Introduction

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In most mammalian cells, stimulation of G_q-couple or tyrosine kinase-type receptors with an agonist leads to the activation of phospholipase C (PLC) followed by a biphasic increase in the cytosolic Ca²⁺ concentration. The first transient phase is due to inositol 1,4,5-trisphosphate (IP₃) that causes the release of Ca²⁺ from intracellular stores, and the second late phase is caused by a sustained Ca²⁺ entry across the plasma membranes. Particularly in nonexcitable cells, the Ca²⁺ entry can be activated without receptor stimulation experimentally by depleting the intracellular Ca²⁺ stores under Ca²⁺-free conditions or in the presence of inhibitors for endoplasmic reticulum Ca²⁺-ATPase pump, such as thapsigargin. This is known as capacitative Ca²⁺ entry, which is mediated by store-operated nonselective cation channels or by Ca²⁺-release-activated Ca²⁺ (CRAC) channels with a high selectivity of channel pores to Ca²⁺. However, depending on cell types, Ca²⁺ entry is observed by activation of G_q-couple receptors even under Ca²⁺ store depletion or by diffusible second messengers, which may be generally referred to as receptor-activated Ca²⁺ channels (RACCs) (Fasolato et al., 1994).

Mammalian homologs of the Drosophila transient receptor potential (TRP) protein form Ca²⁺-permeable cation channels putatively having six transmembrane segments. Since the original Drosophila TRP and TRP-like (TRPL) channels are involved in the light-induced signal transduction via G protein-mediated activation of PLC, the mammalian homologs have been considered as the candidates for Ca²⁺ channels activated downstream of phosphoinositide (PI) metabolism. Although previous studies have demonstrated that mammalian TRP channels are sensitive to store depletion in exogenously expressed cells, recent studies have provided growing evidence for the differences between recombinant TRP channels and native CRAC channels (Harteneck et al., 2000). Most significantly, none of the TRP channels has been shown to encode a channel with the precise ion-conduction properties or selectivity to Ca²⁺ expected of CRAC channels. Recently, recombinant CaT1, a protein distantly related to the classical TRPs, has been shown to manifest the CRAC channel properties (Yue et al., 2001).

The activation mechanism of TRP channels remains controversial and appears not to be universal for all TRP channels. Based on phylogenetic sequence similarity, the TRP family can be classified into four subfamilies: TRP1, TRP2, TRP3/6/7, and TRP4/5. For TRP3/6/7, diacylglycerols (DAGs) have been identified as a common, membrane-anchored activator of the recombinant channels (Hofmann et al., 1999; Okada et al., 1999). In addition, the protein-protein interaction between IP₃ receptor and TRP3 (or TRP6) (Boulay et al., 1999) causes the opening of TRP3 channels by interrupting the inhibitory action of calmodulin bound to TRP3 channels (Ma et al., 2000; Zhang et al., 2001). TRP4/5 are abundantly expressed in neural tissues and colocalized in some neurons (Philipp et al., 1998). Although potentiation of store-operated Ca²⁺ entry has been observed in cells expressing TRP4 or TRP5 (Philipp et al., 1998; Kinoshita et al., 2000), recent studies have shown that TRP4/5 channels are activated after stimulation of G_q/11-couple receptors independently of Ca²⁺ store depletion (Okada et al., 1998; Schaefer et al., 2000). However, different from TRP3/6/7 channels, TRP4 and TRP5 channels are not activated by DAGs or DAG-lipase inhibitor (Hofmann et al., 1999; Schaefer et al., 2000). Thus, it remains unclear what is needed for the opening of TRP4/5 channels.

In the present study, we coexpressed mouse TRP5 (mTRP5) channels with muscarinic M₁ receptors in Xenopus oocytes and recorded ACh-evoked RACC responses that were clearly distinguishable from the native CRAC channel response of oocytes. We have investigated the opening and modulatory mechanism of mTRP5 channels using oocyte model in combination with antisense oligodeoxynucleotides (ODNs) to Xenopus G_q/11, a potent IP₃ receptor agonist adenophostin A (AdA), and several enzyme inhibitors. The present findings suggest that mTRP5 channels are opened by activation of IP₃ receptors.

	Experimental Procedures

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Materials. The mTRP5 cDNA fragment isolated by digesting pCI-neo-mTRP5 (Okada et al., 1998) with NotI and SalI was blunted and inserted into the SmaI site of pGEMHE (Liman et al., 1992), which contains the 5'- and 3'-untranslated regions of the Xenopus laevis -globin gene for yielding a high expression in X. laevis oocytes. The plasmid mTRP5/pGEMHE was linearized by SphI, blunted, and used as the template for in vitro RNA transcription using T7 RNA polymerase. Plasmid pSPM10 carrying porcine muscarinic M₁ receptor (Fukuda et al., 1987) was linearized by XbaI and used as the template for in vitro RNA transcription using SP6 polymerase. Antisense ODNs to X. laevis G_i1 protein (XAG_i1, 5'-CCCATGGCGACGGTTCTCCG-3'), G_q protein (XAG_q, 5'-GTCATGCCTCCTTGACTAGT-3'), and G₁₁ protein (XAG₁₁, 5'-GTCATCCCTTCCCCCCGGCA-3') were designed to span the start codons of the published cDNA sequences for X. laevis G proteins (Olate et al., 1989; Shapira et al., 1994). Peptides mimicking the IP₃ binding site (488-498, NRERQKLMREQ) of X. laevis IP₃ receptor (Kume et al., 1993) and the N-terminal region (2-17, GCTLSAGERAALERSK) of X. laevis G_o protein (Olate et al., 1989) were synthesized and designated as IP₃RF and PGON1, respectively. IP₃RF and PGON1 were dissolved at 135 and 100 µM, respectively, in water and stored at 80°C. Xestospongin C (XeC; Calbiochem-Novabiochem, San Diego, CA), thapsigargin (Wako Pure Chemicals, Osaka, Japan), and 1-oleyl-2-acetyl-sn-glycerol (OAG; Calbiochem-Novabiochem) were dissolved in dimethylsulfoxide as stock solutions at 1, 2, and 10 mM, respectively, and stored at 20°C. U73122 and U73343 (BIOMOL Research Laboratories, Plymouth Meeting, PA) were dissolved at 10 mM in chloroform and stored at 20°C. D-myo-IP₃ K⁺ salt (Sigma, St. Louis, MO), AdA phosphate (a gift from Dr. M. Takahashi, Sankyo Co., Tokyo, Japan), and guanosine 5'-3-O-(thio)triphosphate (GTPS) Li⁺ salt (Roche, Tokyo, Japan) were dissolved in water at 1, 10, and 50 mM, respectively, and stored at 80°C.

Preparation of RNA-Injected Oocytes. Small pieces of ovarian lobes were dissected out from cold-anesthetized X. laevis and shaken gently at 22°C for 90 min in a solution (88 mM NaCl, 1 mM KCl, 1 mM MgSO₄, 2.4 mM NaHCO₃, and 7.5 mM Tris-HCl, pH 7.6) containing 1.1 mg/ml collagenase (Wako Pure Chemicals). Defolliculated oocytes were selected and incubated overnight at 20°C in modified Barth's saline (MBS; 88 mM NaCl, 1 mM KCl, 0.41 mM CaCl₂, 0.33 mM Ca(NO₃)₂, 0.82 mM MgSO₄, 2.4 mM NaHCO₃, and 7.5 mM Tris-HCl, pH 7.6, supplemented with 10 U/ml penicillin and 10 µg/ml streptomycin). Healthy-looking oocytes were injected in Ca²⁺-free MBS with 50 nl of sterile solution containing mRNA for mTRP5 together with M₁ receptor mRNA (each 13.3 ng/oocyte) and incubated further for 1 to 3 days. When needed, oocytes were injected either with one of the antisense ODNs to X. laevis G proteins (50 ng/oocyte), synthetic peptides IP₃RF (10 ng/oocyte), or PGON1 (8 ng/oocyte) on the next day of mRNA injection.

Electrophysiological Recordings. Oocytes were voltage-clamped at a holding potential of 80 mV in Ca²⁺-free frog Ringer (FR) solution (115 mM NaCl, 2 mM KCl, 2 mM MgCl₂, 10 mM Hepes, and 1 mM EGTA, pH 7.4, with NaOH) with two intracellular glass electrodes (1-2 M with 3 M KCl) connected to an OC-725C amplifier (Warner Instrument, Hamden, CT). The current and voltage outputs were monitored using a flat pen recorder and a MacLab A/D converter with Scope software (ADInstruments Pty Ltd., Castle Hill, Australia). The I-V relationship was evaluated by applying a voltage ramping command ranging between 120 mV and + 60 mV for a duration of 1 s using the D/A output from MacLab. For the detection of Ca²⁺ influx, the perfusion line was switched for 30 s to Ca²⁺-containing FR solution (1 mM EGTA was substituted to 2 mM CaCl₂). ACh was dissolved in Ca²⁺-free FR to be 100 µM and applied similarly by perfusion. GTPS (250 pmol) and AdA (0.5 fmol-50 pmol) were injected into oocytes during recording with another glass micropipette in 1% oocyte volume. Depletion of the intracellular Ca²⁺ store was done by pretreatment of the oocytes at room temperature with 2 µM thapsigargin in a Ca²⁺-free FR solution for 2 h before recording. Treatments of oocytes with XeC, U73122, and U73343 were done in a Ca²⁺-containing FR for 30 min before current recording. The amplitude of current trace was measured as the maximal deflection from the baseline to the peak of response. Values are shown as means ± S.E.M. where n is the number of experiments. Statistical significance was evaluated by one-way analysis of variance with post hoc tests using Prism 3.04 software (GraphPad Software, San Diego, CA).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Mouse TRP5 Channels Were Opened after Stimulation of Muscarinic M₁ Receptors. In the present study, mTRP5 was coexpressed with muscarinic M₁ receptors stimulation of which with ACh evokes a G protein-mediated PI turnover in X. laevis oocytes (Fukuda et al., 1987). To evaluate the ACh-induced RACC-related response, we recorded the whole-cell current at a holding potential of 80 mV from the oocytes perfused with Ca²⁺-free FR and applied 2 mM Ca²⁺-containing FR for 30 s before and after a stimulation of coexpressed M₁ receptors with ACh (100 µM).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Activation of mTRP5 channels after stimulation of coexpressed muscarinic M1 receptors with ACh. Whole-cell current traces were recorded at a holding potential of 80 mV in Ca2+-free FR from noninjected (A), M1-mRNA-injected (B), and mTRP5-plus-M1-mRNA-injected (C) oocytes. Two millimolar Ca2+-containing FR (closed bar) or Ca2+-free ACh solution (100 µM, hatched bar) was applied for 30 s as indicated by bars. Vertical spikes on the traces, of which outward (upward) deflections were truncated, indicate the times at which the I-V relation was examined by applying a 1-s ramping voltage command. D, average (±S.E.M., n = 6) amplitudes of the current responses of oocytes to the first application of Ca2+-containing FR (open column), the application of 100 µM ACh in Ca2+-free FR (closed column), and the subsequent application of Ca2+-containing FR (cross-hatched column). E, current-voltage relationship of the responses to Ca2+-containing FR before and after ACh stimulation indicating the mediation by ICl(Ca). Leak-subtracted I-V traces are shown.

Mouse TRP5 Channels Were Not Opened by Depletion of Ca²⁺ Stores. In several studies (Philipp et al., 1998; Kinoshita et al., 2000), TRP proteins have been shown to form Ca²⁺-permeable channels linked to a depletion of intracellular Ca²⁺ stores with endoplasmic reticulum Ca²⁺-ATPase inhibitors. To test whether the opening of mTRP5 channels is triggered or modified by the depletion of Ca²⁺ stores, we pretreated oocytes for 2 h in Ca²⁺-free FR with 2 µM thapsigargin, and tested the effects on ACh-Ca²⁺-induced I_Cl(Ca) response.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   No effect of Ca2+ store depletion on the ACh-induced ICl(Ca) response of oocytes coexpressing mTRP5 and M1 receptors. Representative current traces were recorded from thapsigargin (2 µM, 2 h)-treated noninjected (A) or mTRP5-plus-M1-mRNA-injected (B) oocytes. The peak of the large current response of the mTRP5-expressing oocyte was truncated. C, average (n = 6) amplitudes of ICl(Ca) evoked by exposure to external 2 mM Ca2+ before (open column) and after (cross-hatched column) stimulation of M1 receptors with ACh (100 µM), which in itself evoked no ICl(Ca) response.

Mouse TRP5 Channels Were Opened by Direct Activation of G Proteins with GTPS. Heterotrimeric G proteins may have participated in the TRP5 channel response not only as transducers of the activation signal but also as regulators of the channel activity, because Drosophila TRPL channels have been shown to be activated by constitutively active G₁₁ protein (Obukhov et al., 1996). To evaluate the effects of direct G protein activation, we injected GTPS, which causes irreversible activation of G proteins, and recorded the GTPS-induced change in the I_Cl(Ca) response to Ca²⁺ perfusion (Fig. 3).

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Activation of mTRP5 channels after injection of GTPS. A, native responses of a noninjected oocyte to an injection of GTPS (250 pmol) and the following Ca2+ perfusion 2 min after the injection. B, representative current responses to the GTPS injection recorded from an oocyte coexpressing mTRP5 and M1 receptors. Functional expression of mTRP5 was confirmed by application of ACh (100 µM) and the subsequent perfusion with Ca2+. C, average (n = 6) amplitudes of ICl(Ca) in response to the first application of Ca2+ (open column), the second application of Ca2+ 2 min after injection of GTPS (hatched column), and the third application of Ca2+ after ACh stimulation (cross-hatched column).

Antisense ODNs to X. laevis G_q and G₁₁ Inhibited the Opening of mTRP5 Channels by GTPS and ACh. From X. laevis oocytes, cDNAs for G protein -subunits G_q, G₁₁ (Shapira et al., 1994), G₁₄ (Shapira et al., 1998), G_s, G_i1, G_i3 (Olate et al., 1990), and G_o (Olate et al., 1989) have been cloned. In the oocytes, however, activation of PLC- and subsequent PI turnover are known to be caused mainly by G-subunits (Stehno-Bittel et al., 1995), whereas G_q/11 family proteins determine the specificity of coupling between muscarinic receptors and the PI signaling (Berstein et al., 1992). To distinguish the roles of G subtypes in the transduction of the GTPS- and ACh-evoked opening of mTRP5 channels, we synthesized 20-mer antisense ODNs to X. laevis G_i1 (XAG_i1), G_q (XAG_q), and G₁₁ (XAG₁₁), and 50 ng/oocyte antisense ODN was injected on the following day of mRNA injection to reduce the amount of a specific subtype of endogenous G (Kaneko et al., 1992; Shapira et al., 1998).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Effects of preinjection with antisense ODNs to X. laevis G proteins on GTPS-induced activation of mTRP5 channels. Representative current traces were recorded from oocytes coexpressing mTRP5 and M1 receptors and preinjected with 50 ng of anti-Gi1 antisense ODN, XAGi1 (A) or 50 ng of anti-Gq antisense ODN, XAGq (B). GTPS (250 pmol) was injected, and Ca2+-containing FR was perfused 2 min after the injection. C, average (n = 6) amplitudes of ICl(Ca) in response to the first application of Ca2+ (open column), the second application of Ca2+ 2 min after injection of GTPS (hatched column), and the third application of Ca2+ after ACh stimulation (cross-hatched column). *, P < 0.05 versus control GTPS-Ca2+. #, P < 0.05 versus control ACh-Ca2+. NS, not significant.

Mouse TRP5 Channels Were Opened by Stimulation of IP₃ Receptors with Adenophostin A. AdA, a compound isolated from the culture broth of Penicillium brevicompactum, is the most potent known agonist for the IP₃ receptor (Takahashi et al., 1994), which causes Ca²⁺ mobilization in X. laevis oocytes when injected (Hartzell et al., 1997). To evaluate the contribution of IP₃ receptors in the opening of mTRP5 channels, we injected various amounts of AdA into oocytes and recorded I_Cl(Ca) responses to the subsequent external Ca²⁺ perfusion (Fig. 5).

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Activation of mTRP5 channels after injection of AdA (chemical structure is indicated). Control, noninjected oocytes (A) and oocytes coexpressing mTRP5 and M1 receptors (B) were voltage-clamped at 80 mV in Ca2+-free FR and injected with AdA at a dose of 50 pmol/oocyte. Ten minutes after the injection of AdA, 2 mM Ca2+ was perfused externally. The functional expression of mTRP5 was confirmed by application of ACh (100 µM) and the subsequent perfusion with Ca2+. C, dose-response effect of AdA on the amplitude of ICl(Ca) response to the external perfusion with 2 mM Ca2+ after injection of AdA into oocytes coexpressing mTRP5 and M1 receptors. n = 6.

Antisense ODNs to X. laevis G_q and G₁₁ Did Not Affect the Opening of mTRP5 Channels by Activation of IP₃ Receptors. Since the above findings strongly indicated that direct activation of IP₃ receptors with AdA evoked the opening of mTRP5 channels, we further tested whether the reduction in the amount of X. laevis G proteins by antisense ODNs might affect the AdA-induced opening of mTRP5 channels. As shown in Fig. 6, preloading with XAG_i1 showed no inhibitory effect on the mTRP5-mediated I_Cl(Ca) responses evoked after AdA or ACh compared with the control group. In oocytes preloaded with XAG_q, XAG₁₁, or both at the same total amount, a significant reduction in the ACh-induced mTRP5 response was observed, as previously shown in Fig. 4. However, unlike GTPS, the AdA-evoked mTRP5 opening was not affected by the pretreatments of oocytes with XAG_q, XAG₁₁, or both. These findings demonstrated that direct activation of mTRP5 channels by an activation of IP₃ receptors was not modified by G proteins and also disprove the direct activation of mTRP5 channels by activated G proteins, which was implicated by analogy to Drosophila TRPL channels (Obukhov et al., 1996).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effects of the preinjection of antisense ODNs to X. laevis G proteins on AdA-induced activation of mTRP5 channels. Representative current traces were recorded from oocytes coexpressing mTRP5 and M1 receptors and preinjected with 50 ng of anti-Gi1 ODN, XAGi1 (A) or 25 ng + 25 ng of mixture of anti-Gq XAGq and anti-G11 XAG11 (B). AdA (50 pmol) was injected and Ca2+-containing FR was perfused 10 min after the injection. C, average (n = 6) amplitudes of ICl(Ca) in response to the first application of Ca2+ (open column), the second application of Ca2+ 10 min after injection of AdA (hatched column), and the third application of Ca2+ after ACh stimulation (cross-hatched column). #, P < 0.05 versus control ACh-Ca2+. NS, not significant.

The Opening of mTRP5 Channels by AdA Was Inhibited by an Allosteric Antagonist of IP₃ Receptor, Xestospongin C, but Not by a PLC Inhibitor, U73122. To characterize the differences in the opening of mTRP5 channels induced by ACh, GTPS, and AdA, we utilized a potent membrane-permeable blocker of the IP₃-induced Ca²⁺ release, XeC (Gafni et al., 1997), and a potent PLC blocker, U73122 with its inactive analog U73343 (Thompson et al., 1991). When oocytes coexpressing mTRP5 and M₁ receptors were preincubated for 30 min in Ca²⁺-free FR with 1 µM XeC (Fig. 7A), 10 µM U73122 (Fig. 7B), or 10 µM U73343 (Fig. 7C), both GTPS- and ACh-induced mTRP5 responses of oocytes were almost completely abolished in XeC- and U73122-treated cells, but not in U73343-treated cells (Fig. 7D), indicating that the PLC-mediated PI turnover is required for the ACh- and GTPS-induced opening of mTRP5 channels. In contrast, the opening of mTRP5 by injection of AdA was blocked by XeC, but not by U73122 or U73343 (Fig. 7E). These findings demonstrate that the allosteric antagonist of IP₃ receptor XeC is capable of blocking the stimulation with AdA, and that the upstream PLC activation is not involved in the activation of mTRP5 channels by direct stimulation of IP₃ receptors with AdA.

View larger version (24K): [in this window] [in a new window]   Fig. 7.   Effects of oocyte pretreatments with intracellular signaling inhibitors on the activation of mTRP5 channels induced by GTPS, AdA, and ACh. Current recordings were made from oocytes coexpressing mTRP5 and M1 receptors after 30-min pretreatment with 1 µM XeC (A), 10 µM U73122 (B), or 10 µM U73343 (C) in Ca2+-free FR. D, effects of the pretreatments on the average (n = 6) amplitudes of ICl(Ca) in response to the first application of Ca2+ (open column), the second application of Ca2+ 2 min after injection of 250 pmol of GTPS (hatched column), and the third application of Ca2+ after 100 µM ACh stimulation (cross-hatched column). *, P < 0.05 versus control GTPS-Ca2+. #, P < 0.05 versus control ACh-Ca2+. NS, not significant. E, effects of the pretreatments on the average (n = 8) amplitudes of ICl(Ca) in response to the first application of Ca2+ (open column), the second application of Ca2+ 10 min after injection of 50 pmol of AdA (hatched column), and the third application of Ca2+ after ACh (cross-hatched column). *, P < 0.05 versus control AdA-Ca2+. #, P < 0.05 versus control ACh-Ca2+. NS, not significant.

The Opening of mTRP5 Channels by AdA Was Inhibited by a Synthetic Peptide for the IP₃ Binding Site. To clarify the role of the IP₃ binding site for the activation of mTRP5 channels, we synthesized a polypeptide, IP₃RF, mimicking the IP₃ binding site of X. laevis IP₃ receptors and injected it into oocytes coexpressing mTRP5 and M₁ receptors. As shown in Fig. 8, injection of IP₃RF significantly inhibited both AdA- and ACh-induced openings of mTRP5 channels. The incomplete suppression of ACh-induced response may reflect that the quantitatively insufficient antagonism against massive amounts of IP₃ produced after stimulation of M₁ receptors with ACh. In the control oocytes injected with a peptide with the N-terminal sequence of X. laevis G_o, PGON1, which was effective for the ablation of oocyte-endogenous G_o protein in the study of voltage-dependent Ca²⁺ channel regulation by G_o (Kinoshita et al., 2001), there was no change in the amplitude of AdA- or ACh-induced mTRP5 responses compared with the noninjected oocytes coexpressing mTRP5 and M₁ receptors.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effects of the preinjection of decoy peptides IP3RF and PGON1 on the AdA-induced activation of mTRP5 channels. Synthetic peptides IP3RF and PGON1 were injected at a dose of 10 ng (putative cytosolic concentration of 10 µM) into oocytes coexpressing mTRP5 and M1 receptors 1 day before the recording. Representative responses of oocytes to external perfusion with Ca2+ before and after injection with AdA (50 pmol) and perfusion with ACh (100 µM) are shown in the control, nonpeptide-injected (A) and IP3RF-injected (B) cells. C, average (n = 6) amplitudes of ICl(Ca) in response to the first application of Ca2+ (open column), the second application of Ca2+ 10 min after injection of AdA (hatched column), and the third application of Ca2+ after ACh (cross-hatched column). *, P < 0.05 versus control AdA-Ca2+. #, P < 0.05 versus control ACh-Ca2+. NS, not significant.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

With regard to the opening mechanisms of TRP5 channels, it is still controversial whether these channels are store-operated or dependent on other components of PLC pathway. Evidence supportive of the store-operated story has been provided by the potentiation of native CRAC-like responses of cells used in the expression study (Philipp et al., 1998). However, although independence of store depletion and the inhibitory effects of the PLC inhibitor are strongly suggestive of the direct involvement of the PLC pathway (Okada et al., 1998), no TRP5 channel opening was induced either by DAG or IP₃ (Schaefer et al., 2000), and thus mediators downstream from the activation of PLC remained unclear.

Three findings of the present study demonstrate that activation of IP₃ receptors triggers the opening of recombinant mTRP5 channels: 1) activation of the mTRP5 channel response by a potent IP₃ receptor agonist AdA, 2) inhibition of the mTRP5 channel response by an IP₃ receptor antagonist XeC, and 3) inhibition of the mTRP5 channel response by a peptide IP₃RF mimicking the IP₃ binding site of IP₃ receptor. The I_Cl(Ca) responses of oocytes coexpressing G_q-couple muscarinic M₁ receptors and mTRP5 channels were evoked either by ACh, GTPS, or AdA independently of Ca²⁺ store depletion and were clearly distinguishable from the oocyte-native CRAC channel response by the marked difference in their amplitudes. The upstream signals from muscarinic receptors or direct G protein stimulation with GTPS were abolished either by knock down of endogenous G_q/11 with antisense ODNs or by PLC inhibitor. In contrast, the direct activation of IP₃ receptors with AdA was insensitive to these treatments, indicating that the activation of IP₃ receptor is essential for the opening of mTRP5 channels. The present findings also suggest that neither G proteins nor PI metabolism modifies the opening of mTRP5 channels evoked by activation of IP₃ receptors. However, we cannot rule out the possibility that several factors are needed to act in concert to maximally activate TRP channels, since the ACh-induced response was more resistant to the treatments disrupting the G protein-PLC pathway than the AdA-induced response.

Previously, activation of the TRP4/5 channel response was not observed by activation of IP₃ receptors by IP₃. In the present study using IP₃ itself, the potency of IP₃ in evoking an mTRP5 response was weak and inconsistent in oocytes from batch to batch. However, the potent IP₃ receptor agonist AdA could evoke a Ca²⁺ influx in mTRP5-expressing oocytes in a dose-dependent manner. The half-maximal dose of AdA (5 × 10¹³ mol) corresponded to a putative cytosolic concentration of 1 µM in oocytes, which was comparable with the effective dose of AdA in evoking slow Ca²⁺ release from stores in Xenopus oocytes (DeLisle et al., 1997; Hartzell et al., 1997). Since AdA has a potency about 100-fold greater than that of IP₃ in receptor binding (Takahashi et al., 1994), AdA, but not IP₃, may evoke the opening of mTRP5 channels functionally coupling to IP₃ receptor activation. It is also likely that AdA overcomes the Ca²⁺-dependent inactivation of IP₃ receptors. In type 1 and type 2 IP₃ receptors, IP₃-induced Ca²⁺ release from stores is biphasically dependent on the cytosolic Ca²⁺ concentration, of which the declining phase at a higher Ca²⁺ concentration is responsible for negative feedback regulation of Ca²⁺ release and causes cytosolic Ca²⁺ oscillation (Miyakawa et al., 1999). Because the primary amino acid sequence of IP₃ receptors expressed in Xenopus oocytes is equivalent to that of mammalian type 1 (Kume et al., 1993), IP₃ applied without [Ca²⁺]_i buffering only causes a transient Ca²⁺ release followed by quick inactivation of IP₃ receptors, which may not be sufficient to signal mTRP5 channel opening. In this context, a recent study using rat basophilic leukemia cells has shown the ability of AdA to activate the CRAC current under minimal [Ca²⁺]_i buffering conditions where inactivation of IP₃ receptor was caused after the activation of receptors with IP₃ and the resulting Ca²⁺ influx (Broad et al., 1999).

XeC is a noncompetitive blocker of the IP₃ receptor which inhibits IP₃-induced Ca²⁺ release without interacting with the IP₃ binding site (Gafni et al., 1997). The fact that XeC was effective in inhibiting all the ACh-, GTPS-, and AdA-evoked mTRP5 responses indicates that activation of IP₃ receptor is essential for the mTRP5 channel opening. However, XeC was shown to not only block the IP₃ receptors but also inhibit the endoplasmic reticulum Ca²⁺ pump at an equivalent concentration (De Smet et al., 1999). Although the mTRP5 response was not affected by the filling state of Ca²⁺ stores, we have synthesized a peptide mimicking the IP₃ binding site of the IP₃ receptor and shown that the peptide IP₃RF is effective in inhibiting the ACh- and AdA-evoked mTRP5 channel responses. In mammalian type 1 IP₃ receptor, the inner surface of the IP₃ binding domain was suggested to be lined with 10 basic amino acid residues for which substitutions to neutral amino acids cause significant reduction of the binding activity, and among them, three amino acids are critical residues for the specific IP₃ binding (Yoshikawa et al., 1996). Since these amino acids are well conserved in X. laevis IP₃ receptors (Kume et al., 1993), IP₃RF (NRERQKLMREQ) was designed to include 4 of 10 amino acids for the IP₃ binding pocket (underlined) and 2 of 4 critical amino acids (boldface). A decrease in the mTRP5 response as well as a decrease in the AdA-induced slow increase in the baseline current may reflect that IP₃RF acts as a "decoy" peptide whose basic amino acids interrupt IP₃ binding to the binding pocket.

Recent studies have shown the direct interactions of TRP proteins including TRP5 with mammalian IP₃ receptors, which indicate a direct coupling model in the activation of all TRP channels (Tang et al., 2001). Although the present findings do not demonstrate the direct interaction of mTRP5 channels with X. laevis IP₃ receptors, physical coupling of TRP5 with IP₃ receptor is likely in oocytes since X. laevis oocyte IP₃ receptors are present in the cortical layer as well as cytoplasm of the animal hemisphere and perinuclear layer (Kume et al., 1993). Coexpression of TRP1 and TRP5 forms a nonselective cation channel with different channel properties from the original TRP5 or TRP1 channels (Strübing et al., 2001). X. laevis TRP1 proteins are abundantly expressed in native oocytes (Bobanovic et al., 1999), which is, however, unlikely to form CRAC channels intrinsically present in oocytes (Brereton et al., 2000). Exogenously expressed mTRP5 proteins may form heteromeric channels with endogenous TRP1 proteins, which are functionally coupled to activated IP₃ receptors. In the brain, TRP5 is colocalized with TRP1 in hippocampal neurons (Strübing et al., 2001), and the heteromeric complex forms nonselective cation channels activated by G_q-couple receptors with similar cytosolic Ca²⁺ sensitivity to recombinant TRP1/5 channels (Congar et al., 1997). Since recombinant TRP5 channels also require cytosolic Ca²⁺ for maintaining the activity (Okada et al., 1998; Philipp et al., 1998), Ca²⁺ may act as a coactivator for the activation of IP₃ receptors, which results in opening TRP5 channels.