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

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 alpha1 or alpha2 isoform of the Na(+) pump catalytic (alpha) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that alpha2(f)GCaMP2, like native Na(+) pumps with alpha2-subunits, sorted to PM domains that colocalized with subjacent S/ER; alpha1(f)GCaMP2, like Na(+) pumps with alpha1-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 alpha2(f)GCaMP2 detected the local, store-operated Ca(2+) channel-mediated Ca(2+) entry signal. Thus, alpha1 Na(+) pumps are apparently excluded from the PM microdomains occupied by alpha2 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.