Role of the store-operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor-stimulated calcium oscillations in human embryonic kidney cells.
We have investigated the nature of the Ca2+ entry supporting [Ca2+]i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store-operated Ca2+ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca2+]i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca2+. However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)-expressing cells and did not affect Ca2+ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca2+ extrusion with a high concentration of Gd3+, or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca2+]i oscillations, and this was reversed by use of high concentrations of Gd3+; however, knockdown of Orai1 did not affect arachidonic acid-activated entry. RNAi targeting 34 members of the transient receptor potential (TRP) channel superfamily did not reveal a role for any of these channel proteins in store-operated Ca2+ entry in HEK293 cells. These findings indicate that the Ca2+ entry supporting [Ca2+]i oscillations in HEK293 cells depends upon the Ca2+ sensor, Stim1, and calcium release-activated Ca2+ channel protein, Orai1, and provide further support for our conclusion that it is the store-operated mechanism that plays the major role in this pathway.
Wedel
Barbara
B
Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, NC 27709, USA.
Boyles
Rebecca R
RR
Putney
James W
JW
Bird
Gary S
GS
D001426:Bacterial Proteins / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007668:Kidney / Q000166:cytology*; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000502:physiology*; D016210:Methacholine Chloride / Q000494:pharmacology; D018721:Muscarinic Agonists / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000235:genetics / Q000502:physiology*; D000071740:ORAI1 Protein; D034622:RNA Interference; D011976:Receptors, Muscarinic / Q000235:genetics / Q000502:physiology*; D000071737:Stromal Interaction Molecule 1; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection
D001426:Bacterial Proteins; D015220:Calcium Channels; D004791:Enzyme Inhibitors; D008164:Luminescent Proteins; D008565:Membrane Proteins; D018721:Muscarinic Agonists; D009363:Neoplasm Proteins; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D011976:Receptors, Muscarinic; C103530:STIM1 protein, human; D000071737:Stromal Interaction Molecule 1; C054562:yellow fluorescent protein, Bacteria; D016210:Methacholine Chloride; D019284:Thapsigargin; D002118:Calcium