A key role for reverse Na+/Ca2+ exchange influenced by the actin cytoskeleton in store-operated Ca2+ entry in human platelets: evidence against the de novo conformational coupling hypothesis.
We have previously demonstrated a role for the reorganization of the actin cytoskeleton in store-operated calcium entry (SOCE) in human platelets and interpreted this as evidence for a de novo conformational coupling step in SOCE activation involving the type II IP(3) receptor and the platelet hTRPC1-containing store-operated channel (SOC). Here, we present evidence challenging this model. The actin polymerization inhibitors cytochalasin D or latrunculin A significantly reduced Ca2+ but not Mn2+ or Na+ entry into thapsigargin (TG)-treated platelets. Jasplakinolide, which induces actin polymerization, also inhibited Ca2+ but not Mn2+ or Na+ entry. However, an anti-hTRPC1 antibody inhibited TG-evoked entry of all three cations, indicating that they all permeate an hTRPC1-containing store-operated channel (SOC). These results indicate that the reorganization of the actin cytoskeleton is not involved in SOC activation. The inhibitors of the Na+/Ca2+ exchanger (NCX), KB-R7943 or SN-6, caused a dose-dependent inhibition of Ca2+ but not Mn2+ or Na+ entry into TG-treated platelets. The effects of the NCX inhibitors were not additive with those of actin polymerization inhibitors, suggesting a common point of action. These results indicate a role for two Ca2+ permeable pathways activated following Ca2+ store depletion in human platelets: A Ca2+-permeable, hTRPC1-containing SOC and reverse Na+/Ca2+ exchange, which is activated following Na+ entry through the SOC and requires a functional actin cytoskeleton.
Harper
Alan G S
AG
Department of Physiology, Development and Neuroscience, University of Cambridge, Cambridge CB2 3EG, UK.
Sage
Stewart O
SO
D000199:Actins / Q000378:metabolism*; D001593:Benzyl Compounds / Q000494:pharmacology; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism*; D047630:Depsipeptides / Q000494:pharmacology; D006801:Humans; D008345:Manganese / Q000378:metabolism; D011188:Potassium / Q000378:metabolism / Q000494:pharmacology; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology; D013890:Thiourea / Q000031:analogs & derivatives / Q000494:pharmacology
C101670:2-(2-(4-(4-nitrobenzyloxy)phenyl)ethyl)isothiourea methanesulfonate; C487681:2-(4-(4-nitrobenzyloxy)benzyl)thiazolidine-4-carboxylic acid ethyl ester; D000199:Actins; D001593:Benzyl Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D047630:Depsipeptides; D019831:Sodium-Calcium Exchanger; D053778:Thiazolidines; C057531:jasplakinolide; D015638:Cytochalasin D; D008345:Manganese; D019284:Thapsigargin; D012964:Sodium; D013890:Thiourea; D011188:Potassium; C037067:latrunculin A; D002118:Calcium