Local subplasma membrane Ca 2+ signals detected by a tethered Ca 2+ sensor

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Abstract
Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent “junctional” sarcoplasmic/endoplasmic reticulum (jS/ER) constitute specialized Ca 2+ signaling complexes in many cell types. We examined the possibility that some Ca 2+ signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The Ca 2+ sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the α1 or α2 isoform of the Na + pump catalytic (α) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that α2(f)GCaMP2, like native Na + pumps with α2-subunits, sorted to PM domains that colocalized with subjacent S/ER; α1(f)GCaMP2, like Na + pumps with α1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca 2+ signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca 2+ channel-mediated Ca 2+ entry after S/ER unloading with cyclopiazonic acid (in Ca 2+ -free medium). When cytosolic Ca 2+ diffusion was markedly restricted with EGTA, however, only α2(f)GCaMP2 detected the local, store-operated Ca 2+ channel-mediated Ca 2+ entry signal. Thus, α1 Na + pumps are apparently excluded from the PM microdomains occupied by α2 Na 2+ pumps. The jS/ER and adjacent PM may communicate by Ca 2+ signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca 2+ signals in other subPM microdomains and signals associated with other organelles.