Stim1 and Orai1 mediate CRAC currents and store-operated calcium entry important for endothelial cell proliferation.
Recent breakthroughs in the store-operated calcium (Ca(2+)) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore forming subunit of the highly Ca(2+)-selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca(2+) release-activated Ca(2+) current (I(CRAC)), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na(+) currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I(CRAC) in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I(CRAC). Ectopic expression of Stim1 in ECs increased their I(CRAC) to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.
Cardiovascular Sciences, MC8, Albany Medical College, 47 New Scotland Avenue, MC-8, Albany, NY 12208, USA.
Iskandar F
IF
Abdullaev
Jonathan M
JM
Bisaillon
Marie
M
Potier
Jose C
JC
Gonzalez
Rajender K
RK
Motiani
Mohamed
M
Trebak
D000818:Animals; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D002455:Cell Division; D017931:DNA Primers; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D015471:Leukemia, Basophilic, Acute / Q000503:physiopathology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology; D000071740:ORAI1 Protein; D018408:Patch-Clamp Techniques; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology; D014471:Umbilical Veins
D015220:Calcium Channels; D017931:DNA Primers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; C532493:Orai1 protein, rat; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C103530:STIM1 protein, human; C518857:Stim1 protein, rat; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D019284:Thapsigargin; D002118:Calcium