期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2006
卷号：103
期号：35
页码：13232-13237
DOI：10.1073/pnas.0605757103
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent "junctional" sarcoplasmic/endoplasmic 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 ({alpha}) 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.
