Molecular Bases of Multimodal Regulation of a Fungal Transient Receptor Potential (TRP) Channel*

Background: Multimodality of TRP channels underlies their diverse physiological functions. Results: We identified a fungal multimodal TRP channel whose cytosolic domain (CTD) mediates various channel regulation. Conclusion: CTD has an oligomerization module critical for osmoreception, yet its flexible structure allows dynamic regulations with other functional modalities. Significance: This work proposes structural and biophysical principles for multimodality of a TRP channel family member. Multimodal activation by various stimuli is a fundamental characteristic of TRP channels. We identified a fungal TRP channel, TRPGz, exhibiting activation by hyperosmolarity, temperature increase, cytosolic Ca2+ elevation, membrane potential, and H2O2 application, and thus it is expected to represent a prototypic multimodal TRP channel. TRPGz possesses a cytosolic C-terminal domain (CTD), primarily composed of intrinsically disordered regions with some regulatory modules, a putative coiled-coil region and a basic residue cluster. The CTD oligomerization mediated by the coiled-coil region is required for the hyperosmotic and temperature increase activations but not for the tetrameric channel formation or other activation modalities. In contrast, the basic cluster is responsible for general channel inhibition, by binding to phosphatidylinositol phosphates. The crystal structure of the presumed coiled-coil region revealed a tetrameric assembly in an offset spiral rather than a canonical coiled-coil. This structure underlies the observed moderate oligomerization affinity enabling the dynamic assembly and disassembly of the CTD during channel functions, which are compatible with the multimodal regulation mediated by each functional module.

Transient receptor potential (TRP) 3 channels are tetrameric non-selective Ca 2ϩ -permeable cation channels existing in a wide variety of fungi and animals (1). The TRP channels exhibit relatively diverse primary sequences and are divided into seven subfamilies: TRPC, TRPV, TRPM, TRPML, TRPN, TRPP, and TRPA (2). With regard to their physiological functions, TRP channels serve as sensors for various environmental stimuli, such as temperature, chemical substances, and osmolarity (3). The physiological relevance of TRP channels has long been implicated in store-operated calcium entry (4), although this aspect still remains controversial. TRP channels also act as voltage-gated cation channels (5). Notably, many important physiological functions are achieved by a single type of TRP channel that can respond to both physical and chemical stimuli. For example, TRPV1 is activated by both high temperature (ϳՆ42°C) and capsaicin (6); TRPV4 is activated by both warm temperature (ϳ27-42°C) (7,8) and high osmolarity (9,10); and TRPA1 is activated by noxious cold temperature (ϳՅ17°C) (11), pungent chemical substances, such as mustard oils (12,13), and O 2 (14). The "multimodality" of the channel regulation is one of the most remarkable features of the TRP channel functions.
The TRP channel core domain consists of six transmembrane (TM) regions, with a central channel pore composed of a re-entrant loop between TM5 and TM6 from each protomer in the tetramer, which is conserved among not only the TRP channels but also the voltage-gated channel superfamily members (15). On the other hand, the molecular architectures of the N and C termini of the cytosolic domains are highly diverse among the TRP family members. The domains usually consist of multiple functional domains or regions for channel regulation (16), such as ankyrin repeats as bait for protein or ligand interactions (17), a coiled-coil region that probably functions in channel assembly (18), an EF-hand motif (19) or a calmodulin binding motif (20) for Ca 2ϩ -mediated regulation, and a phosphatidylinositol phosphate (PIP) binding domain for regulation (21). The structural information for TRP channels is limited to a few individual functional modules (22)(23)(24)(25)(26)(27)(28), and low resolution electron microscopy structural studies of entire TRP channels revealed large cytosolic domains with chamber-like structures (29 -32, 77). However, the molecular mechanisms by which the functional modules in the cytosolic domains regulate the channel activity have remained unclear. Furthermore, in order to attain the multimodality, each module should be functionally compatible in a single channel; for example, in some cases, several modules should operate simultaneously. The molecular bases of the module integration in the cytosolic domains, enabling the mutual compatibility between the modules, have not been investigated extensively.
Among the TRP channel family members, those possessing cytosolic domains with relatively small and thus simple structures were identified in fungi (33)(34)(35). The TRP channel from the yeast Saccharomyces cerevisiae, Yvc1 or TRPY1, has been most extensively characterized (33,34,36,37). TRPY1 is a Ca 2ϩ release channel in yeast vacuoles that functions upon hyperosmotic shock (33,34). TRPY1 is reportedly activated by cytosolic Ca 2ϩ elevation (34,38,39) and membrane stretching (37), and its function is affected by the vacuolar PIPs level (40). Therefore, the fungal TRP channels are also likely to possess similar channel regulation multimodality as the other mammalian TRP channels, despite their smaller cytosolic domains.
In this study, we addressed the multimodality of TRP channels by analyzing the fungal TRP channels. We identified a TRP channel homolog from Gibberella zeae, which shares sequence similarity with TRPY1 but exhibits substantially higher responses to multiple stimuli, as a good model for investigation. The integrated approaches with biochemical, physiological, crystallographic, and NMR spectroscopic analyses revealed the structural and functional correlations between the modular characteristics of the cytosolic domain and the multimodal regulation of the channel activities.

EXPERIMENTAL PROCEDURES
Materials-Ultrapure water from MilliQ synthesis (Millipore) with resistance of Ͼ18.3 megaohms/cm and Ͻ3 ppb total organic carbon was used for all research, including preparation of culture media, if not mentioned otherwise. For general purposes, the highest purity chemical reagents available from either Nacalai Tesque, Wako Pure Chemical, Kanto-Chemical, or Sigma-Aldrich Japan were used. The peptide TRPGz-CC, corresponding to residues 600 -620, was obtained from either Hokkaido System Science or RIKEN Research Resource Center, and its selenomethionine derivative was purchased from Operon.
All functional experiments were performed using the TRPY1 knock-out Saccharomyces cerevisiae strains, YKO11863 (with the BY4742 background) and SH1007 (with the BJ5458 background; for details, see Hamamoto et al. (41)). The TRPGz gene from G. zeae (XM_384354.1) was cloned by PCR amplification using first-strand cDNA prepared from G. zeae total RNA, generously provided by Dr. Yoshitaka Takano (Kyoto University). The gene encoding TRPY1 (NM_001183506.1) (34) was provided by Prof. Ching Kung (University of Wisconsin).
Transformation and Culture of Yeast Cells-Transformation of S. cerevisiae cells was performed according to the method optimized by Akada et al. (44). Briefly, S. cerevisiae cells were cultured on a YPD agar plate overnight, the day before transformation. Immediately before the transformation, the yeast cells were collected from the YPD agar plate by suspension in sterilized water and pelleting by a tabletop centrifuge for 30 s. The cells were washed once with transformation buffer composed of 667 l of 60% PEG 4000, 100 l of 1 M DTT, 50 l of 4 M lithium acetate, and 183 l of MilliQ water, and they were resuspended in transformation buffer. Usually, the cells from an overnight culture on one 90-mm YPD plate were suspended into 1 ml of transformation buffer, and the volume of the yeast cell pellet was usually about 200 -400 l. A 50-l portion of the competent yeast cells was added to the of DNA mixture (50 g of heat denatured carrier DNA and 50 -500 ng of DNA for transformation) and incubated for 2-3 h at 42°C. After this period of incubation, the yeast cells were diluted 2-fold in the sterile water. The diluted cells were spread on agar plates containing synthetic complete medium lacking specific nutrients, such as L-leucine for the pEVP11/AEQ vector (see below) or uracil for the pDR195 and p416GALL vectors, for transformant selection.
The transformants were inoculated in the appropriate culture media and cultured at 30°C. Unless otherwise stated, the YKO11863 strain transformed with p416GALL-based expression vectors was cultured in the Sc-dropout medium (ϪUra, ϪLeu) supplemented with 1% (w/v) galactose and raffinose. The SH1007 strain transformed with pDR195-based expression vectors was cultured in the same medium supplemented with 2% (w/v) glucose.
Confocal Microscopic Observations of Localization in S. cerevisiae Cells-The yeast TRPY1 knock-out strain YKO11863 was used for microscopic observations. The yeast cells were transformed with plasmids encoding either p416GALL-TRPGz-GFP or p416GALL-TRPY1-GFP. Yeast vacuoles were stained with CellTracker Blue CMAC dye (Invitrogen) according to the manufacturer's recommendations, and the fluorescent signals from both the GFP and CMAC dye were monitored with a Zeiss LSM510 system. The GFP and CMAC dye were excited at 488 and 364 nm, respectively. In order to visualize the yeast cells, transmitted light images were also acquired at the same time.
Luminometric Measurements of Cytosolic Ca 2ϩ Changes-The aequolin-Ca 2ϩ reporter system was used to measure the cytosolic Ca 2ϩ changes. The yeast strain lacking TRPY1, SH1007, was transformed with the aequolin expression vector, pEVP11/AEQ (45), which was kindly provided by Prof. P. H. Masson (University of Wisconsin), and pDR195 constructs (the His tag fusion constructs). The transformants were cultured overnight in the presence of 5 M coelenterazine, and the A 600 was adjusted to 1.0 by resuspension in the culture medium. The luminescence evoked by increased cytosolic Ca 2ϩ upon various stimulations was recorded by a Varioskan Flash microplate reader (Thermo Scientific). A 40-l aliquot of the yeast cell suspension was mixed with 10 l of 50 mM O,OЈ-bis(2-aminophenyl)ethylene glycol-N,N,NЈ,NЈ-tetraacetic acid in the same medium used for cell suspension for 30 s before adding the stimulant. Various concentrations of stimulants at 2ϫ concentrations were added, in a volume of 50 l, using an autoinjector equipped with a Varioskan Flash microplate reader. For luminometric measurements under temperature-controlled conditions, a high sensitivity photometer (Hamamatsu Photonics) connected to a pen chart recorder was used. To measure the response upon temperature shock, a thin walled PCR tube, containing 50 l of the yeast cell suspension, was placed in the thermomodule of the thermal cycler for PCR, and after the base-line luminescence was recorded, heat shock was applied at the appropriate time.
For comparative analyses, the maximum luminescence intensity observed for the response of each sample was normalized by the expression level analyzed by Western blotting. A 100-l portion of the yeast cell suspension, from exactly the same culture used for the luminescence measurements, was pelleted by a tabletop centrifuge at ϳ2,000 ϫ g for 1 min and resuspended into an aqueous 0.2 M NaOH solution. After a 2-min incubation at ambient laboratory temperature, the yeast cells were pelleted in the same manner as above, suspended in 20 l of SDS-PAGE sample buffer, and subjected to SDS-PAGE. The proteins on the gels were electroblotted onto nitrocellulose membranes, using an iBlot system (Invitrogen). The proteins of interest were probed with an anti-penta-His antibody conjugated with HRP (Qiagen) and were detected with Immobilon Western HRP substrate (Millipore). Luminescence images were obtained by a ChemiDoc gel documentation system (Bio-Rad), and luminescence intensities were measured by the Quantity One software (Bio-Rad).
Preparation of Giant Yeast Cells and Patch Clamp Recording-The yeast SH1007 cells, transformed with the pDR195-TRPGz constructs, were enlarged using the spheroplast incubation method, as described previously (41,46,47). The giant yeast cells were transferred to the recording chamber of the patch clamp device and then were subjected to hypotonic conditions (100 mM KCl, 150 mM sorbitol, 10 mM Tris-MES, pH 7.5) to disrupt the plasma membrane and release the intact vacuoles. The disrupted plasma membrane and organelles were removed with washing buffer (100 mM KCl, 1 mM MgCl 2 , 200 mM sorbitol, 10 mM Tris-MES, pH 7.5), to facilitate the access of the patch pipette to the vacuole membrane. The whole-cell configuration was achieved by ZAP pulse (20 ms, 1.2 V). The pipette solution contained 100 mM KCl, 1 mM MgCl 2 , 200 mM sorbitol, and 10 mM Tris-MES, pH 7.5. The bath solution was the same unless otherwise stated. To determine the Ca 2ϩ dependences of TRPGz, patch clamp recording in the cytoplasmic side-out excised patch configuration was performed. Measurements were performed with the same pipette solutions as described above, and the bath solution was the same except for the Ca 2ϩ concentration. To prepare a low concentration of free Ca 2ϩ , a Ca 2ϩ -EGTA buffered solution was employed. For [Ca 2ϩ ] ϭ 10 M, 1 mM EGTA and 1.0058 mM CaCl 2 were used, for [Ca 2ϩ ] ϭ 1 M, 1 mM EGTA and 0.96 mM CaCl 2 were used, and for [Ca 2ϩ ] ϭ 0.1 M, 1 mM EGTA and 0.701 mM CaCl 2 were used. The currents were amplified by a patch clamp amplifier (CEZ2400, Nihon-Koden) and recorded by a digital data recorder (EX-RP10, Sony). All recording was performed using the standard patch clamp technique at 25°C.
Fluorescence-Detection Size-Exclusion Chromatography (FSEC) (48)-Yeast cells (SH1007) transformed with pDR195-TRPGz-GFP and pDR195-TRPGz⌬CC-GFP were cultured overnight in conventional YPD medium and then harvested by centrifugation at 5,000 ϫ g for 2 min at room temperature. The cells were resuspended in 500 l of lysis buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM KCl, supplemented with cOmplete protease inhibitor mixture (Roche Applied Science)) in a 2.0-ml polypropylene test tube. After approximately the same volume of prechilled glass beads was added, the mixture was vortexed for 15 min at 4°C and then centrifuged at 15,000 ϫ g for 5 min at 4°C. After the supernatant was recovered, a 500-l portion of lysis buffer was added, and the previous procedure was repeated. The supernatants were combined and ultracentrifuged at 50,000 rpm for 30 min, using a TLA55 rotor at 4°C, to obtain the yeast total membrane fraction.
The total membrane fractions were solubilized with 40 mM digitonin by an incubation for 3 h at 4°C and then subjected immediately to the FSEC analyses. FSEC was performed with a SEC-5 column (500-Å pore size, 4.6 ϫ 300 mm, Agilent), using a Shimadzu isocratic HPLC system with FSEC buffer, composed of 50 mM Tris, pH 7.5, 200 mM NaCl, 20 mM KCl, and 1 mM digitonin. The fluorescence of yEGFP was monitored, with excitation at 480 nm and detection at 520 nm. The elution peaks provided an estimation of the molecular masses as 1,020 (95% confidence interval of 1,280 to 815) kDa and 1,200 (95% confidence intervals of 1580 to 912) kDa for TRPGz-GFP (protomer mass of 107 kDa) and TRPGz-⌬CC-GFP (105 kDa), respectively. The estimated molecular masses of membrane proteins obtained by FSEC are often about 2-3-fold larger than the predictions, probably because of bound detergent micelles (49). Therefore, the results were interpreted to mean that both the wild type and the coiled-coil (CC) deletion mutant of TRPGz form tetramers. The even larger estimated molecular mass of the CC deletion mutant was considered to reflect the larger hydrodynamic volume of its cytosolic part, due to the absence of CC-mediated assembly.
In Vivo Cross-linking-Overnight cultures of yeast cells (SH1007), transformed with pDR195-TRPGz and pDR195-TRPGz⌬CC, were harvested from agar plates by suspension in MilliQ water and centrifuged at 2,500 ϫ g for 10 min at room temperature. The cells were resuspended in 15 ml of spheroplast buffer (1.2 M sorbitol, 0.1 M potassium phosphate, pH 7.5, supplemented with cOmplete protease inhibitor mixture). Zymolyase 20T (Seikagakukogyo) was added at 0.1 mg/ml, and then 1.0-ml aliquots were treated with 0.1 mM disuccinimidyl glutarate for 0.5 h at ambient temperature. After this incubation, the reaction mixtures were centrifuged at 13,000 ϫ g for 5 min to harvest the yeast cell pellets. The cell pellets were then mixed with SDS-PAGE sample loading buffer and separated on 3-8% NuPAGE Tris-acetate SDS-polyacrylamide gels (Invitrogen). The gels were subjected to electroblotting onto nitrocellulose membranes with an iBlot system (Invitrogen), and the protein bands of interest were detected using an HRP-conjugated anti-penta-His antibody (Qiagen) and Immobilon Western HRP substrate (Millipore).
Protein Preparation-GST fusion proteins were expressed in BL21(DE3) using pET42-TRPGz-CTD, pET42-TRPGz-CTD N , and pET42-TRPGz-CTD C and purified as follows. Briefly, E. coli cells expressing the proteins of interest were lysed in lysis buffer (50 mM Tris, 200 mM NaCl, 20 mM KCl, and 1 mM TCEP (pH 7.5)), supplemented with cOmplete protease inhibitor mixture. The lysate was loaded onto a 5-ml GSTrap HP column (GE Healthcare), washed with the same buffer without protease inhibitor mixture, and eluted with following buffer: 50 mM Tris, 10 mM glutathione, and 1 mM TCEP (pH 8.0). The concentration of each protein sample was quantified by the Bradford method.
TRPGz-CTD and its mutants with C-terminal hexahistidine tags were expressed in E. coli Rosetta2(DE3), using the expression vector pET-19b-TRPGz-CTD, TRPGz-CTD⌬CC, TRPGz-CTD-M607D/L610D, TRPGz-CTD-L610R, or TRPGz-CTD-E606P in NZCYM medium supplemented with 0.5% glucose and appropriate antibiotics. Protein expression was induced by 1 mM isopropyl 1-thio-␤-D-galactopyranoside for 3-4 h after the A 600 reached 0.6 -1.0. For NMR studies, proteins were expressed in modified M9 medium with [ 15 N]NH 4 Cl supplemented by 0.5 g/liter 15 N-labeled algal amino acids and 50 mg/liter The proteins were purified by Ni 2ϩ -NTA metal ion affinity chromatography, His tag cleavage by TEV protease, and anion exchange chromatography as follows. The E. coli cells (1 liter of culture) expressing TRPGz-CTD were disrupted in phosphatebuffered saline composed of 50 mM sodium phosphate, 250 mM NaCl, 250 mM KCl, and 2 mM TCEP (pH 8.0) supplemented with cOmplete protease inhibitor mixture. The lysate was applied to a Ni 2ϩ -NTA Superflow (Qiagen) column packed in house (ϳ10 ml in bed volume), which was washed with the same buffer without protease inhibitor mixture. The proteins of interest were eluted by elution buffer, composed of 20 mM sodium phosphate, 250 mM NaCl, 250 mM KCl, 1 mM TCEP, and 250 mM imidazole (pH 8.0). The eluted protein was treated with TEV protease, prepared in house according to van den Berg et al. (50), at 4°C for 8 -12 h, and then dialyzed against ϳ20 volumes of the dialysis buffer (20 mM Tris, 1 mM TCEP, pH 8.0) for 3-4 h to reduce the salt concentration of the samples for the following anion exchange chromatography. The partially desalted proteins were applied to a Source Q (GE Healthcare) anion exchange column and purified by elution with a 0 -40% (B) linear gradient of the following buffer combinations (A, 20 mM Tris, 1 mM TCEP, pH 8.0; B, 20 mM Tris, 500 mM NaCl, 500 mM KCl, 1 mM TCEP, pH 8.0). The identities of the purified proteins were confirmed by MALDI-TOF or electrospray ionization-Q-TOF-MS.
Lipid Binding Assays-For the lipid overlay assays, membrane lipid strips and PIP Strips (Echelon) were used, according to the supplier's recommended protocols. The strips were blocked by Blocking One (Nacalai Tesque) for 1 h at room temperature and then probed with the GST fusion of the test protein at a concentration of 0.75 g/ml in 5 ml of lysis buffer. The strips were incubated overnight (ϳ12 h) at 4°C with gentle shaking. Detection of lipid binding was performed using an HRP-conjugated anti-GST antibody (MBL) at a 1:5,000 dilution and Immobilon Western Chemiluminescent HRP substrate (Millipore).
For the liposome co-sedimentation experiments, PolyPIPosome (Echelon) was used. A mixture of 5 g of the GST-fused test proteins, 15 l of PolyPIPosome, and 1 ml of Tris-buffered saline (50 mM Tris (pH 7.5), 200 mM NaCl, and 20 mM KCl) containing 0.05% Igepal CA-630 was incubated for 10 min with gentle agitation at 25°C. The mixture was then centrifuged for 10 min at 21,500 ϫ g and 25°C. The supernatant was recovered for analysis, and the precipitate was mixed with 250 l of Trisbuffered saline and centrifuged for 10 min at 21,500 ϫ g and 25°C. This washing procedure was performed three times, and the precipitate obtained after the washing step was recovered for analysis. For Western blotting analyses, 20 l of 2ϫ loading buffer for SDS-PAGE was added to the precipitated fractions. The mixtures were heated at 95°C for 5 min, and a 10-l portion was subjected to SDS-PAGE (SuperSepAce 15-20% Tris-Tricine gels, Wako). For the supernatant fractions, an 8-l portion of the supernatant fractions was mixed with 2 l of 5ϫ SDS-PAGE loading buffer and treated similarly. The gels were then subjected to electroblotting and detection, as described above.
X-ray Crystallography-The peptide TRPGz-CC, corresponding to residues 600 -620, was dissolved in MilliQ water at a concentration of 10 mg/ml (w/v) and was crystallized by the sitting drop vapor diffusion method. Crystals were grown at 20°C in 18 -25% PEG 3350, 0.2 M sodium acetate, and 0.1 M HEPES (pH 8.0). X-ray diffraction intensity data were collected using the MAR225 and MAR225HE detectors at the BL26B2 and BL41XU beamlines of SPring-8, respectively, and were processed with the HKL2000 package (51). The phase calculations were performed by the multiwavelength anomalous dispersion method, using three data sets collected at wavelengths of 0.97909, 0.97944, and 0.99159 Å from a crystal prepared from the selenomethionine-substituted peptide. The determination of the selenium sites, the initial phase determination, and the initial model building were performed using autoSHARP (52,53) with the ccp4i package (54). The structure model was manually rebuilt using COOT (55) and refined with Refmac5 (56). The data collection and structure refinement statistics are summarized in Table 1.
Analytical Ultracentrifugation-The sedimentation equilibrium experiment was performed at 4°C using an Optima XL-I system (Beckman Coulter), equipped with an An-60 Ti rotor. TRPGz-CTD and its mutants were dialyzed against 50 mM Tris (pH 7.5), 200 mM NaCl, 20 mM KCl, and 1 mM TCEP. The absorbance of the test protein solutions at 280 nm was adjusted to 0.5, 1.0, and 2.0, corresponding to ϳ0.5, 1.0, and 2.0 mg/ml protein, respectively. A 100-l portion of the test protein solution was placed in the six-channel Epon charcoal-filled centerpiece, with 110 l of optical blank. Centrifugation was performed at speeds of 8,000, 12,000, and 16,000 rpm. Data were collected using interference optics and processed using the XL-A/XL-I data analysis software version 6.04 (Beckman Instruments) using the self-association model. The obtained association constants were converted to molar units with a conversion factor of 3.26 fringes/mg/ml/1.2 cm, with the molecular weight calculated from the primary sequence of the test protein. Partial specific volumes and solvent density were calculated by the Ultrascan 3 package.

TRPGz Is a TRP Channel Homolog in the Fungal Vacuolar
Membrane That Responds to Various Extracellular and Cytoplasmic Stimuli-G. zeae is a plant pathogenic ascomycete responsible for Fusarium head blight on wheat and barley as well as being an important model organism for biological studies (57). The G. zeae-derived TRP channel homolog, hypothetical protein FG04178.1, which we named TRPGz, consists of 692 amino acid residues and shares sequence similarity with TRPY1 (with 40% amino acid identity among 533 core residues; Fig. 1). A BLAST search using the homolog sequence against human proteins produced TRPC6, -7, and -3 as the highest scoring hits, with 23% sequence identity (among 280 core residues) with TRPC6. The important residues for TRPY1 channel gating that are conserved in other TRP channels, such as Phe 380 in the putative TM5 and Tyr 458 in the putative TM6 (58), are also conserved in this homolog (Fig. 1). These results implied that TRPGz shares a similar architecture and functions with other TRP channels, especially TRPY1.
We cloned the TRPGz gene and analyzed its molecular functions by expressing the protein in TRPY1 knock-out strains of S. cerevisiae. Fluorescent microscopy revealed that TRPGz localized at the vacuolar membrane ( Fig. 2A). Furthermore, TRPGz responded to extracellularly applied hyperosmotic shock, resulting in an increase in the cytosolic calcium concentration to an even larger extent than that observed with TRPY1 (Fig. 2B). The response was observed upon the addition of 1.2 M sorbitol and became saturated at about 1.4 M (Fig. 2C). These results strongly indicated that, like TRPY1 (33), TRPGz is a vacuolar membrane protein responsible for Ca 2ϩ release from the vacuole to the cytosol upon osmotic upshock.
Whole-yeast vacuole patch clamp recording (41) revealed that TRPGz evoked a voltage-dependent cation current. The current amplitude was also increased by the supplementation of Ca 2ϩ to the cytoplasmic side (Fig. 2D). The channel activation by cytosolic Ca 2ϩ was observed in a concentration-dependent manner, with an effective concentration of at least 1 M Ca 2ϩ (Fig. 2E). We also found that TRPGz responded to an extracellularly applied oxidizing reagent, hydrogen peroxide, which evoked cytosolic Ca 2ϩ elevation (Fig. 2F) in a concentration-dependent manner, with a similar range between 0.2 and 5.0 mM (Fig. 2G), as reported by Popa et al. (59). The level of responses was not saturated within the range of the tested H 2 O 2 concentration. Interestingly, TRPGz-expressing yeast cells showed a marked cytosolic Ca 2ϩ increase upon rapid temperature elevation from 25 to 40°C (Fig. 2H). It should be noted that the responses were not significant in the cases of stepwise temperature elevation (Fig. 2H), implying the possibility that the observed response is not necessarily derived from the direct temperature sensing but also a consequence of the effect caused by rapid temperature increase.
These results indicated that the TRPGz channel is essential for responses to various multiple cytoplasmic and extracellular stimuli, such as cytosolic Ca 2ϩ , membrane potential, extracellular oxidizer application, and temperature change as well as osmotic upshock. Therefore, TRPGz probably plays key roles in Ca 2ϩ signaling, inducing reactions against various cell stresses, as proposed for TRPY1 in S. cerevisiae (33). Importantly, the above mentioned stimuli are the ones commonly known to activate other eukaryotic TRP channels. Therefore, we expected TRPGz to serve as a prototypic TRP channel and addressed its molecular bases for multimodal regulation.
The Putative Coiled-coil Region in TRPGz-CTD Is Essential for the Hyperosmotic and Temperature Responses but Not for Channel Formation and Responses to Other Stimuli-We searched for the essential regions for the above described various regulations of the TRPGz channel activities. We focused on the CTD located downstream of the channel pore-forming TM helix S6, the region from Ala 539 to the C terminus, and assessed the channel functions of serial deletion mutants (Fig. 1). We first confirmed that most of the tested deletion mutants of TRPGz displayed the proper vacuolar localization, although the deletion of the entire CTD (from Ala 539 to the C terminus) resulted in defective trafficking (data not shown). The mutants exhibiting the proper localization were subjected to further functional analyses.
Notably, deletion mutants lacking the region between residues 600 and 621 (⌬CC and ⌬600Ϫ) did not show any response to hyperosmotic shock, in contrast to the mutant retaining the  (74) with the BLOSUM 62 matrix using Geneious software version 5.5 (75), where initial multiple sequence alignment was built with the default parameter setting given by the software, and then the multiple sequence alignment was divided into three parts (N-terminal, core TM, and C-terminal parts) and realigned separately in the same manner as described above. The combined multiple sequence alignment was further adjusted manually. Conserved residues with 100% similarity based on PAM250 matrix were colored yellow with a red background. The TRPGz-CTD is highlighted in green, where the symbols indicating its structural characteristics are shown above or below the TRPGz sequence as follows. The presumed coiled-coil region (residues 600 -621; the CC region) is depicted by the pink cylinder, the residues subjected to mutation in this study are marked with closed triangles, and the three methionine residues, Met 575 , Met 607 , and Met 613 , which were analyzed by NMR (Fig. 7, C-E), are boxed in green. The residues referred to in this work are pinpointed with blue stars. Positions of the estimated transmembrane and pore helix domain are shown as reported elsewhere (34,76) region from residue 600 to 621 (⌬621Ϫ) (Fig. 3A). Furthermore, the responses to rapid temperature increase were also diminished by the deletion of the region from residue 600 to 621 (Fig.  3B), in a manner similar to the hyperosmotic shock responses (Fig. 3A). The amino acid sequence analysis revealed that the region from residue 600 to 621 is predicted to be a coiled-coil domain (Fig. 1). These results suggested that the CC region in the CTD is required for osmotic and temperature shock reception by TRPGz.
Because coiled-coil modules are present in numerous TRP channels and are considered to be important for homo-or heterotetrameric channel assembly in many cases (18), we examined whether the CC region is responsible for the oligomerization of the entire channel region of TRPGz. FSEC analyses of the C-terminal GFP fusion protein of wild type TRPGz and its CC deletion mutant (TRPGz-⌬CC) revealed that both proteins exclusively eluted as presumable tetramers (Fig. 3C) without any apparent additional elution peaks for lower molecular weight species resulting from oligomer decomposition. The tetramerization of the entire channel molecule was also verified by in vivo cross-linking experiments using protein samples without GFP, in which both the wild type and CC deletion mutant generated similar cross-linking profiles, with protein bands corresponding to the tetramer (Fig. 3D). Therefore, the tetramerization of TRPGz itself is most likely to occur independently of the CC region.
Interestingly, whole-vacuole patch clamp measurements revealed both Ca 2ϩ and voltage-dependent activations, even for the mutant lacking the CC region, to similar extents as those for the wild type protein (Fig. 3E). Furthermore, the activation by H 2 O 2 application was not significantly affected by the deletion of the CC region (Fig. 3F). These results verified that TRPGz lacking the CC region retains the intact channel architecture and suggested that the CC region is not required for the cytosolic Ca 2ϩ , membrane potential change, and H 2 O 2 responses. The results also implied that the Ca 2ϩ , membrane potential, and hyperoxic activations of the TRPGz channel occur by a modality different from those for the hyperosmotic and temperature change activations, which require the CC region.
The C-terminal Region of the TRPGz-CTD, Downstream from the CC Region, Is Responsible for Channel Regulation by PIPs-We next addressed the roles of the other regions in the CTD for the TRPGz functions. It is notable that the C-terminal deletion mutant of TRPGz, ⌬621Ϫ, gave significantly higher responses upon all stimuli in the assays described above, such as osmotic upshock, temperature increase, and H 2 O 2 treatment (Fig. 3, A,   B, and F). We found a cluster of basic residues in the C terminus ( Fig. 1), which is expected to interact with acidic phospholipids, such as PIPs. To clarify the interaction between the TRPGz-CTD and phospholipids, we performed protein-lipid overlay assays. The results revealed that the TRPGz-CTD interacted strongly with all PIPs and phosphatidic acid, whereas it showed weaker interactions with other acidic lipids, such as cardiolipin, lysophosphatidic acid, and sphingosine 1-phosphate (Fig. 4A). Among the PIPs, no binding specificity for the phosphorylation sites in the inositol moiety was observed. Similar reaction patterns were observed for the C-terminal half of the CTD, whereas the N-terminal half lacked reactivity to any phospholipids. The PIP binding by the CTD was also confirmed by liposome co-precipitation assays (Fig. 4B). These results indicated that the C-terminal basic cluster in the TRPGz-CTD specifically binds to general PIPs.
Furthermore, the electrophysiology also revealed that the application of water-soluble phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-diphosphate analogues to the cytoplasmic side significantly reduced the whole-vacuole currents of TRPGz (Fig. 4C), whereas the currents from TRPGz-D621 were not affected by the application of PIPs (Fig. 4D). These results clearly indicated that the interaction between the C-terminal basic cluster of TRPGz-CTD and the PIPs on the vacuolar membrane is responsible for the channel regulation, by inhibiting the channel activity. It should be noted that hyperosmotic shock dynamically increases the PIP levels in S. cerevisiae, mediated by proteins including vacuole-associated Vac14p, resulting in changes in vacuolar morphology (60,61). Therefore, the interactions of PIPs with the C-terminal TRPGz-CTD are likely to play important roles for channel regulation under cellular stress conditions. Taken together, the TRPGz-CTD is essential for the multimodal regulation of its channel activities, and each module in the CTD is responsible for different modalities, such as the CC region in the middle for activation by hyperosmotic shock and the basic cluster region at the C terminus for channel inhibition.

The TRPGz-CTD Is Primarily Composed of Intrinsically Disordered Regions with a Central Tetrameric Parallel Helix
Bundle-In order to elucidate the structural basis of the channel regulation by the CTD, we analyzed its structural characteristics. Unfortunately, our attempts to crystallize the CTD failed, for not only the entire region but also several patterns of deletions in the N-or C-terminal regions. The 1 H-15 N HSQC NMR spectrum of the CTD revealed that most of the CTD regions showed the characteristics of random coil structures (Fig. 5) and are considered to be intrinsically disordered regions. Nevertheless, we successfully crystallized the presumed coiled-coil region, TRPGz-CC, consisting of Val 600 -Thr 620 , and solved the x-ray crystal structure at 1.25 Å resolution (Table 1).
TRPGz-CC formed a left-handed parallel coiled-coil-like tetrameric assembly with an approximate length of 40 Å (Fig.  6A). At the intermolecular interface inside the molecular assembly, Val 600 , Leu 603 , Met 607 , Leu 610 , Leu 614 , and Leu 617 , the residues at the canonical "a" and "d" positions in the presumed coiled-coil region (Figs. 1 and 6B) form extensive hydrophobic interactions devoid of water molecules. At the surface, numerous salt bridges and hydrogen-bond networks were observed, such as between Glu 606 and Glu 609 from one protomer and Arg 604 , Glu 608 , and Lys 611 from another protomer, further reinforcing the interprotomer interaction (Fig. 6C).
TRPGz-CTD Exists in Association and Dissociation Equilibrium in the Physiological State-We next asked whether the tetrameric assembly observed in the TRPGz-CC crystal structure actually corresponds to the native state of the protein and is not simply achieved by the crystal packing of the small fragment. To address this, we first analyzed the oligomerization state of the entire CTD, including not only the CC region but also the upstream/downstream flanking regions (Fig. 1), by sedimentation equilibrium analytical ultracentrifugation experiments. The results indicated that the entire CTD is in equilibrium between monomer, dimer, and tetramer, with an apparent molecular mass of 56,000 Da, corresponding to a 3.1-mer (Table 2). On the other hand, the CTD lacking the CC region (⌬CC) was observed as a monomer (Table 2). In order to clarify the relationship between CTD oligomerization and assembly at the CC region, we introduced point mutations in the CC region to disrupt the intermolecular interactions. Based on the crystal structure, residues such as Met 607 and Leu 610 , lining the hydrophobic intermolecular interface (Fig. 6B), and Glu 606 , on the hydrophilic interface (Fig. 6C), were replaced with either charged amino acids or proline as a helix breaker. As expected, the oligomerization states of the CTD were significantly affected by the mutations to various extents; almost all of the mutants, including the double mutant M607D/L610D, exhibited diminished multimer formation, and L610R displayed weakened oligomerization, whereas E606P had less influence on oligomerization (Table 2). These results clearly indicated that the oligomerization observed in the CC crystallographic structure is responsible for the oligomerization in the entire CTD and thus the native state of the full-length protein. In addition, the moderate interprotomer affinity of the CTD is consistent with the weaker packing characteristics observed in the crystal structure, as compared with the cases of canonical four-stranded coiled coils, which exclusively exist as tetramers judged by analytical centrifugation experiments with lower protein concentrations than those used in this study (63,64).  The dynamic oligomerization state, between association and dissociation, is further supported by NMR analyses of the entire CTD. In the 1 H-15 N HSQC spectrum, the number of observed amide peaks was smaller than that expected from the primary sequence (Fig. 5). In the HSQC spectrum of M607D/L610D, extra amide peaks were detected in addition to those observed for the wild-type CTD (Fig. 7A), which were undetectable for a deletion mutant lacking the CC (Fig. 7B). Taken together, the amide signals in the CC region undergo exchange broadening, in agreement with the dynamic oligomerization. The same trend was also observed for the methyl signals of methionine residues. Significant line broadening was observed for the peaks of Met 607 and Met 613 in the CC region ( Fig. 1) in the 1 H-13 C HMQC spectrum of the wild type (Fig. 7C), whereas the line broadening was no longer manifested in the M607D/L610D mutant (Fig. 7D) or the CC deletion mutant (Fig. 7E). In summary, the CTD exists in equilibrium between monomer, dimer, and tetramer association and dissociation, mediated by the central CC region with weak assembly characteristics.
The CC Region-mediated TRPGz-CTD Oligomerization Positively Correlates with the Levels of Hyperosmotic Responses-To further clarify the relationship between the CC region-mediated CTD assembly and the channel activities, we examined the hyperosmotic responses of the above-described point mutants with disrupted CTD assembly at the CC region. Almost all of the mutants exhibited either weak or minimal responses. Notably, the amplitudes of the responses of the mutants clearly exhibited a positive correlation with the association constants of the CTDs, as determined by the sedimentation equilibrium experiments ( Fig. 8A and Table 2). These results strongly indicated that the hyperosmotic activation (and probably also the temperature increase activation) occurs on the TRPGz molecules with the CTD regions oligomerized through the CC regions (Fig. 8B).

DISCUSSION
Multimodal regulation is one of the most characteristic functions of TRP channels. In this study, we determined that a fungal TRP channel, TRPGz, is activated by various extracellular and cytosolic stimuli, such as hyperosmolarity, H 2 O 2 application, temperature increase, cytosolic Ca 2ϩ elevation, and membrane potential change. We identified the multiple regulatory modules of TRPGz within its C-terminal cytosolic domain that mediate at least part of the multimodal channel functions, such as the middle presumed coiled-coil region necessary for activa-tion by hyperosmolarity and temperature increase and the C-terminal phospholipid binding module for PIP-dependent channel inhibition. From another point of view, the results clarified that the TRPGz activities are regulated by multiple independent modalities, including at least those that require the CTD assembly at the CC region (hyperosmotic and temperature activations) and those independent from the CTD assembly (Ca 2ϩ , membrane potential, and H 2 O 2 activations) (Fig. 8B).
Coiled-coil modules are present in numerous TRP channels and are considered to be important for homo-or heterotetrameric channel assembly in many cases (18). However, we found that the assembly at the CC region, the presumable coiled-coil region of TRPGz, is not necessary for tetrameric channel formation and some channel modalities, such as Ca 2ϩ activation (Fig. 3E). Nevertheless, the CC region is indispensable for the other modalities, such as hyperosmotic responses (Fig. 3A). The results clearly implied that the CTD oligomerization through the CC region positively correlates with the channel opening for hyperosmotic and temperature increase activation (Figs. 3 (A and B) and 8A). There are several reported examples indicating that the assemblies of the cytosolic domains mediated by structural modules, including coiled-coils, are not necessary for tetrameric channel formation but are required for some channel functions. For example, cyclic AMP binding to the C-terminal domain of a hyperpolarization-activated, cyclic nucleotide-modulated channel, which reportedly shares structural similarity with the TRPV1 channel (68), was suggested to shift the association equilibrium of the domain to the tetramer, with the resultant conformational change responsible for channel opening (69).
In the case of TRPGz, what is the relevance of the CTD assembly to the channel activities arising from hyperosmotic shock or temperature increase? It is noteworthy that both are physical stimuli, and we hypothesize that one of the possible candidates underlying the activities is mechanosensitivity, arising from membrane stretching. Zhou et al. reported the mechanosensitive channel activities of TRPY1 and other fungal TRPY1 orthologs and suggested that they can explain the response to hyperosmotic shock, which causes dehydration of the cytosol and vacuole, resulting in transient osmotic pressure on the vacuolar membrane (35,37). With regard to the responses to rapid temperature increase observed with TRPGz, the responses could also be caused by vacuolar membrane tension resulting from thermal expansion of the solutions in the cytosol and vacuoles upon rapid temperature elevation, in addition to the possibilities of direct temperature sensing and/or changes in the physical properties of the vacuolar membrane. Further analyses are necessary to clarify the mechanism. Because the tetrameric channel pore exists between the TM helices S5 and S6, the CTD assembly just beneath the pore-lining S6 might serve as a fulcrum for the pore opening by membrane tension or as an anchor to maintain the appropriate topology and positioning for the pore opening even under conditions with membrane tension. We attempted to measure the force-activated vacuolar currents, but no significant conductance was observed by applying pressure up to ϳ0.5 kilopascal (data not shown). Because a pressure threshold for TRPGz activation is likely to exist (Fig. 2C), further analyses are needed, although the application of higher pressure is not fea- sible under the current experimental conditions, due to the fragility of the yeast vacuolar membrane (37).
What is the relevance of the CTD structure to the TRPGz channel activities that are independent of the CC region, such as cytosolic Ca 2ϩ elevation, membrane potential shift, and H 2 O 2 activation or PIP-dependent inhibition? The offset spiral assembly at the CC region observed in the x-ray crystallographic structure disrupts the interstrand interactions between the uppermost and lowermost protomers and explains the moderate interstrand affinity observed by analytical ultracen-   Table 2). The y axis plots show the means of the peak heights of the responses from each sample, with the error bars representing the S.E. values (n ϭ 4). B, hypothetical scheme of TRPGz channel activation and inhibition. Based on the results in this study and the previous knowledge about TRPY1 and other TRP channels, TRPGz is considered to permeate cations, including Ca 2ϩ , as its channel functions. The activation by hyperosmolarity and rapid temperature increase is an independent modality from the activation by cytosolic Ca 2ϩ , membrane potential, and hyperoxic shock. The CTD exists in dissociation and association equilibrium, mediated by the CC region. The former activities require CTD assembly mediated by the CC region, whereas the CC region is unnecessary for the latter activities. In both cases, PIP binding to the C-terminal basic cluster in the CTD inhibits the channel activities. The dynamic assembly/ disassembly characteristics of the CC region and the intrinsically disordered characteristics outside of the CC region enable multiple conformational substates of CTD during the channel functions. In the figure, the N-terminal cytosolic region is removed for clarity. trifugation and confirmed by NMR analyses. The weaker assembly, as compared with the canonical coiled-coil, enables the association and dissociation of the CTD during various functions and thus is considered to be compatible with the multimodal regulation mechanisms. Some require the assembly for activation, as in the cases of hyperosmotic shock and temperature increase, whereas others, supported by other regulatory modules, might require sufficient structural flexibility with less restraint, allowing access to their stimulants/ligands and the resultant conformational change for regulation. The structural characteristics of the entire CTD, which primarily consists of intrinsically disordered regions, are also probably favorable for regulation by multiple stimulants/ligands. In TRPY1, the calcium binding sites for channel activation are reportedly located in the C-terminal domain (70), and a similar acidic residue cluster also exists in TRPGz upstream of the CC region (Fig. 1). Several TRP channels are considered to act as sensors for the cellular redox state and are equipped with oxidizing/reducing sites (e.g. Cys residues) in cytosolic domains (e.g. as described by Takahashi et al. (14)). The PIP-binding module was also found in the TRPGz-CTD downstream of the coiled-coil, as a regulatory module that probably functions in suppressing channel gating and maintaining Ca 2ϩ homeostasis under conditions of cell stress, which in many cases induces elevated PIP levels.
These dynamic interaction properties of the CTD with various ligands suggest that multiple conformational substates of CTD exist (Fig. 8B). With regard to the oligomerization of the CTD, the ⌬G for TRPGz-CTD oligomerization is Ϫ7.9 kcal/ mol (estimated from the equilibrium constant in Table 2). On the other hand, the nonspecific PIP-binding motif, such as the MARCKS effector domain, probably exhibits strong electrostatic interaction with PIPs, with a ⌬G of Ϫ8 kcal/mol (estimated from the value reported by Arbuzova et al. (71)). Notably, the activation energy of TRPV1 was reported as ϳ7 kcal/ mol (72), and all of these are comparable energy levels. These facts imply relevance of the conformational diffusion of the CTD to the conformational diffusion in ion channel gating (73). The structural characteristics of the TRPGz-CTD described above, with its intrinsically disordered region and the assembly region with moderate association affinity in the middle, allow dynamic association and dissociation with various binding partners and thus are considered to be favorable for the multimodal activation commonly observed in TRP channels.