title: Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ 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.
authors.affiliation: Department of Physiology, University of Maryland School of Medicine, Baltimore, 21201, USA.
authors.forename: Moo Yeol
authors.initials: MY
authors.lastname: Lee
authors.forename: Hong
authors.initials: H
authors.lastname: Song
authors.forename: Junichi
authors.initials: J
authors.lastname: Nakai
authors.forename: Masamichi
authors.initials: M
authors.lastname: Ohkura
authors.forename: Michael I
authors.initials: MI
authors.lastname: Kotlikoff
authors.forename: Stephen P
authors.initials: SP
authors.lastname: Kinsey
authors.forename: Vera A
authors.initials: VA
authors.lastname: Golovina
authors.forename: Mordecai P
authors.initials: MP
authors.lastname: Blaustein
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D004533:Egtazic Acid / Q000494:pharmacology; D016257:Fura-2; D006801:Humans; D008297:Male; D051379:Mice; D015335:Molecular Probes / Q000032:analysis / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012701:Serotonin / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism
chemical_list: D015335:Molecular Probes; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D012701:Serotonin; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium; D016257:Fura-2