Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor

Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent junctional sarcoplasmic/encloplasmic reticulum (jS/ER) constitute specialized Ca2+ signaling complexes in many cell types. We examined the possibility that some Ca2+ signals arising in the junctional space between the PM and jS/ER may represent cross-talk between the PM and jS/ER. The Ca2+ sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the alpha 1 or alpha 2 isoform of the Na+ pump catalytic (a) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that alpha 2(f)GCaMP2, like native Na+ pumps with alpha 2-subunits, sorted to PM domains that colocalized with subjacent S/ER; alpha 1(f)GCaMP2, like Na+ pumps with alpha 1-subunits, was more uniformly distributed. The GCaMP2 moieties in both constructs were tethered just beneath the PM. Both constructs detected global Ca2+ signals evoked by serotonin (in arterial smooth muscle cells) and ATP, and by store-operated Ca2+ channel-mediated Ca2+ entry after S/ER unloading with cyclopiazonic acid (in Ca2+-free medium). When cytosolic Ca2+ diffusion was markedly restricted with EGTA, however, only alpha 2(f)GCaMP2 detected the local, store-operated Ca2+ channel-mediated Ca2+ entry signal. Thus, alpha 1 Na+ pumps are apparently excluded from the PM microdomains occupied by alpha 2 Na2+ pumps. The jS/ER and adjacent PM may communicate by Ca2+ signals that are confined to the tiny junctional space between the two membranes. Similar methods may be useful for studying localized Ca2+ signals in other subPM microdomains and signals associated with other organelles.