Abstract
SummaryIn polarized cells or cells with complex geometry, clustering of plasma-membrane (PM) ion channels is an effective mechanism for eliciting spatially restricted signals. However, channel clustering is also seen in cells with relatively simple topology, suggesting it fulfills a more fundamental role in cell biology than simply orchestrating compartmentalized responses. Here, we have compared the ability of store-operated Ca2+ release-activated Ca2+ (CRAC) channels confined to PM microdomains with a similar number of dispersed CRAC channels to activate transcription factors, which subsequently increase nuclear gene expression. For similar levels of channel activity, we find that channel confinement is considerably more effective in stimulating gene expression. Our results identify a long-range signaling advantage to the tight evolutionary conservation of channel clustering and reveal that CRAC channel aggregation increases the strength, fidelity, and reliability of the general process of excitation-transcription coupling.
Highlights
Clustering of ion channels is commonly observed in the cell-surface membrane (Hille, 2002)
By comparing a Ca2+ release-activated Ca2+ (CRAC) channel mutant that is active in the absence of STIM1 and does not aggregate at ER-plasma membrane (PM) junctions with channels that re-localize to these sites, we have examined whether CRAC channel proximity imparts a signaling advantage to excitation-transcription coupling
CRAC Channels Activate Both c-fos and NFAT Transcription Factors Ca2+ microdomains near open CRAC channels in RBL-1 cells activate two transcription factors: c-fos, through enhanced protein expression (Ng et al, 2009), and cytoplasmic NFAT, which can be followed functionally through an NFAT-dependent GFP reporter gene (Kar et al, 2011). To confirm that both responses depended on Orai1, we first used a small interfering RNA knockdown approach to reduce expression of channel protein
Summary
Clustering of ion channels is commonly observed in the cell-surface membrane (Hille, 2002). Multimeric STIM complexes aggregate in regions of peripheral ER, located only $10–20 nm from the PM, forming clusters or ‘‘puncta’’ when fluorescently tagged STIM1 is expressed (Wu et al, 2006). At these sites, STIM activates PM Orai proteins (Feske et al, 2006), identified through site-directed mutagenesis as the pore-forming subunits of the CRAC channel (Prakriya et al, 2006; Vig et al, 2006; Yeromin et al, 2006). STIM traps and gates open Orai channels through binding of its CRAC activation domain or STIM1 Orai activation region to intracellular C- and N-terminal sites on the Orai channel (McNally et al, 2013; Park et al, 2009; Yuan et al, 2009), which leads to a conformational change at the external entrance to the pore (Gudlur et al, 2014)
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