resistant to cAMP. dTRP was expressed in HEK293 cells, and currents were measured as described in the legend to Fig. 4A. A, application of 2 µM 8-Br-cAMP did not reduce the current, while application of 100 µM 2-APB (5 min) caused strong current reduction (2-APB). B, dTRP currents obtained by voltage ramps as described in the legend to Fig. 4B. The dTRP current shows outward rectification, and the reversal potential is near 0 mV. The currents registered before (Control) and 5 min after 8-Br-cAMP application appear identical, by contrast to the diminished current measured in the presence 2-APB. C, effect of 8-Br-cAMP (cAMP, 2 µM) and 8-Br-cGMP (cGMP, 2 µM) on dTRP and pTRP currents measured at +100 mV. Data represent means ± S.E. of n = 6–10 cells.

Does pTRP Contribute to the NCCE Conductance in DUM Neurons?—Our immunocytochemical investigations have shown that DUM neurons express pTRP channels, and the properties of the pTRP current in the heterologous expression system are compatible with the suggestion that this channel may be involved in forming the NCCE channel in DUM neurons. To obtain further support for this hypothesis, we tested whether LOE-908, 2-APB, and La³⁺, which attenuated the pTRP current in HEK293 cells (Fig. 7B), would also affect NCCE in DUM neurons. Using a bath solution containing only 2 mM Ca²⁺ as charge carrier, LOE-908 (10 µM) and 2-APB (100 µM) reduced the resting current density at –90 mV by 0.43 ± 0.15 pA/pF (n = 5) and 0.54 ± 0.09 pA/pF (n = 5), respectively (Fig. 9A). This reduction is similar to the effect of 8-Br-cAMP (2 µM) that caused a reduction by 0.42 ± 0.06 pA/pF (n = 7). This shows that the NCCE channel in DUM neurons, in addition to cAMP and LOE-908 (8), is sensitive to 2-APB. However, the relative effect of 2-APB appears to be weaker than on pTRP channels expressed in HEK293 cells (Fig. 7A). While 2-APB produced a stronger reduction of the TRP current than cAMP and LOE-908, there was no significant difference in the effect of all three compounds in DUM neurons. To confirm that these findings in DUM neurons reflect inherent properties of NCCE channels, we repeated the experiments using 3 mM Sr²⁺ as charge carrier to exclude Ca²⁺-dependent artifacts; the effects of cAMP, LOE-908, and 2-APB were not different either (Fig. 9A). With both charge carriers, La³⁺ (1 mM) was the most potent blocker of resting current; it reduced the current density by 1.7 ± 0.5 pA/pF (n = 4) for Ca²⁺ and by 1.6 ± 0.4 pA/pF (n = 7) for Sr²⁺. Taken together, the NCCE channel in DUM neurons is sensitive to those compounds that inhibit heterologously expressed pTRP channels, but we find differences in the quantitative effect of some compounds in the two systems.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. The cAMP effect on pTRP is not mediated by activation of PKA. A, pTRP currents obtained by voltage ramps as described in Fig. 4B under the indicated conditions. The sequence of conditions is given at the bottom. B, effect of 8-Br-cAMP (2 µM)on the current measured at +100 mV under control conditions (without PKA inhibitor) and after preincubation with KT5720 (10 nM), Rp-cAMPS (100 µM), or PKI 1422 (1 µM). Data represent means ± S.E. of n = 6 cells.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. Pharmacology of pTRP (A) and dTRP (B) channels. Currents were measured at +100 mV in the whole-cell mode using symmetric CsCl solutions before (Control) and 3 min after application of 8-Br-cAMP (2 µM), LOE-908 (10 µM), 2-APB (100 µM), SKF96365 (10 µM), and La3+ (1 mM). The data were normalized to control currents and represent means ± S.E. (n = 7–11).

View larger version (15K): [in this window] [in a new window]   FIGURE 8. The truncated splice form pTRPb is insensitive to cAMP. A, pTRPb was expressed in HEK293 cells, and current families were measured as described in the legend to Fig. 4A. Currents measured 5 min after application of 2 µM 8-Br-cAMP are not reduced with respect to the control records. B, pTRPb currents obtained by voltage ramps as described in Fig. 4B. The pTRPb current shows outward rectification, and the reversal potential is near 0 mV. Currents registered before (Control) and 5 min after 8-Br-cAMP application are identical. C, effect of 8-Br-cAMP (2 µM), LOE-908 (10 µM), and 2-APB (100 µM) on pTRPb currents measured at +100 mV. Data represent means ± S.E. of n = 8–10 cells.

View larger version (17K): [in this window] [in a new window]   FIGURE 9. The inhibitors of pTRP affect the non-capacitative Ca2+ entry current in Periplaneta DUM neurons. A, reduction of current density at –90 mV with 2 mM Ca2+ (white)or 3 mM Sr2+ (black) as charge carrier by 8-Br-cAMP (cAMP,2 µM), LOE-908 (10 µM), and 2-APB (100 µM). Data represent means ± S.E. of n = 5–9 cells. B, current density at –90 mV with 2 mM Ca2+ before (white bars) and upon application of 10 µM ETYA (black bars). The effect of ETYA alone (Control) is reduced after preincubation of cells with 8-Br-cAMP (cAMP,2 µM), LOE-908 (10 µM), and 2-APB (100 µM). Data represent means ± S.E. of n = 5–7 cells.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The immunocytochemical findings have indicated that the Periplaneta TRP protein is frequently expressed within the nervous system. Most prominently, it occurs in nerve fibers involved in processing/conducting sensory, particularly olfactory and optic, information. The role of pTRP in these fibers remains elusive. In hippocampal neurons the insertion of TRPC5 channels in neurites terminates their outgrowth (18). An intriguing finding in the Periplaneta brain was the fact that branching regions of fibers do not express pTRP (e.g. in the central body, Fig. 3D). Furthermore, we cannot exclude the possibility that pTRP is also expressed in glial cells surrounding the neurites. This point needs to be analyzed in more detail, e.g. by using glial cell markers and by performing electron microscopy. The most important result of the immunocytochemical approach for the present study was to see that the pTRP protein is located in the somata of DUM neurons.

Drosophila TRP, but also the mammalian counterparts that form the subfamily of canonical TRPs (TRPC), function as receptor-operated channels (3). Activation of PLC was found to be a key element in the signal transduction process leading to dTRP activation (19). On the other hand, heterologously expressed dTRPL and dTRP proteins form constitutively active channels. However, coexpression of dTRPL with dTRP produces regulated channels that can be activated by agonists for receptors that stimulate PLC (3).

In Periplaneta DUM neurons we previously found a Ca²⁺-permeable conductance that was voltage-independent and constitutively active (8, 20). This Ca²⁺-background conductance was up-regulated by the peptide hormone AKH I via PLC activation. It was further established that the regulation of this conductance was independent of intracellular stores, i.e. that it contributes to NCCE (8). One might speculate whether the channel responsible for the Ca²⁺-background conductance might belong to the TRP family. A detailed analysis of the AKH I-signal transduction process yielded a complex picture. The diacyglycerol produced by PLC activation is metabolized by diacyglycerol lipase to AA. This activates, by an unknown mechanism, NO-sensitive guanylyl cyclase. The resulting increase in cGMP level activates the phosphodiesterase 2, which in turn lowers the cAMP level (8). The final step in the up-regulation of Ca²⁺-background conductance was thus the reduction of an inhibition. Such kind of regulation is akin to that of ATP-sensitive K⁺ channels, which open upon a drop in ATP concentration (21).

Searching for the molecular substrate of the Ca²⁺-background conductance we found pTRP, a homolog of the Drosophila TRP. This protein is expressed in DUM cells, and we have seen that channels formed by pTRP in HEK293 cells show some properties expected for a channel responsible for the Ca²⁺-background conductance in DUM neurons.

It has to be kept in mind that overexpression of TRP proteins in HEK293 cells may lead to the formation of new TRP heteromultimers including endogenous TRPCs (13) or to up-regulation of TRPC expression. However, the difference in 8-Br-cAMP sensitivity of currents produced by expression of pTRP and its C-terminally truncated version pTRPb (compare Fig. 4, C and D, with Fig. 8) clearly demonstrates that the overexpressed and not an endogeneous channel protein determines the channel properties. Moreover, expression of dTRP, the Drosophila homolog to pTRP, results in the generation of currents similar to those produced by pTRP but with partially different pharmacological properties (cf. Fig. 7).

With respect to a putative role of pTRP in forming Ca²⁺-background channels, the most important feature, besides its permeability to Ca²⁺, is the sensitivity to cAMP. Similarly important is the insensitivity of pTRP to cGMP, since in DUM neurons the phosphodiesterase 2-mediated decrease in cAMP concentration requires an increase in cGMP concentration (8). Sensitivity of pTRP to both cAMP and cGMP would thus not be compatible with the regulation of the NCCE channel in DUM neurons. Furthermore, both pTRP and the background conductance are inhibited by LOE-908, 2-APB, and by La³⁺. Taken together, our results are compatible with the hypothesis that pTRP is involved in forming channels that conduct the Ca²⁺-permeable background conductance conferring NCCE in DUM neurons.

If our assumption was correct we had to propose a new functional role for a TRP channel, namely to act as a pacemaker channel. In DUM neurons the firing frequency is regulated by a variety of ion currents including the Ca²⁺-background current (22). Depression of this current, e.g. by FMRF-related peptides, decreases the firing rate (23), while potentiation, e.g. by AKH I, enhances the firing rate (8). Although AKH I modulates a set of DUM cell currents, i.e. Na⁺ and P/Q-type Ca²⁺ current as well as Na⁺- and Ca²⁺-dependent K⁺ currents, solely the up-regulation of the Ca²⁺-background current produces faster spiking (24). Furthermore, the Ca²⁺-background current in DUM neurons is involved in controlling the intracellular Ca²⁺ concentration (20) and in filling of intracellular Ca²⁺ stores (25). Up-regulation of this current can induce local as well as global Ca²⁺ signals (8).

From the viewpoint of ion channel regulation the most intriguing result of the present study is the down-regulation of pTRP current by cAMP. Although the binding site of cAMP on pTRP is currently unknown, two conclusions can be drawn from sequence comparisons, pattern searches, and electrophysiological measurements of the short pTRPb splice variant. (i) The distal C-terminal 400 amino acids of pTRP seem to contain the cAMP-binding site as the truncated pTRPb splice variant does not show any cAMP effect, and (ii) no consensus cAMP-binding sites were found in pTRP indicating an unusual cAMP binding. Further work will be dedicated to analyze the cAMP effect on the level of single channels and to identify putative cAMP-binding sites in the channel protein. The latter will not exclude a search for N-terminal cAMP-binding sites, since it might also be possible that cAMP binds at the N terminus, while the distal C terminus is necessary to transduce the conformational change to channel closure.

	FOOTNOTES

 
^* This work was supported in part by Deutsche Forschungsgemeinschaft Grant Wi 1422/2-4,5. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, D-07745 Jena, Germany. Tel.: 49-3641-949360; Fax: 49-3641-571002; E-mail: dwicher{at}ice.mpg.de.

² The abbreviations used are: TRP, transient receptor potential; AKH I, adipokinetic hormone I; 8-Br-cAMP, 8-bromo-cAMP; 8-Br-cGMP, 8-bromo-cGMP; dTRP, Drosophila transient receptor potential; DUM, dorsal unpaired median; ETYA, 5,8,11,14-eicosatetraynoic acid; KLH, keyhole limpet hemocyanin; LOE-908, (R,S)-2-(3,4-dihydroxy-6,7-dimethoxyisochinolin-1-yl)-2-phenyl-N,N-di(2-(2,3,4-trimethoxyphenyl)ethyl)-acetamide; NCCE, non-capacitative Ca²⁺ entry; PLC, phospholipase C; PKI, myristoylated PKA-inhibiting peptide 14–22 amide; pTRP, Periplaneta transient receptor potential; Rp-cAMPS, adenosine 3',5'-cyclic phosphorothioate-Rp; RT, reverse transcription; RACE, rapid amplification of cDNA ends.

	ACKNOWLEDGMENTS

 
We thank S. Arendt, S. Kaltofen, A. Rossner, A. Schmidt (Jena, Germany), and S. Ünsal (Berlin, Germany) for technical assistance; Dr. C. Montell (Baltimore) and Dr. L. Salkoff (St. Louis, MO) for the gift of dTRP DNA; and Boehringer Ingelheim (Germany) for the gift of LOE-908.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Clapham, D. E. (2003) Nature 426, 517–524[CrossRef][Medline] [Order article via Infotrieve]
2. Minke, B. (1977) Biophys. Struct. Mech. 3, 59–64[CrossRef][Medline] [Order article via Infotrieve]
3. Xu, X. Z., Chien, F., Butler, A., Salkoff, L., and Montell, C. (2000) Neuron 26, 647–657[CrossRef][Medline] [Order article via Infotrieve]
4. Clapham, D. E., Montell, C., Schultz, G., and Julius, D. (2003) Pharmacol. Rev. 55, 591–596[Abstract/Free Full Text]
5. Montell, C. (2005) Science's STKE 2005, re3
6. Baumann, E., and Penzlin, H. (1984) Biomed. Biochim. Acta 43, K13–K16[Medline] [Order article via Infotrieve]
7. Wicher, D., Walther, C., and Penzlin, H. (1994) J. Comp. Physiol. A 174, 507–515[Medline] [Order article via Infotrieve]
8. Wicher, D., Messutat, S., Lavialle, C., and Lapied, B. (2004) J. Biol. Chem. 279, 50410–50419[Abstract/Free Full Text]
9. Achenbach, H., Walther, C., and Wicher, D. (1997) Neuroreport 8, 3737–3741[Medline] [Order article via Infotrieve]
10. Derst, C., Messutat, S., Walther, C., Eckert, M., Heinemann, S. H., and Wicher, D. (2003) Eur. J. Neurosci. 17, 1197–1212[CrossRef][Medline] [Order article via Infotrieve]
11. Hille, B. (2001) Ion Channels of Excitable Membranes, Third Ed., Sinauer Associates, Sunderland, MA
12. Bugaj, V., Alexeenko, V., Zubov, A., Glushankova, L., Nikolaev, A., Wang, Z., Kaznacheyeva, E., Bezprozvanny, I., and Mozhayeva, G. N. (2005) J. Biol. Chem. 280, 16790–16797[Abstract/Free Full Text]
13. Zagranichnaya, T. K., Wu, X., and Villereal, M. L. (2005) J. Biol. Chem. 280, 29559–29569[Abstract/Free Full Text]
14. Clapham, D. E., Runnels, L. W., and Strubing, C. (2001) Nat. Rev. Neurosci. 2, 387–396[Medline] [Order article via Infotrieve]
15. Merritt, J. E., Armstrong, W. P., Benham, C. D., Hallam, T. J., Jacob, R., Jaxa-Chamiec, A., Leigh, B. K., McCarthy, S. A., Moores, K. E., and Rink, T. J. (1990) Biochem. J. 271, 515–522[Medline] [Order article via Infotrieve]
16. Shuttleworth, T. J., and Thompson, J. L. (1996) Biochem. J. 316, 819–824
17. Moneer, Z., and Taylor, C. W. (2002) Biochem. J. 362, 13–21[CrossRef][Medline] [Order article via Infotrieve]
18. Bezzerides, V. J., Ramsey, I. S., Kotecha, S., Greka, A., and Clapham, D. E. (2004) Nat. Cell Biol. 6, 709–720[CrossRef][Medline] [Order article via Infotrieve]
19. Montell, C. (1999) Annu. Rev. Cell Dev. Biol. 15, 231–268[CrossRef][Medline] [Order article via Infotrieve]
20. Heine, M., and Wicher, D. (1998) Neuroreport 9, 3309–3314[Medline] [Order article via Infotrieve]
21. Dunne, M. J., Cosgrove, K. E., Shepherd, R. M., and Ammala, C. (1999) Trends Endocrinol. Metab. 10, 146–152[CrossRef][Medline] [Order article via Infotrieve]
22. Grolleau, F., and Lapied, B. (2000) J. Exp. Biol. 203, 1633–1648[Abstract]
23. Predel, R., Neupert, S., Wicher, D., Gundel, M., Roth, S., and Derst, C. (2004) Eur. J. Neurosci. 20, 1499–1513[CrossRef][Medline] [Order article via Infotrieve]
24. Wicher, D., Berlau, J., Walther, C., and Borst, A. (2006) J. Neurophysiol. 95, 311–322[Abstract/Free Full Text]
25. Messutat, S., Heine, M., and Wicher, D. (2001) Cell Calcium 30, 199–211[CrossRef][Medline] [Order article via Infotrieve]

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