SERCA2b and 3 play a regulatory role in store-operated calcium entry in human platelets.
Two agonist-releasable Ca(2+)stores have been identified in human platelets differentiated by the distinct sensitivity of their SERCA isoforms to thapsigargin (TG) and 2,5-di-(tert-butyl)-1,4-hydroquinone (TBHQ). Here we have examined whether the SERCA isotypes might be involved in store-operated Ca(2+)entry (SOCE) activated by the physiological agonist thrombin in human platelets. Ca(2+)-influx evoked by thrombin (0.01 U/mL) reached a maximum after 3 min, which was consistent with the decrease in the Ca(2+)content in the stores; afterwards, the extent of SOCE decreased with no correlation with the accumulation of Ca(2+)in the stores. Inhibition of SERCA2b, by 10 nM TG, and SERCA3, with 20 microM TBHQ, individually or simultaneously, accelerated Ca(2+) store discharge and subsequently enhanced the extent of SOCE stimulated by thrombin. In addition, TG and TBHQ modified the time course of thrombin-evoked SOCE from a transient to a sustained increase in Ca(2+) influx, which reveals a negative role for SERCAs in the regulation of SOCE. This effect was consistent under conditions that inhibit Ca(2+) extrusion by PMCA or the Na(+)/Ca(2+) exchanger. Coimmunoprecipitation experiments revealed that thrombin stimulates direct interaction between SERCA2b and 3 with the hTRPC1 channel, an effect that was found to be independent of SERCA activity. In summary, our results suggest that SERCA2b and 3 modulate thrombin-stimulated SOCE probably by direct interaction with the hTRPC1 channel in human platelets.
Redondo
Pedro C
PC
Department of Physiology, Development and Neuroscience of University of Cambridge, Cambridge, CB3 9ET, United Kingdom. pcr@unex.es
Salido
Ginés M
GM
Pariente
José A
JA
Sage
Stewart O
SO
Rosado
Juan A
JA
D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology*; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D006801:Humans; D047468:Immunoprecipitation; D053496:Inositol 1,4,5-Trisphosphate Receptors / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D050052:TRPC Cation Channels / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology; D013997:Time Factors
D053496:Inositol 1,4,5-Trisphosphate Receptors; D007527:Isoenzymes; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D013917:Thrombin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506946:ATP2A2 protein, human; C506949:ATP2A3 protein, human; D002118:Calcium