Heat shock protein 90 inhibitors reduce trafficking of ATP-gated P2X1 receptors and human platelet responsiveness

Abstract

We have used selective inhibitors to determine whether the molecular chaperone heat shock protein 90 (HSP90) has an effect on both recombinant and native human P2X1 receptors. P2X1 receptor currents in HEK293 cells were reduced by ∼70-85% by the selective HSP90 inhibitor geldanamycin (2 μM, 20 min). This was associated with a speeding in the time course of desensitization as well as a reduction in cell surface expression. Imaging in real time of photoactivatable GFP-tagged P2X receptors showed that they are highly mobile. Geldanamycin almost abolished this movement for P2X1 receptors but had no effect on P2X2 receptor trafficking. P2X1/2 receptor chimeras showed that the intracellular N and C termini were involved in geldanamycin sensitivity. Geldanamycin also inhibited native P2X1 receptor-mediated responses. Platelet P2X1 receptors play an important role in hemostasis, contribute to amplification of signaling to a range of stimuli including collagen, and are novel targets for antithrombotic therapies. Platelet P2X1 receptor-, but not P2Y1 receptor-, mediated increases in intracellular calcium were reduced by 40-45% following HSP90 inhibition with geldanamycin or radicicol. Collagen stimulation leads to ATP release from platelets, and calcium increases to low doses of collagen were also reduced by ∼40% by the HSP90 inhibitors consistent with an effect on P2X1 receptors. These studies suggest that HSP90 inhibitors may be as effective as selective antagonists in regulating platelet P2X1 receptors, and their potential effects on hemostasis should be considered in clinical studies.

FIGURE 1.

Effects of the HSP90 inhibitor geldanamycin on P2X1 receptor-mediated currents and receptor expression. A, α,β-meATP-evoked (10 μm, application indicated by bar) reproducible inward currents in the permeabilized patch recording configuration from human P2X1 receptors expressed in HEK293 cells (left). The HSP90 inhibitor geldanamycin (2 μm) reduced the peak amplitude of the currents with maximal effect after 15 min. B, mean normalized data of P2X1 receptor-mediated currents in control conditions and in the presence of geldanamycin (n = 5–7). Inset, time course of the P2X1 receptor current in control following geldanamycin. Traces have been normalized to the peak current amplitude to show the increased rate of decay with geldanamycin. Values are shown as means ± S.E. (error bars). C, Western blotting of P2X1 receptors expressed in HEK293 cells. Results show that 2 μm geldanamycin (30 min) has no effect on total levels of the receptor, but reduced surface expression of the P2X1 receptor assessed by cell surface biotinylation results in reduction of P2X1 receptors on membrane surface (by 36.3 ± 6.7%, n = 8).

FIGURE 2.

HSP90 promotes trafficking of P2X1 but not P2X2 receptors. A, representative snapshots of HEK293 cells expressing P2X1 receptors C-terminally tagged with PAGFP. Fluorescent images represent the cell in control conditions or treated with 2 μm geldanamycin (>30 min) before P2X1-PAGFP activation (dotted circle indicates region to be photoactivated), immediately after activation (indicated by star), and 250 s after photoactivation. Right panel, brightfield images of the cell. Under control conditions the P2X1-PAGFP fluorescence moves away from the site of illumination; however, following geldanamycin treatment the P2X1-PAGFP remains predominantly within the area of photoillumination. B, averaged changes in fluorescence in control cells and cells treated with geldanamycin as fraction of maximal fluorescence observed immediately after activation of P2X1-PAGFP (indicated by star) or P2X2-PAGFP, within the cell; size of activated area, 6 μm² (n = 5–9). Images were taken on an Olympus inverted microscope with a confocal laser scanning module (OlympusFluoView1000). Cells were imaged with a 60× oil immersion objective (UPLASAPO 60×, NA 1.35). Cells were bathed in standard extracellular solution at room temperature. Fluorescent intensities of regions of interest were obtained using FluoView software. Values are shown as means ± S.E.

FIGURE 3.

Effects of the HSP90 inhibitor geldanamycin on P2X1 and P2X2 receptor currents and identification of the region of P2X1 receptor responsible for sensitivity to HSP90 inhibition. A, representative traces of P2X1 and P2X2 currents in control conditions and after treatment with 2 μm geldanamycin (>30 min). Bar represents period of agonist application, 10 μm α,β-meATP and 100 μm ATP for P2X1 and P2X2, respectively. B, schematic representation of P2X1/2 chimeras swapping portions of the intracellular N terminus (upper panel) and transmembrane segments and C terminus (lower panels). The histogram shows the effects of 2 μm geldanamycin treatment (30 min) treatment on currents mediated by P2X1, P2X2, and P2X1/P2X2 chimeric receptors expressed as a fraction of the response to 100 μm ATP in control nontreated cells. Values are shown as means ± S.E. (error bars; n = 6–14). **, p < 0.01; ***, p < 0.001 significantly different from P2X1.

FIGURE 4.

HSP90 inhibitors reduced P2X1-dependent calcium transients in platelets. Washed platelets loaded with fura-2 were treated with 0.2% dimethyl sulfoxide, 2 μm geldanamycin, or 1 μg ml⁻¹ radicicol for 20 min prior to stimulation with 1 μm α,β-meATP or 1 μm ADP; in addition the bottom left panel shows effects of 1-min preincubation with geldanamycin on α,β-meATP responses. Intracellular calcium concentration was measured at 37 °C under stirring conditions in the presence of 2 mm CaCl₂. A, representative calcium traces. B, summary data displayed as a percentage of the control calcium response. Values are shown as means ± S.E. (error bars; n = 3–4; ***, p < 0.001).

FIGURE 5.

Collagen-evoked platelet calcium responses were perturbed by HSP90 inhibitors through disruption of P2X1 function. A, washed platelets loaded with fura-2 were treated with 0.2% dimethyl sulfoxide, 2 μm geldanamycin, or 1 μg ml⁻¹ radicicol for 20 min, and calcium transients measured in response to 0.5 μg ml⁻¹ collagen before and after P2X1 desensitization in the presence of 2 mm CaCl₂. B, summary data are displayed as a percentage of the control Ca²⁺ response. Values are shown as means ± S.E. (error bars) response (n = 4; *, p < 0.05; **, p < 0.01).