1Department of Physiology and Biophysics, Seoul National University College of Medicine; and 2Department of Urology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea
Submitted 3 September 2004 ; accepted in final form 12 April 2005
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ABSTRACT |
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nonselective cation channels; Ca2+-permeable cation channels
TRPC4 and TRPC5 are rapidly desensitized after activation by GPCR, and this desensitization does not depend on extracellular monovalent cations such as Na+ or Cs+. Under both monovalent cation conditions, TRPC4 and TRPC5 were found to be desensitized after the activation of muscarinic receptors. Even when intracellular GTPS was used to activate TRPC4 and TRPC5, TRPC4 and TRPC5 currents were rapidly desensitized. Activated G proteins stimulate the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) through PLC-
, and PIP2 is hydrolyzed into inositol 1,4,5-trisphosphate (IP3) and DAG. This hydrolysis of PIP2 was suggested to be associated with the inactivation of TRP melastatin 7 (TRPM7), although it activates other TRP channels such as TRPC3TRPC7 and TRP vanilloid 1 (TRPV1). In the case of TRPCs, protein kinase C (PKC) was found to be involved in the desensitization process, whereas DAG activated TRPC3, TRPC6, and TRPC7 but not TRPC1, TRPC4, and TRPC5 (35). When 1-oleoyl-2-acetyl-sn-glycerol, a membrane-permeable analog of DAG, was applied before GPCR stimulation by acetylcholine, it inhibited TRPC5 activation (35). Intracellular Ca2+ concentration ([Ca2+]i) measurements showed that PKC inhibited all TRPC channels, whereas DAG activated TRPC3, TRPC6, and TRPC7. We have been studying the desensitization mechanism of TRPC5 using whole cell patch-clamp techniques. In our previous studies (12, 38), when we increased the extracellular Ca2+ concentration ([Ca2+]o) from 0 to 10 mM, it caused initial facilitation and a subsequent faster desensitization; when intracellular GTP
S was used to activate TRPC4 and TRPC5, a similar phenomenon was observed. These results suggest that Ca2+-dependent processes are involved in the desensitization of TRPC5.
We recorded TRPC5 current electrophysiologically and used characteristic current-voltage (I-V) relationships as markers of TRPC5 expression (12, 38; see also Ref. 5). The desensitization of TRPC5 by PKC was found to be dependent on extracellular and intracellular Ca2+, intracellular MgATP or EGTA, and PKC inhibitors. To identify the PKC phosphorylation sites responsible for the desensitization, we mutated 11 putative PKC phosphorylated TRPC5 sites. Seven sites had no or only a slight effect on the desensitization process, whereas mutations of the other three sites caused TRPC5 not to be expressed on the plasma membrane. However, only the T972A mutant slowed the desensitization process.
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EXPERIMENTAL PROCEDURES |
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Cell culture and transient transfection. Human embryonic kidney (HEK)-293 cells (American Type Culture Collection, Manassas, VA) were maintained according to the suppliers recommendations. For transient transfection, cells were seeded in 12-well plates. The next day, 0.52 µg/well of pcDNA vector containing the cDNA of TRPC5 or point mutants of TRPC5 were mixed with 50100 ng/well of pEGFP-C1 (Clontech) and transfected into cells using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturers protocol. After 1824 h, cells were trypsinized and used for whole cell recordings.
Electrophysiology.
Whole cell currents were recorded using an Axopatch 200A amplifier (Axon Instruments). Currents were filtered at 5 kHz (3 dB, 4-pole Bessel), digitized using a Digidata 1200 Interface (Axon Instruments), and analyzed using a personal computer equipped with pClamp 9.0 software (Axon Instruments). Patch pipettes were made from borosilicate glass and had resistances of 36 M when filled with standard intracellular solutions. For whole cell experiments, we used an external bath medium (normal Tyrode solution) of the following composition (in mM): 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), with pH adjusted to 7.4 using NaOH. Cs+-rich external solution was made by replacing NaCl and KCl with equimolar CsCl. CaCl2 was simply omitted from the external bath medium to produce Ca2+-free PSS. The pipette solution contained (in mM) 140 CsCl, 10 HEPES, 0.2 Tris-GTP, 0.5 EGTA, and 3 MgATP, with pH adjusted to 7.3 using CsOH. The calculated junction potential between the pipette and bath solutions used for all cells during sealing was 4 mV (pipette negative, using pClamp 9.0 software). No junction potential correction was applied. Experiments were performed at room temperature (1822°C). Gravity was used to superfuse the solution and apply the drugs. The chamber volume was 400 µl, and the time required to reach the chamber was
30 s. Latency was the time from arrival time of solution to the chamber to the peak activation of current.
Statistics. Results are expressed as means ± SE. Where appropriate, results were compared using Students t-test.
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RESULTS |
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When we increased the concentration of EGTA in the pipette solution, latency to activation increased and the rate of desensitization slowed (Fig. 1, B and C). This latency increased to 128 ± 18 s (n = 10) at 1 mM [EGTA]i and 123 ± 10 s (n = 8) at 2 mM [EGTA]i. Under the control condition of 0.5 mM [EGTA]i, the current decayed to one-half of its peak value at 36 s after CCh application, and this time required to reach half peak value increased to 225 s at 1 mM [EGTA]i. No desensitization was observed with 2 mM [EGTA]i in the pipette. Desensitization data increased from 0.06 ± 0.01 (n = 11) under the control condition of 0.5 mM [EGTA]i to 0.59 ± 0.02 (n = 10) at 1 mM [EGTA]i and 0.91 ± 0.03 (n = 8) at 2 mM [EGTA]i (Fig. 1D), suggesting that the desensitization process depends on intracellular Ca2+. However, when we increased [EGTA]i to 10 mM, no TRPC5 currents showing typical I-V relationships were observed. In addition, CCh could not activate TRPC5 current after pretreatment with 10 µM cyclopiazonic acid (CPA) or 30 µM 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), and when 10 µM CPA or 30 µM BHQ were applied alone to deplete Ca2+ stores, TRPC5 current was not induced either. Thus, to activate TRPC5 by muscarinic stimulation, optimal [Ca2+]i levels are needed.
When we omitted intracellular MgATP, latency to activation increased and the rate of desensitization slowed (Fig. 2A), and latency to activation was 63 ± 9 s (n = 10). During the application of CCh, the TRPC5 current did not decay (n = 6). Desensitization scores increased to 0.96 ± 0.01 (n = 11) under the MgATP-free pipette condition (Fig. 2B). These results suggest that the desensitization process depends on the phosphorylation of intracellular TRPC5.
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DISCUSSION |
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In Drosophila, PKC is involved in the termination of light-induced response. The Drosophila TRP channel exists in a functional complex containing photoreceptor PLC, PKC, and calmodulin held within the PDZ domain-containing INAD (inactivation-no afterpotential D) scaffold protein (16, 17). There is evidence that the PKC within this complex directly phosphorylates and inhibits the TRP channel in a negative feedback loop that controls phototransduction (8, 14). In vertebrate systems, TRPC channels may be organized within similar regulatory complexes via PDZ domain-containing proteins such as the Na+/H+ exchanger regulatory factor (NHERF), which is known to interact with and organize TRPC4 and TRPC5 channels and PLC- isoforms (33). The mutation site T972 resides in the VTTRL motif and binds to the PDZ domain. PKC phosphorylation of T972 might induce TRPC5 desensitization by disrupting the interaction motif of TRPC5 (TTRL) with the PDZ domain of NHERF or other PDZ domain-containing proteins. Such disruption causes TRPC5 to be disconnected from NHERF, PLC-
, or the cytoskeleton and to be desensitized via the endocytosis or degradation of TRPC5. The mutation of T972 to alanine might effect desensitization by maintaining the interaction motif of TRPC5 (TTRL) with the PDZ domain of NHERF or other PDZ domain-containing proteins. There is also evidence that PDZ-ligand interactions are disrupted by phosphorylation, which typically occur on COOH-terminal amino acids. These interactions were disrupted by PKC phosphorylation in the GluR2 receptor (4) or by PKA phosphorylation in stargazin (2). It is equally possible that PKC phosphorylates the PDZ domain of NHERF or other PDZ domain-containing proteins that interact with the PDZ binding motif of TRPC5 (TTRL). Although less prevalent than phosphorylation of the COOH-terminal motif, PDZ-peptide interactions also can be regulated by the phosphorylation of the PDZ domain. Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent phosphorylation of the synapse-associated protein 97 (SAP97) PDZ1 domain disrupted the interaction between SAP97 and the NMDA receptor 2A subunit, but not with the glutamate receptor type 1 (GluR1) AMPAR [
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor] subunit (7).
The PDZ-interacting domain of TRPC4, which belongs to the same subfamily as TRPC5, controls TRPC4 localization and surface expression in transfected HEK-293 cells (15). Moreover, TRPC4 mutant lacking the TRL motif accumulated in cell outgrowth and exhibited a distribution unlike that of the wild-type channel, and deletion of the TRL motif reduced the amount of channel associated with the plasma membrane. According to our results (Fig. 7), YVTTRL mutant of mTRPC4 or
QVTTRL mutant of mTRPC5 is not expressed well at the plasma membrane. To the contrary,
QVTTRL mutants of rat TRPC5 showed properties similar to those of the wild-type rat TRPC5 (20).
QVTTRL mutants of rat TRPC5 were expressed well and showed PKC desensitization, although the desensitization process seemed a little slower than that of the wild-type form. In mouse TRPC4 and TRPC5, the PDZ domain-binding motif might also be important for controlling TRPC localization and surface expression in transfected HEK-293 cells.
In gastric guinea pig myocytes, a nonselective cation channel was activated by muscarinic stimulation. Desensitization of this channel was found to depend on PKC activity (11), to be dependent on [Ca2+]i, and to be reversed by PKC inhibitors. Conventional PKC was involved in the desensitization process. When [Ca2+]i was increased to 200 nM, PKC- was activated and induced desensitization. However, at <200 nM [Ca2+]i, PKC-
was not activated and the current was not desensitized. We also have shown that TRPC5 is a molecular candidate for the nonselective cation channel that is activated by muscarinic stimulation in murine gastric myocytes (12, 38). The desensitization of TRPC5 might correspond to the desensitization of the nonselective cation channels activated by muscarinic stimulation in native tissues, such as in the gastric myocytes of guinea pigs and murine gastric myocytes.
On the other hand, PKC sensitizes TRPV1 channel activity by augmenting the channel open probability (1, 27). Furthermore, PKC-mediated phosphorylation also reduces the heat threshold of channel activation (27, 34). Thus PKC sensitizes TRPV1 and induces inflammatory hyperalgesia. A recent report showed that PKC sensitizes TRPV1 in association with the rapid recruitment of vesicular channels to the cell surface by regulated exocytosis (18). TRPV1 also exhibits a time- and Ca2+-dependent desensitization, a long-lasting refractory state during which the receptor does not respond to vanilloids or to other stimuli (9). The association of the COOH terminus of TRPV1 with PIP2 inhibits TRPV1 channel activity (3, 28), and its interaction with calmodulin promotes channel desensitization (19). Although PIP2 hydrolysis activates both TRPV1 and TRPC5, their desensitization processes differ. In TRPC5, PKC phosphorylation is involved in desensitization, whereas calmodulin is involved in the desensitization of TRPV1.
The PKC-mediated inhibition of receptor-induced PLC provides an important feedback loop mediated by DAG and Ca2+ on PLC enzyme (6, 13, 29, 37). If this process is involved in the desensitized process, all TRPC5 mutants should have been desensitized. However, the T972A mutant was not desensitized. This result suggests that TRPC5 itself might become phosphorylated by PKC and desensitized. In addition, pretreatment with PKC inhibitor should activate TRPC5 if PKC-mediated inhibition of receptor-induced PLC provides an important feedback loop mediated by DAG and Ca2+ on PLC enzyme. To the contrary, pretreatment with PKC inhibitor did not have any effect at lower concentrations and sometimes blocked TRPC5 current activation at a concentration >1 µM.
We conclude that the desensitization of TRPC5 occurs via PKC phosphorylation and that threonine at residue 972 might be important for the PKC phosphorylation of mTRPC5, although direct phosphorylation of the mTRPC5 channel itself was not shown.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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