Dr. Stewart Sage
University of Cambridge

Mechanism of agonist-evoked store-mediated calcium entry in human platelets

In many cells the release of Ca²⁺ from intracellular stores leads to the activation of Ca²⁺ entry across the plasma membrane. The mechanism of this store-mediated Ca²⁺ entry (SMCE) is not fully understood. In platelets as well as other cells evidence has been presented that the actin cytoskeleton participates in the mechanism, which may resemble secretory events (Yao et al, 1999; Patterson et al, 1999). We have recently obtained evidence for the participation of small GTP-binding proteins in SMCE in platelets, and for their role involving the actin cytoskeleton (Rosado & Sage, 2000a; Rosado, Jenner & Sage, 2000). Furthermore, we have shown that that the type II inositol 1,4,5-trisphosphate (IP₃) receptor found in the intracellular store membrane couples to the putative Ca2+ channel protein hTrp1 in the plasma membrane when the intracellular stores are depleted (Rosado & Sage, 2000b). Thus there is strong evidence in platelets that SMCE is activated by a conformational coupling event involving a secretion-like mechanism (Patterson et al., 1999). Compatible with this, we have demonstrated an requirement for basal levels of IP₃ (using Li⁺) and for functional IP3 receptors (using xestospongin C) in the activation of SMCE (Rosado & Sage, 2000b).
We have shown that the physiological agonist thrombin also evokes coupling between the type II inositol 1,4,5-trisphosphate (IP₃) receptor and hTrp1, but have found surprising results with thrombin when we distrupt the actin cytoskeleton. There is now a need to test the effects of cytoskeletal disruption on weak and intermediate strength agonists to investigate whether the results we have obtained with thrombin hold for other agonists.

Patterson, R.L., van Rossum, D.B. and Gill, D.L. (1999). Store-operated Ca2+ entry; Evidence for a secretion-like coupling mechanism. Cell 98, 487-499.
Rosado, J.A., Jenner, S. & Sage, S.O. (2000). A role for the actin cytoskeleton in the initiation and maintenance of store-mediated calcium entry in human platelets. Evidence for conformational coupling. Journal of Biological Chemistry 275, 7527-7533.
Rosado, J.A. & Sage, S.O. (2000a). Farnesylcysteine analogues inhibit store-regulated calcium entry in human platelets. Evidence for involvement of small GTP-binding proteins and actin cytoskeleton. Biochemical Journal 347, 183-192.
Rosado, J.A. & Sage, S.O. (2000b). IP3 receptors couple with hTrp1 channels when Ca2+ stores are depleted in human platelets. Biochemical Journal 350, 631-635.
Sage, S.O. (1997). The Wellcome Prize Lecture: Calcium Entry Mechanisms In Human Platelets. Experimental Physiology 82, 807-823.
Yao, Y., Ferrer-Montiel, M.M. & Tsien, R.Y. (1999). Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger. Cell 98, 475-485.