TY - JOUR
T1 - Local subplasma membrane Ca2+ signals detected by a tethered Ca2+ sensor
AU - Lee, Moo Yeol
AU - Song, Hong
AU - Nakai, Junichi
AU - Ohkura, Masamichi
AU - Kotlikoff, Michael I.
AU - Kinsey, Stephen P.
AU - Golovina, Vera A.
AU - Blaustein, Mordecai P.
PY - 2006/8/29
Y1 - 2006/8/29
N2 - 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 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 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 Ca 2+ diffusion was markedly restricted with EGTA, however, only α2(f)GCaMP2 detected the local, store-operated Ca2+ channel-mediated Ca2+ entry signal. Thus, α1 Na+ pumps are apparently excluded from the PM microdomains occupied by α2 Na2+ 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.
AB - 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 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 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 Ca 2+ diffusion was markedly restricted with EGTA, however, only α2(f)GCaMP2 detected the local, store-operated Ca2+ channel-mediated Ca2+ entry signal. Thus, α1 Na+ pumps are apparently excluded from the PM microdomains occupied by α2 Na2+ 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.
KW - Arterial smooth muscle
KW - Astrocytes
KW - Na pump
KW - PLasmERosome
KW - Store-operated Ca entry
UR - http://www.scopus.com/inward/record.url?scp=33748366036&partnerID=8YFLogxK
U2 - 10.1073/pnas.0605757103
DO - 10.1073/pnas.0605757103
M3 - Article
C2 - 16924099
AN - SCOPUS:33748366036
SN - 0027-8424
VL - 103
SP - 13232
EP - 13237
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 35
ER -