Negatively-charged Amino Acids near and in Transient Receptor Potential (TRP) Domain of TRPM4 Channel Are One Determinant of Its Ca

Transient Receptor Potential (TRP) channel Melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca 2+ , which is essential for the activation. The Ca 2+ sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as PI(4,5)P2. Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin binding sites have suggested that Ca 2+ binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch-clamp and molecular biological techniques, we show that http://www.jbc.org/cgi/doi/10.1074/jbc.M114.606087 The latest version is at JBC Papers in Press. Published on November 6, 2014 as Manuscript M114.606087 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on Jauary 1, 2018 hp://w w w .jb.org/ D ow nladed from Amino acids in TRP domain of TRPM4 affect Ca 2+ sensitivity 2 there are at least two functionally different divalent cation binding sites and the negatively charged amino acids near and in the TRP domain in C-terminal tail of TRPM4 (D1049 and E1062 of rat TRPM4) are required for maintaining the normal Ca 2+ sensitivity of one of the binding sites. Applications of Co 2+ , Mn 2+ , or Ni 2+ to the cytosolic side potentiated TRPM4 currents, increased the Ca 2+ sensitivity, but were unable to evoke TRPM4 currents without Ca 2+ . Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca 2+ sensitivity in the presence of Co 2+ or PI(4,5)P2 but hardly affected the sensitivity to Co 2+ and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca 2+ sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca 2+ . Transient Receptor Potential Channel Melastatin (TRPM), a subfamily of the TRP ion channel, consists of eight channels, TRPM1 to TRPM8. Among the TRPM channels, TRPM4, as well as TRPM5, forms a Ca 2+ -activated nonselective monovalent cation channel, which does not conduct divalent cations such as Ca 2+ (1) although other TRPM channels permeate them (2). TRPM4 does not differentiate Na + and K + (3,4), and its opening affects cell functions through membrane depolarization. Unlike TRPM5, TRPM4 has a relatively broad tissue expression pattern (4). In those tissues, TRPM4 has been implicated in several physiological functions, for example, immune response (5-7) and constriction of cerebral arteries (8,9). Additionally, its mutations in the TRPM4 gene have been associated with cardiac conduction dysfunction in human patients (4,10-12). Furthermore, it has been shown that TRPM4 mediates axonal and neuronal degeneration in experimental autoimmune encephalomyelitis and multiple sclerosis (13). TRPM4 channel activity is increased by membrane depolarization, but it absolutely requires intracellular Ca 2+ (14). Thus, the most important regulator of TRPM4 activity is the intracellular Ca 2+ . However, the activation mechanisms of TRPM4 by Ca 2+ have not been completely clarified. Calmodulin is thought to play an important role in the activation of TRPM4 by Ca 2+ through its binding to the C-terminal tail of TRPM4 because it has been reported that deletion mutants of calmodulin binding sites showed strongly impaired current activation by reducing Ca 2+ sensitivity (14). For example, although wild-type TRPM4 has shown large currents in the presence of 100 μM Ca 2+ , the mutants have shown negligible currents under the same conditions (14). However, the mutants were still able to be activated by very high concentrations (e.g. 1 mM) of Ca 2+ and positive voltages (14). Furthermore, TRPM5, which shows the highest homology to TRPM4 (4), has been suggested to be activated by Ca 2+ directly rather than through calmodulin because calmodulin modulators did not affect TRPM5 (15). These by gest on Jauary 1, 2018 hp://w w w .jb.org/ D ow nladed from Amino acids in TRP domain of TRPM4 affect Ca 2+ sensitivity 3 findings imply that there are unidentified intrinsic Ca 2+ binding sites in TRPM4 as mentioned elsewhere (4,14). Moreover, a membrane phospholipid, Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2, PIP2), has been shown to restore the Ca 2+ sensitivity of TRPM4 after desensitization (16,17). For example, the EC50 for Ca 2+ after desensitization was reported to be 520 μM, and that after application of PIP2 was 120 μM (16). Positively charged amino acids in a C-terminal pleckstrin homology (PH) domain were identified as important determinants of PIP2 action (17). However, the mechanism of how PIP2 increases the Ca 2+ sensitivity of TRPM4 has not been revealed. The C-terminal cytosolic tail of TRPM4, which is important for the regulation of its activity, contains the TRP domain and TRP box. The TRP domain refers to a homologous block of about 25 residues immediately C-terminal to S6 that is loosely conserved in almost all TRP mammalian subfamilies (18). The TRP domain encompasses a highly conserved 6-amino acid TRP box (18). The TRP domain of TRPM8, TRPM5, and TRPV5 has been suggested to serve as a PIP2-interacting site (19). However, it has been shown that the TRP box and TRP domain of TRPM4 are not the main determinants of PIP2 action (17). Therefore, the functional role of the TRP domain and TRP box in TRPM4 remains elusive. Ca 2+ binding sites of Ca 2+ -regulated proteins exhibit diverse divalent cation selectivities. Thus, the divalent cation selectivities of binding sites have been used as a powerful tool for distinguishing properties of different Ca 2+ binding sites in conjunction with the molecular biological approaches. For example, it has been shown that the large-conductance Ca 2+ -activated K + channel, BK channel, has three divalent cation binding sites, the so-called Ca 2+ -bowl, RCK1 domain, and E399-related low-affinity sites, of which divalent cation selectivities are different (20,21). On the basis of such an idea, it has been shown that Sr 2+ and Ba 2+ do not substitute for Ca 2+ in TRPM4 activation (22). However, not much has been done to reveal the overall mechanisms of the activation of TRPM4 by Ca 2+ , such as the number of binding sites in TRPM4 and their roles in the activation by Ca 2+ . The objective of this paper is to obtain further understanding of the mechanisms underlying the activation of TRPM4 by intracellular Ca 2+ . In order to reveal the properties of divalent cation binding sites of TRPM4, we firstly examined the effects of larger variety of divalent cations applied to the cytosolic side of the channel. Secondly, we explored the amino acid residues responsible for the activation by Ca 2+ using single amino acid mutagenesis approaches. Among several mutants of the amino acid residues in the cytosolic C-terminal tail of TRPM4, we found two negatively-charged amino acids near and in the TRP domain of TRPM4 to be important determinants of Ca 2+ sensitivity. EXPERIMENTAL PROCEDURES Animal Ethics Approval − All animal experiments by gest on Jauary 1, 2018 hp://w w w .jb.org/ D ow nladed from Amino acids in TRP domain of TRPM4 affect Ca 2+ sensitivity 4 were performed in accordance with guidelines from and protocols approved by the Institutional Animal Care and Use Committee (IACUC), Graduate School of Veterinary Medicine, Hokkaido University and the Committee on Animal Experimentation, Niigata University School of Medicine. Molecular cloning and Site-directed Mutagenesis − TRPM4b, the long form of TRPM4, forms a functional channel and considered to be the significant variant (3). Therefore, we refer to TRPM4b as TRPM4 in this paper. Rat TRPM4 was cloned from mRNA of the stria vascularis in the cochlea because Ca 2+ -activated nonselective currents were recorded from the apical membrane of marginal cells in freshly isolated stria vascularis using inside-out mode of patch clamp technique (data not shown) as similarly reported from those of guinea pig (23) and gerbil (24). Additionally, the expression of TRPM4 in the marginal cells has been confirmed by immunohistochemistry more recently (25). RNA was extracted from stria vascularis in the cochleae of BN/SsNSlc male rats (5-6 weeks old) using NucleoSpin RNA XS (Takara Bio, Otsu, Japan). cDNA was synthesized using PrimeScript II Reverse Transcriptase (Takara Bio). The full length of the open reading frame of TRPM4 cDNA was amplified as two overlapped N-terminal and C-terminal fragments by PCR using a high fidelity polymerase (PrimeSTAR GXL, Takara Bio) and the following primers: 5’ GGC GGC TGA GAG AAA TAC ACG GAG C 3’ (N-terminal Forward) and 5’ GTC ACT CCA GGG GGC TTG TTC AAA G 3’ (N-terminal Reverse), 5’ AAC TTT TCC GTG GGG ACA TCC AGT G 3’ (C-terminal Forward) and 5’ CAT GGG GTC TAC GGT GAG GAC AAG G 3’ (C-terminal Reverse), respectively. The primers were designed based on the reported sequence of rat TRPM4 cDNA (Genbank accession #NM_001136229.1). The two fragments of TRPM4 were cloned in TA-vector (Takara Bio) and sequenced. The nucleic acid and amino acid sequences of the cloned rat TRPM4 were identical to those recorded in database (#NM_001136229.1 and #NP_001129701.1, respectively (26)). The N-terminal and C-terminal fragments, which contained no PCR errors, were subcloned and combined in a bicistronic expression vector pIRES2-EGFP (Takara Bio). Site-directed mutagenesis of TRPM4 cDNA in pIRES2-EGFP was accomplished using the PrimeSTAR Mutagenesis Basal kit (Takara Bio). Mutations were verified by DNA sequencing. Cell culture and transfection − HEK 293T cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium; Sigma-aldrich, St. Louis, MO) supplemented with 10% FBS (Moregate Biotech, Bulimba, Australia, or Thermo Fisher Scientific, Waltham, MA. Their local distributors were Hana-nesco Bio, Tokyo, Japan and Thermo Fisher Scientific K.K., Yokohama, Japan, respectively.) and penicillin/streptomycin (1000 U/ml and 1000 μg/ml, respectively, Thermo Fisher Scientific) at 37 °C in a 5% CO2 incubator. Cells were by gest on Jauary 1, 2018 hp://w w w .jb.org/ D ow nladed from Amino acids in TRP domain of TRPM4 affect Ca 2+ sensitivity 5 transiently transfected with plasmids using TransIT-293 Transfection Reagent (Takara Bio). The cells were plated on coverslips the following day. Patch-clamp recordings were made two days after transfection from EGFP positive cells, which were identified with an inverted microscope (Diaphot 300, Nikon, Tokyo, Japan) equipped with a super high-pressure mercury lamp light source (C-SHG, Nikon) for excitation of green fluorescence from EGFP. Electrophysiology − HEK 293T cells on coverslips were transferred to a bath mounted on the stage of the inverted microscope and superfused with a standard NaCl rich solution containing 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM D-glucose, and 10 mM HEPES (pH = 7.4 with NaOH). The pipette solution contained 145 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES (pH = 7.4). Before patch excision, the bath solution was changed to mainly a solution containing 145 mM NaCl, 10 mM HEPES (pH = 7.4), and 1 mM CaCl2. In many cases, a divalent cation-free solution contained 5 mM EGTA to chelate divalent cations but in some experiments, a nominally divalent cation-free solution was also used, which was made simply without adding divalent cations. Dichloride salts of divalent cation were used. The concentration of free Ca 2+ in solutions less than 10 μM was adjusted by adding an appropriate amount of CaCl2, calculated using a software webmaxc (http://www.stanford.edu/~cpatton/webmaxcS.htm ), to solutions containing 5 mM EGTA. In some experiments, o-phenylenedioxydiacetic acid (o-PDDA, Sigma-aldrich) was used as a divalent cation chelator and free divalent cation concentrations were calculated with the stability constants reported elsewhere (27). Na-fluoride (NaF, 145 mM) was also used as a Ca 2+ -chelator (21,28). Osmolality of solutions were measured using Vapro Vapor Pressure 5600 (Wescor Inc, Logan, UT, USA). The osmolality of most solutions were near physiological range e.g. that of the pipette solution was 283 ±2 mOsm/kg, that of nominal Ca 2+ -free solution was 276 ±1 mOsm/kg, and that of the solution containing 10 mM CaCl2 was 301 ±0 mOsm/kg. However, the osmolality of the solution containing 30 mM CaCl2 was 352 ±1 mOsm/kg. The results of the present study might be partially affected by the change in osmolality as a background effect. The speed of perfusion was about 1.5 ml/min. The bath solution around a patch membrane was cleared within 10 sec in most cases or within 15 sec at the latest. Axopatch 200B patch-clamp amplifier, digidata 1322A, and the pCLAMP8 software (Axon Instruments, Union City, CA) were used to perform voltage clamp, data storage, and analysis. A reference Ag–AgCl electrode was connected into the bathing solution via an agar bridge filled with the aforementioned standard NaCl-rich bath solution. Patch electrodes had a resistance between 3 and 5 megaohms. The currents were filtered at 1 kHz with an internal four-pole Bessel by gest on Jauary 1, 2018 hp://w w w .jb.org/ D ow nladed from Amino acids in TRP domain of TRPM4 affect Ca 2+ sensitivity 6 filter, and sampled at 5 kHz. Current–voltage (I-V) relations for the currents were studied using voltage ramps. The membrane potential held at −60 mV, and the command voltage was varied from −100 to +100 mV over a duration of 400 ms following a prepulse of −100 mV for 50 ms every 5 s. Because TRPM4 is activated by membrane depolarization, we analyzed the current amplitudes at −100 mV and +100 mV as the least and the most activated current among the currents evoked by the applied pulse, respectively. Furthermore, because the current amplitudes at −100 mV in some mutants were negligible, the current amplitudes at +100 mV were used for the analysis of dose-response. All experiments were performed at room temperature. Data analysis − Dose-response curves were fits of the averages with the Hill equation: I = Imax × C EC50 n × Cn where Imax is the maximum currents, C is the concentration of substance being tested, EC50 is the concentration for half-maximal effect, n is the Hill coefficient. The relationship between EC50 for Ca 2+ and the concentration of Co 2+ was analyzed with the pseudo Hill equation: y = ymax + (ymin − ymax) × C EC50 n × Cn where ymax is the EC50 for Ca 2+ in the absence of Co 2+ and ymin is the minimal EC50 for Ca 2+ among those estimated in the presence of Co 2+ , C is the concentration of Co 2+ , EC50 is the concentration of Co 2+ for half-maximal effect, n is the pseudo Hill coefficient. Chord conductance-voltage curves were fitted with the Bolzmann equation: G = Gmax × (1 − 1 − f 1 + e(m1 2 ⁄ ) dx ⁄ ) where Gmax is fixed to the normalized conductance at +100 mV, Vm is the membrane potential, V1/2 is the membrane potential at which the conductance is half of Gmax, dx is the slope factor, and f is the voltage-independent conductance fraction. All curves were obtained by fitting the data averages. The results are reported as means ±S.E. of independent experiments (n), where n refers to the number of cells patched. Statistical significance was evaluated using Student’s two-tailed paired or unpaired t test or Dunnett’s test as appropriate. A value of P < 0.05 was considered significant.