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Role of the store‐operated calcium entry proteins Stim1 and Orai1 in muscarinic cholinergic receptor‐stimulated calcium oscillations in human embryonic kidney cells
Author(s) -
Wedel Barbara,
Boyles Rebecca R.,
Putney James W.,
Bird Gary S.
Publication year - 2007
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2006.125641
Subject(s) - orai1 , gene knockdown , trpc3 , hek 293 cells , microbiology and biotechnology , stim1 , transient receptor potential channel , trpc5 , trpc1 , trpc , chemistry , biology , receptor , biochemistry , endoplasmic reticulum , apoptosis
We have investigated the nature of the Ca 2+ entry supporting [Ca 2+ ] i oscillations in human embryonic kidney (HEK293) cells by examining the roles of recently described store‐operated Ca 2+ entry proteins, Stim1 and Orai1. Knockdown of Stim1 by RNA interference (RNAi) reduced the frequency of [Ca 2+ ] i oscillations in response to a low concentration of methacholine to the level seen in the absence of external Ca 2+ . However, knockdown of Stim1 did not block oscillations in canomical transient receptor potential 3 channel (TRPC3)‐expressing cells and did not affect Ca 2+ entry in response to arachidonic acid. The effects of knockdown of Stim1 could be reversed by inhibiting Ca 2+ extrusion with a high concentration of Gd 3+ , or by rescuing the knockdown by overexpression of Stim1. Similarly, knockdown of Orai1 abrogated [Ca 2+ ] i oscillations, and this was reversed by use of high concentrations of Gd 3+ ; 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 Ca 2+ entry in HEK293 cells. These findings indicate that the Ca 2+ entry supporting [Ca 2+ ] i oscillations in HEK293 cells depends upon the Ca 2+ sensor, Stim1, and calcium release‐activated Ca 2+ 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.