Polarized but differential localization and recruitment of STIM1, Orai1 and TRPC channels in secretory cells.
Polarized Ca(2+) signals in secretory epithelial cells are determined by compartmentalized localization of Ca(2+) signaling proteins at the apical pole. Recently the ER Ca(2+) sensor STIM1 (stromal interaction molecule 1) and the Orai channels were shown to play a critical role in store-dependent Ca(2+) influx. STIM1 also gates the transient receptor potential-canonical (TRPC) channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca(2+) influx and frequency of Ca(2+) oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% colocalization. Consequently, STIM1 shows higher colocalization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher colocalization with the tight junction protein ZO1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP(3) receptors (IP(3) Rs)/TRPCs is enhanced by cell stimulation and disrupted by 2-aminoethoxydiphenyl borate (2APB). The polarized localization and recruitment of these proteins results in preferred Ca(2+) entry that is initiated at the apical pole. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca(2+) permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca(2+) ] oscillations and thus impact critical cellular functions.
Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Jeong Hee
JH
Hong
Qin
Q
Li
Min Seuk
MS
Kim
Dong Min
DM
Shin
Stefan
S
Feske
Lutz
L
Birnbaumer
Kwong Tai
KT
Cheng
Indu S
IS
Ambudkar
Shmuel
S
Muallem
D000818:Animals; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D000071740:ORAI1 Protein; D021381:Protein Transport; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels / Q000378:metabolism*
D015220:Calcium Channels; D008562:Membrane Glycoproteins; D000071740:ORAI1 Protein; C520430:Orai1 protein, mouse; C100757:Stim1 protein, mouse; D000071737:Stromal Interaction Molecule 1; D050052:TRPC Cation Channels; C096351:transient receptor potential cation channel, subfamily C, member 1; D002118:Calcium