TRPC4 expression determines sensitivity of the platelet-type capacitative Ca2+entry channel to intracellular alkalosis

The present study was designed to analyze the molecular basis of the intracellular pH-dependent capacitative Ca²⁺ entry (CCE) of human platelets and megakaryocytic cells, specifically to test the hypothesis that members of the classical transient receptor potential (TRPC) protein family are involved in the CCE pathway that is promoted by intracellular alkalosis. Human platelets as well as the tested megakaryocytic cell lines (CMK cells, MEG-01 cells) and HEK293 cells displayed thapsigargin-induced CCE and responded to monensin with comparable elevation in intracellular pH. Promotion of CCE by monensin-induced intracellular alkalosis, however, was profound in mature platelets, moderate in CMK cells and lacking in MEG-01 cells as well as in HEK293 cells. Analysis of the TRPC expression pattern by immunoblotting revealed that mature platelets and CMK cells express TRPC4 along with TRPC1 and TRPC3, while TRPC4 is lacking in MEG-01 cells. HEK293 cells displayed CCE characteristics as well as lack of TRPC4 expression similar to MEG–01 cells. Over-expression of TRPC4 in HEK293 cells was found to result in a gain of pH-sensitivity of CCE with clearly detectable promotion of CCE in response to monensin. These results suggest that platelet CCE channel complexes contain TRPC4 as a molecular component that determines sensitivity of CCE to intracellular alkalosis.

Keywords: Platelets; megakaryocytes; intracellular alkalosis; transient receptor potential channels

Store-operated or capacitative Ca2+ entry (CCE) is a mechanism indispensable for generation of Ca2+ signals in non-excitable cells, including platelets. Since the very initial identification of mammalian homologues of the Drosophila trp gene product, members of this superfamily of transient receptor potential cation channels have been considered as candidates of CCE channels. Although substantial evidence has been accumulated for a role of some classical transient receptor potential (TRPC) species as components of CCE channel complexes, the exact molecular structure of TRPC-related CCE complexes remains elusive. Recent evidence supports the concept of heteromeric TRPC complexes as the basis of CCE [1]. In platelets, TRPC1 has been suggested as a Ca2+ channel subunit that contributes to CCE by a conformational coupling mechanism [[2], [3]], while TRPC6 has been suggested to form a store-independent nonselective cation entry channel [4].

Little is known about the function of TRPCs and their role in CCE in megakaryocytes, platelet precursor cells. However, expression of TRPCs at the mRNA level has been demonstrated in megakaryocytes [[5], [6]], and, interestingly, CCE and genetic expression of TRPC1, C4 and C6 have been reported to increase with cell differentiation [5]. Thus, it is tempting to speculate that TRPC-related CCE pathways are quantitatively as well as qualitatively different in megakaryocytes and platelets. Such a plasticity of the CCE pathway may be essential for proper differentiation of the precursor cells as well as for function of mature platelets. One characteristic feature of platelet CCE channels is their marked sensitivity to intracellular pH (pHi), which has been considered to be essential for the control of platelet function [7]. Receptor stimulation by agonists activates Na+/H+ exchanger (NHE), resulting in an increase in intracellular pH in a variety of cells. Physiological agonists, such as thrombin, epinephrine, platelet-activating factor, vasopressin and endothelin, cause cytoplasmic alkalinization through NHE activation in platelets [[8], [9], [10], [11], [12]]. The relationship between agonist-induced NHE activation and platelet function such as aggregation, however, is still elusive. Artificial alkalosis induced by NH4Cl, trimethylamine and monensin has been reported to augment CCE in platelets [[13], [14], [15]]. Thus, intracellular alkalosis has been proposed as a mechanism that promotes platelet CCE and aggregation. However, it has not been determined whether intracellular alkalosis affects CCE in megakaryocytes.

Here we present evidence for divergent pHi regulation of the CCE pathways in platelets and their precursor cells and suggest differences in the TRPC expression pattern of TRPC isoforms as the basis of divergent CCE regulation.

CMK cells and MEG-01 cells were obtained from DSMZ (Germany) and ATCC (USA), respectively, and cultured in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum (FCS), 4 mM glutamate, 100 U/mL penicillin and 100 µg/mL streptomycin in a humidified atmosphere at 37°C under 5% CO2–95% air. The cells were spread in a 12-well plate or a 100-mm dish and cultured until reaching a confluent condition. Then, confluent cells were used for the assays.

Blood was obtained from healthy donors who had not been administrated any drugs for at least 10 days before the experiments. The blood (18 mL) was rapidly transferred to a plastic tube containing 2 mL of a potent anti-thrombin inhibitor, D-phenylalanyl-prolyl-arginine chloromethyl ketone (final concentration of 10 µM), and mixed. The blood was then centrifuged at 150 × g for 10 min, and the supernatant was obtained as platelet-rich plasma (PRP). PRP was then mixed with 40 mL of Ca2+-free Tyrode solution that had been buffered by Hepes (pH 7.4) and contained 1 mM EGTA, and the mixture was centrifuged at 150 × g for 10 min. After centrifugation at 400 × g for 5 min, the obtained pellet was suspended with 40 mL of the above Tyrode–Hepes solution and then further centrifuged at 400 × g for 5 min. The pellet obtained was suspended again with 2 mL of the above solution, and the suspension was used for the experiments.

[Ca2+]i was measured using a fluorescent Ca2+ indicator, fura-2. The washed platelets and cultured CMK and MEG-01 cells were loaded with fura-2 by incubation with fura-2/AM (final concentration, 5 µM) at 37°C for 30 min. After loading, CMK cells, MEG-01 cells and HEK293 cells were washed once with physiological salt solution (PSS) buffered by Hepes (mM: NaCl 135, KCl 5, KH2PO4 1, CaCl2 2.5, MgCl2 1, Hepes 10, glucose 10, pH 7.4), and they were re-suspended in Ca2+-free PSS buffered by Hepes and containing 0.01 mM EGTA (nominally Ca2+-free solution). After loading, the platelets were washed once with Ca2+-free PSS buffered by Hepes (pH 7.4) and containing 1 mM EGTA, and they were resuspended in 2 mL of Ca2+ and EGTA-free PSS buffered by Hepes and containing fibrinogen (nominally Ca2+-free solution).

Fluorescence measurements were carried out with a dual-wavelength spectrofluorometer (F2500 Fluorescence Spectrophotometer, Hitachi, Tokyo, Japan) using a 0.4-mL cuvette maintained at 37°C. The wavelengths used for excitation were 340 and 380 nm, and that used for emission was 510 nm. Using the ratio (R) of fluorescence intensity (F) of F340/F380, the baseline-corrected changes in [Ca2+]i were determined. The fluorescence after sequential addition of 0.2% Triton X-100 and EGTA (5 mM) to the platelet suspension provided the maximum fluorescence ratio (Rmax) and minimum fluorescence ratio (Rmin), respectively. [Ca2+]i was calculated using the formula described by Grynkiewicz et al. [16]:

Graph

where β is the ratio of the emission fluorescence values at 380-nm exitation wavelength in the presence of Triton X-100 and EGTA, and Kd, the dissociation constant for Ca2+, is 224.

Intracellular pH was measured using a pH indicator, 2′7′-bis-(2-carboxyethyl)-5(6)-carboxy-fluorescein (BCECF). CMK cells, MEG-01 cells, platelets and HEK293 cells were loaded with BCECF/AM (final concentration, 1 µM) for 30 min at 37°C. After loading, the cells were washed and suspended in 2 mL of nominally Ca2+-free PSS as mentioned above. Fluorescence measurement was carried out with a dual-wavelength spectrophotometer. Excitation wavelengths used were 500 and 440 nm, and emission was collected at 530 nm. Using ratio (R) of fluorescence intensity (F) of F500/F440, the fractional change in pHi was determined. Calibration of pHi measurements was performed in a nigericin (3 µM)-containing high-K+ solution at various extracellular pH values.

Pellets from each suspension of CMK cells, MEG-01 cells and platelets were centrifuged in ice-cold PBS at 1000 × g for 5 min at 4°C. Cell pellets were resuspended in 0.5 mL of ice-cold lysis buffer [50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 60 mM n-octyl-β-D-glucopyranoside, 1 mM PMSF, and a protease inhibitor mixture (complete, Roche Applied Science, Germany)] and incubated for 15 min on ice. Cell lysates were sonicated and centrifuged at 10 000 × g for 10 min at 4°C. After removing the supernatant, the pellet was dissolved in a Laemmli buffer (2×), and the samples were then denaturated at 95°C for 5 min. Proteins from cell lysates were separated by SDS–PAGE and transferred to nitrocellulose sheets. Nitrocellulose membranes were incubated with PBS containing 1% Bio-Rad blocking reagent overnight at 4°C and then incubated with polyclonal antibodies against TRPC1, TRPC4 (1:200, Alomone, Israel) or TRPC3 (generated in our laboratory) for 2 h at room temperature. One set of experiments was performed with an anti-TRPC4 antibody kindly provided by Dr V. Flockerzi. After washing with PBS, the membranes were further incubated with horseradish peroxidase-conjugated anti-rabbit antibody used as a secondary antibody (1:5000) for 1 h at room temperature. Membranes were detected by using Chemi Glow™ West detection system and developed using a Herolab RH-5.2 Darkroom Hood equipped with an E.A.S.Y 1.3 HC camera (Herolab Laborgeräte GmbH, Germany).

HEK293 cells were transiently transfected with TRPC4α using TransfastTM Transfection Reagent (Promega Corporation, Austria) according to the manufacturer's instructions. Some experiments were performed with a HEK293 cell line stably expressing TRPC4α (T4-60), kindly provided by Dr M.X. Zhu.

Thapsigargin (Sigma, St. Louis, MO, USA), fura-2/AM and BCECF/AM (Dojin, Kumamoto, Japan) were dissolved in dimethylsulfoxide to make stock solutions of 1, 5 and 5 mM, respectively, and stored at −30°C. 1-β{[3-4-Methoxyphenyl] propoxy}-4-methoxyphenethyl}-1-H-imidazole hydrochloride (SKF-96365, Calbiochem, La Jolla, CA, USA) and sodium propionate (Wako, Osaka, Japan) were dissolved in distilled water to make stock solutions of 10 mM and 2 M, respectively, and stored at 4°C. Monensin (Sigma, St. Louis, MO, USA) was dissolved in ethanol to make a stock solution of 50 mM and stored at 4°C.

Statistical analysis was done using Student's t-test. P-values less than 0.05 were regarded as significant.

Monensin was used as a tool to elevate intracellular pH and to study the effects of intracellular alkalosis on Ca2+ entry. Figure 1 shows the mean pHi value in basal condition and the peak pHi value obtained after addition of monensin (10 µM) for platelets, megakaryocytic cells and HEK293 cells. Monensin induced comparable cytoplasmic alkalosis in platelets, CMK cells, MEG-01 cells and HEK293 cells.

Graph: Figure 1. Changes in intracellular pH induced by monensin (10 µM) in CMK cells, MEG-01 cells, platelets and HEK293 cells. The mean values of basal pH (control) and peak pH after incubation with monensin are shown. n = 2 − 3.

Ca2+ re-addition after stimulation with thapsigargin in nominally Ca2+-free solution induced a remarkable increase in cytoplasmic Ca2+ of platelets, megakaryocytic cells and HEK293 cells. This Ca2+ re-addition-induced increase in cytoplasmic Ca2+ was taken as a measure of CCE. Figure 2 shows the CCE-mediated increases in cytoplasmic Ca2+ of platelets and megakaryocytes at normal and elevated intracellular pH. Monensin strongly augmented CCE in platelets (Figures 2 and 3A) but induced only a moderate, though significant, promotion of CCE in CMK cells (Figure 2). Notably, the CCE pathway of MEG-01 cells was virtually insensitive to the alkalosis induced by monensin (Figures 2 and 3B). Representative recordings of [Ca2+]i as well as pHi for platelets and MEG-01 cells are also shown in Figure 3A, B, respectively. For a rigorous test of the pHi sensitivity of MEG-01 Ca2+ entry mechanisms to intracellular alkalosis, we performed experiments over a wider range of extracellular Ca2+ concentrations (0.5–3 mM). However, Ca2+ entry measured at extracellular Ca2+ concentrations of 0.5–3 mM was not promoted by monensin in MEG-01 cells (Figure 3B). As shown in Figure 4, the augmentation of CCE by monensin was abolished in the presence of propionate and sensitive to inhibition by a classical CCE inhibitor, SKF-96365, in both CMK cells and platelets.

Graph: Figure 2. Effects of monensin on thapsigargin-induced capacitative Ca2+ entry (CCE) in CMK cells, MEG-01 cells and platelets. CCE was induced by addition of Ca2+ (1 mM) to a nominally Ca2+-free medium after release of intracellular stored Ca2+ by thapsigargin (1 µM). Monensin (10 µM) or a vehicle (control) was added to the cell suspension at 1 min before addition of Ca2+. The cells were treated with thapsigargin for 120 s (for platelets) and 180 s (for MEG-01 cells and CMK cells) in order to stimulate capacitative Ca2+ entry channels. The y-axis values in the figure represents increments of [Ca2+]i from basal levels to peak levels after Ca2+ addition. Asterisks denote significant difference (P < 0.01, n = 5) from the control incubated with a vehicle of monensin.

Graph: Figure 3. Representative time-course recordings of [Ca2+]i (left panel) and intracellular pH (right panel) in platelets (A) and MEG-01 cells (B). CCE was induced by addition of a single dose of Ca2+ (1 mM) (A) or cumulative doses of Ca2+ (0.5–3 mM) (B) to a nominally Ca2+-free medium after release of intracellular stored Ca2+ by thapsigargin (1 µM). Monensin (10 µM) or a vehicle (control) was added to the cell suspension at the indicated time before addition of Ca2+ in order to induce intracellular alkalinization.

Graph: Figure 4. Effects of propionate and SKF-96365 on capacitative Ca2+ entry (CCE) augmented by monensin in CMK cells and platelets. CCE was induced by addition of Ca2+ (1 mM) to a nominally Ca2+-free medium after release of intracellular stored Ca2+ by thapsigargin (1 µM). Monensin (10 µM) or a vehicle (control) was added to the cell suspension at 1 min before addition of Ca2+. The cells were treated with thapsigargin for 120 sec (for platelets) or 180 sec (for CMK cells) in order to stimulate capacitative Ca2+ entry channels. Propionate (20 mM) or SKF-96365 (100 µM) was added to the cell suspension at 1 min before addition of Ca2+. The y-axis values in the figure represent increments of [Ca2+]i from basal levels to peak levels after Ca2+ addition. Asterisks denote significant difference (p < 0.01, n = 5) from the control incubated with a vehicle of monensin.

Figure 5A shows a comparison of TRPC1, C3 and C4 protein expressions in platelets and megakaryocytic cell lines. Platelets displayed prominent expression of TRPC3 and TRPC4 but weak expression of TRPC1. On the other hand, TRPC1 was strongly expressed in CMK cells as well as in MEG-01 cells. In contrast, TRPC4 was barely expressed in CMK cells and not detectable in MEG-01 cells. Moreover, we further confirmed the expression of TRPC4 in human platelelts by use of another antibody, which was kindly provided by Dr V. Flockerzi (data not shown). Since functionally distinct splice forms of TRPC4 (α and β) have been described [17], we analyzed the relative expressions of these splice variants in human platelets. As illustrated in Figure 5B, resolution of two distinct bands corresponding to the α and β forms of TRPC4 was possible for human platelet homogenates in optimized experimental conditions. The prominent species in platelets was a variant as evidenced by a comparison with TRPC4α over-expressed in HEK293 cells. In contrast, the weak immunoreactivity detected in CMK cells corresponded to a smaller protein, most likely TRPC4β. These results prompted us to investigate the relationship between TRPC4α expression and pHi sensitivity of CCE.

Graph: Figure 5. A) Representative Western blot images illustrating expression of TRP proteins in CMK cells, MEG-01 cells and platelets. Proteins (100 µg) from cell lysates were separated on 8% SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted to detect TRPC1, C3 and C4. B) Representative Western blot images showing the expression of α (∼102 kDa) and β (∼97 kDa) variants of TRPC4 in platelets and CMK cells. Results from TRPC4α-transfected HEK293 cells are shown for comparison. Proteins (150 µg) from cell lysates were separated on 6% SDS-PAGE, transferred to a nitrocellulose membrane and immunoblotted against TRPC4.

HEK293 cells display a significant endogenous CCE based on CCE channels that may involve specific endogenous TRPC proteins expressed at low levels [[18], [19]]. It is of note that TRPC4 is typically not detectable by immunoblotting in wild-type HEK293 cells as illustrated in Figure 6A. This endogenous CCE of HEK293 cells was fairly insensitive to monensin-induced intracellular alkalosis (Figure 6A, B), resembling the situation observed in MEG-01 cells (Figure 2). Since HEK293 cells represent a well-established and highly efficient expression system for TRPC4, we tested the hypothesis of pHi sensitivity of CCE channels being controlled by TRPC4α expression levels. Over-expression of TRPC4α failed to promote overall magnitude of CCE in HEK293 cells in the control condition at physiological pHi but generated a significant sensitivity of CCE channels to intracellular alkalosis as illustrated in Figure 6A, B. The lack of detectable TRPC4 expression in wild-type HEK293 cells in comparison to HEK293 cells over-expressing TRPC4 protein is shown by immunoblotting results (insert). Monensin promoted CCE-mediated Ca2+ entry into T4-60 cells [CCE: 305.30 ± 30.1 nM (control) vs. 477.2 ± 51.5 nM (monensin–treated) (P < 0.05, n = 10)].

Graph: Figure 6. (A) Effects of monensin on thapsigargin-induced capacitative Ca2+ entry (CCE) in TRPC4α-transfected HEK293 cells and wild-type HEK293 cells. CCE was induced by addition of Ca2+ (1 mM) to a nominally Ca2+-free medium after release of intracellular stored Ca2+ by thapsigargin (1 µM). Monensin (10 µM) or a vehicle (control) was added to the cell suspension at 150 s before addition of Ca2+. The cells were treated with thapsigargin for 200 s in order to stimulate capacitative Ca2+ entry channels. The y-axis values in the figure represents increments of [Ca2+]i from basal levels to peak levels after Ca2+ addition. An asterisk denotes significant difference (P < 0.01, n = 5) from the control incubated with a vehicle of monensin. Insert: representive Western blot images illustrating TRPC4 immunoreactivity in homogenates from wild-type and TRPC4-transfected HEK293 cells. (B) Representative time-course recordings of [Ca2+]i in HEK293 cells under the same conditions as those in (A). CCE was induced by addition of a single dose of Ca2+ (1 mM) to a nominally Ca2+-free medium after release of intracellular stored Ca2+ by thapsigargin (1 µM). Monensin (10 µM) or a vehicle (control) was added to the cell suspension at the indicated time before addition of Ca2+ in order to induce intracellular alkalinization.

In the present study, we demonstrated that megakaryocytes and platelets express CCE pathways with distinctly different regulatory properties in terms of sensitivity to pHi. Our results suggest that TRPC4 is a component of the platelet CCE pathway and functions as a determinant of pHi sensitivity of CCE channels.

Remarkable Ca2+ entry was observed in platelets as well as in CMK and MEG-01 cells during Ca2+ re-addition protocols utilizing thapsigargin as a tool to accomplish store depletion. These results are in line with results of a recent study demonstrating high CCE in megakaryocytes and platelets [6]. Regulatory increases in cytoplasmic pH of platelets are typically induced by activation of NHE following receptor stimulation by agonists [[8], [9], [10], [11], [12]], and cytoplasmic alkalosis has been proposed as a pivotal mechanism for regulation of CCE in platelets as well as in vascular smooth muscle cells [[14], [20]]. However, the role of this mechanism in cellular control of agonist-induced Ca2+ entry has been considered controversial [[20], [21], [22]]. In contrast to the above reports on promotion of CCE by artificial intracellular alkalosis, this condition has been reported to inhibit CCE in vascular endothelial cells [23]. Thus, regulation of CCE by intracellular alkalosis appears to be highly dependent on the cell system. This variability may reflect the existence of divergent CCE channel complexes in different cell types. So far, pHi sensitivity of megakaryocytes has not been investigated. Based on the known pHi sensitivity of platelet CCE channels, it was expected that a similar pHi regulation in megakaryocytes, the cellular precursors of platelets, would be observed. Monensin, a monovalent ion-selective ionophore, which facilitates the transmembrane exchange of sodium ions for protons, is an established tool for inducing sustained intracellular alkalosis. In the present study, monensin was found to cause comparable intracellular alkalosis in megakaryocytes and platelets but exerted strikingly divergent effects on CCE: monensin markedly augmented CCE in platelets, while CCE in CMK cells was barely augmented by monensin, and CCE in MEG-01 cells remained completely unaffected. Experiments using a classical CCE inhibitor, SKF-96365, showed that Ca2+ entry measured in the presence of monensin displayed typical sensitivity to this blocking agent, ruling out the possibility of involvement of Ca2+ entry pathways other than CCE by monensin. Moreover, experiments using propionate to specifically antagonize intracellular alkalosis showed that the observed promotion of CCE was indeed based on pHi regulation. Monensin-induced augmentation of CCE in CMK cells and platelets was abolished in the presence of propionate, which has been shown to induce cytoplasmic acidification [24] and to cancel monensin-induced increase in cytoplasmic pH [15]. Thus, the observed modulation of CCE by monensin reflects pHi-dependent promotion of CCE channel activity. Importantly, pHi sensitivity of CCE was much smaller in CMK cells than in platelets. Moreover, pHi sensitivity was lacking for the CCE pathway expressed in MEG-01 cells. These findings prompted us to hypothesize that the molecular composition of the CCE channel complex in megakaryocytes undergoes a significant molecular rearrangement before thrombopoiesis, resulting in altered pHi sensitivity of CCE.

In an attempt to test for a role of TRPC proteins in the observed difference in pHi sensitivity of CCE, we investigated the expression of TRPC proteins, which are considered to be putative components of CCE channel complexes. TRPC expression was different in megakaryocytes and platelets in that TRPC4α was highly expressed in platelets but barely detectable in CMK and apparently lacking in MEG-01 cells. These results opened the view that TRPC4 expression may be correlated with pHi sensitivity. As recent experiments have questioned the reliability of the commercially available anti-TRPC4 antibodies [25], at least for mouse tissues, we performed control experiments with the HEK293 expression system. The used TRPC4 antibody recognized an about 100-kDa protein only in TRPC4 overexpressing cells but not in wild-type HEK293 cells (see Figures 5 and 6). It is notable that expression levels of TRPC1 were much higher in CMK and MEG-01 cells than in platelets. In a previous study in which mRNA expression of TRPCs in megakaryocytes was analyzed by PCR amplification, TRPC4 expression was detected in megakaryocytes but not in CD34-positive stem cells, while both megakaryocytes and undifferentiated stem cells showed expression of TRPC1 [6]. However, mRNA expression characterized by PCR amplification does not necessarily reflect protein expression. The results of the present study have provided evidence for substantial differences in TRPC protein expression patterns of platelets and their precursor cells. The TRPC4α splice variant was found to be expressed at high levels in platelets but was not expressed in megakaryocytes. This observation urged us to speculate that expression of TRPC, particularly that of TRPC4α, might be involved in the observed differences in pHi sensitivity to CCE channels. Attempts to evaluate the role of TRPC4α by overexpression in megakaryocytes were not successful due to extremely low transfection efficiency. Thus, we set out to test this hypothesis in a HEK293 expression system. This system was considered suitable since HEK293 cells express endogenous TRPC at a relatively low level and are known to be an efficient expression system for these ion channel proteins. Moreover, CCE of wild-type HEK293 cells was found to be insensitive to intracellular alkalosis. Monensin promoted CCE in TRPC4α-transfected HEK293 cells, suggesting that TRPC4α overexpression determines CCE up-regulation by cytoplasmic alkalosis. TRPC1 has been demonstrated to contribute to CC [[1], [26]] and to form heteromultimers with TRPC4 [27]. For platelets, TRPC1, TRPC4 and TRPC5 have recently been demonstrated to form a heteromultimer that resides in cholesterol-rich membrane domains [28]. Interestingly, TRPC1, as one potential interaction partner of TRPC4, is indeed expressed in megakaryocytes and platelets as well as in HEK293 cells [2]. Thus, it is tempting to speculate that heteromeric complexes of TRPC1/TRPC4 with different subunit stoichiometry may be the basis of the observed divergent pHi sensitivity of CCE pathways. The significance of association of TRPC1 with TRPC4 in platelets and their precursor cells awaits to be clarified.

In conclusion, we demonstrated that regulation of CCE channels of platelets and megakaryocytes displays divergent sensitivity to cytoplasmic alkalosis and suggest TRPC4α as a key component of platelet CCE channels and as a determinant of pHi sensitivity of this Ca2+ entry pathway.

This work was supported by grants for scientific research from the Grant of the Yamagata University 21st Century COE program and from P18280 (FWF to K.G.). The authors wish to thank Dr. M.X. Zhu for providing murine TRPC4α cDNA and T4-60 cells and Dr. V. Flockerzi for providing anti-TRPC4 as well as Mrs. R. Schmidt for her skillful assistance.