A role for SNAP-25 but not VAMPs in store-mediated Ca2+ entry in human platelets

Abstract

Store-mediated Ca2+ entry (SMCE) is a major mechanism for Ca2+ influx in non-excitable cells. Recently, a conformational coupling mechanism allowing coupling between transient receptor potential channels (TRPCs) and IP3 receptors has been proposed to activate SMCE. Here we have investigated the role of two soluble N-ethylmaleimide-sensitive-factor attachment protein receptors (SNAREs), which are involved in membrane trafficking and docking, in SMCE in human platelets. We found that the synaptosome-associated protein (SNAP-25) and the vesicle-associated membrane proteins (VAMP) coimmunoprecipitate with hTRPC1 in platelets. Treatment with botulinum toxin (BoNT) E or with tetanus toxin (TeTx), induced cleavage and inactivation of SNAP-25 and VAMPs, respectively. BoNTs significantly reduced thapsigargin- (TG) and agonist-evoked SMCE. Treatment with BoNTs once SMCE had been activated decreased Ca2+ entry, indicating that SNAP-25 is required for the activation and maintenance of SMCE. In contrast, treatment with TeTx had no effect on either the activation or the maintenance of SMCE in platelets. Finally, treatment with BoNT E impaired the coupling between naturally expressed hTRPC1 and IP3 receptor type II in platelets. From these findings we suggest SNAP-25 has a role in SMCE in human platelets.

Figure 1. Coimmunoprecipitation of SNAP-25 and VAMPs with hTRP1 in human platelets

Resting platelets and platelets treated with 1 μm TG and 50 nm Iono were lysed and whole cell lysates were immunoprecipitated with anti-hTrp1 antibody. Proteins were separated by 15% SDS-PAGE and transferred to a nitrocellulose membrane for subsequent analysis. Membranes were either stained using Ponceau stain (top left panel) or analysed by Western blotting using either anti-SNAP-25 antibody (top middle panel), anti-VAMP antibody (top right panel) or anti-hTRPC1 antibody (bottom panels) as described in Methods. Positions of molecular mass markers are shown on the left. Arrows indicate the position of immunoreactive bands which shown increased detection or are only detected in TG + Iono treated cells. These results are representative of four independent experiments performed in different donors.

Figure 2. Effect of botulinum toxin E and tetanus toxin on SNAP-25 and VAMP cleaving and surface location of hTRPC1

A, human platelets were treated for 1 h at 37°C with 300 nm BoNT E or TeTx and lysed. Whole cell lysate proteins were separated by 15% SDS-PAGE and transferred to a nitrocellulose membrane for subsequent analysis. Membranes were analysed by Western blotting using either anti-SNAP-25 antibody (upper panel) or anti-VAMP antibody (lower panel) as described in Methods. Positions of molecular mass markers are shown on the right. These results are representative of four independent experiments performed in different donors. B, human platelets were incubated for 1 h at 37°C in the presence of 300 nm BoNT E (c), 300 nm TeTx (d) or the vehicle (b) and then fixed. The platelet suspension was then incubated with 1 μg ml⁻¹ anti-hTrp1 antibody for 1 h followed by incubation with 0.02 μg ml⁻¹ FITC-conjugated donkey anti-rabbit IgG antibody for a further 1 h. In (a) fixed platelets were incubated with 0.02 μg ml⁻¹ FITC-conjugated anti-rabbit IgG antibody for 1 h. Values are mean ±s.e.m. of four independent experiments performed in different donors.

Figure 3. Effect of botulinum toxin E on the activation of TG-evoked Ca²⁺ entry in human platelets

A, resting fura-2-loaded platelets (top and bottom panels) or platelets incubated for 1 h at 37°C (middle panel) were stimulated with TG (1 μm; top panel) or the vehicle (HBS; middle and bottom panels), as indicated, in a Ca²⁺-free medium (200 μm EGTA was added). CaCl₂ (final concentration 300 μm) was added to the medium 3 min later. Elevations in [Ca²⁺]_i were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca²⁺]_i. B, platelets were incubated for 1 h with several concentrations of BoNT E (30–300 nm) at 37°C and then stimulated with TG (1 μm) in a Ca²⁺-free medium (200 μm EGTA was added). CaCl₂ (final concentration 300 μm) was added to the medium 3 min later to initiate Ca²⁺ entry. Data are presented as mean values of 4–12 experiments. Inset: bar graph showing the Ca²⁺ entry under different experimental conditions expressed as the percentage of control. Values are mean ±s.e.m. of 4–12 experiments performed using six different donors. *P < 0.01 compared to control.

Figure 4. Effect of botulinum toxin E on the activation of thrombin-induced Ca²⁺ entry in human platelets

Fura-2-loaded human platelets were incubated for 1 h in the presence of 300 nm BoNT E at 37°C and then stimulated with thrombin (1 U ml⁻¹), as indicated, in a Ca²⁺-free medium (200 μm EGTA was added). CaCl₂ (final concentration 300 μm) was added to the medium at the time indicated to initiate Ca²⁺ entry. Elevations in [Ca²⁺]_i were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca²⁺]_i. Data are presented as mean ±s.e.m. of five experiments performed using five different donors.

Figure 5. Effect of botulinum toxin E on the maintenance of TG-induced Ca²⁺ entry in human platelets

Fura-2-loaded human platelets were suspended in a Ca²⁺-free medium (200 μm EGTA added). Cells were then stimulated with TG (1 μm) and 3 min later 300 nm BoNT E or the vehicle (HBS; control) was added. CaCl₂ (final concentration 300 μm) was added to the medium 1 h after the addition of the toxin or the vehicle to initiate Ca²⁺ entry. Data are presented as mean ±s.e.m. of five experiments performed in five different donors.

Figure 6. Effect of tetanus toxin on the activation of TG- or thrombin-induced Ca²⁺ entry in human platelets

Fura-2-loaded platelets were incubated for 1 h in the presence of TeTx (300 nm) at 37°C and then stimulated with TG (1 μm) or thrombin (1 U ml⁻¹), as indicated, in a Ca²⁺-free medium (200 μm EGTA was added). CaCl₂ (final concentration 300 μm) was added to the medium at the time indicated to initiate Ca²⁺ entry. Elevations in [Ca²⁺]_i were monitored using the 340/380 nm ratio and traces were calibrated in terms of [Ca²⁺]_i. Data are presented as mean ±s.e.m. of 17 experiments performed in 10 different donors.

Figure 7. BoNT E reduces the coupling between hTRPC1 and the type II IP₃R in store-depleted human platelets

Human platelets were pre-incubated in the absence or presence of BoNT E (300 nm) for 1 h at 37°C. Cells were then stimulated with TG (1 μm) plus Iono (50 nm) and then lysed. A, whole cell lysates were immunoprecipitated (i.p.) with anti-hTRPC1 antibody and analysed by Western blotting (WB) using anti-IP₃R type II antibody (anti-IP₃R II; top panel) or anti-hTRPC1 antibody (bottom panel). B, whole cell lysates were immunoprecipitated (i.p.) with anti-IP₃R II antibody and analysed by Western blotting (WB) using anti-hTRPC1 antibody (top panel) or anti-IP₃R II antibody (bottom panel). Positions of molecular mass markers are shown on the right. These results are representative of three independent experiments performed using different donors.