Modulation of two functionally distinct Ca2+ stores in astrocytes: Role of the plasmalemmal Na/Ca exchanger

Golovina, Vera A.; Bambrick, Linda L.; Yarowsky, Paul; Krueger, Bruce K.; Blaustein, Mordecai P.

doi:10.1002/(sici)1098-1136(199604)16:4<296::aid-glia2>3.0.co;2-z

Cited by 76 publications

(17 citation statements)

References 42 publications

(71 reference statements)

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“…ASMCs were prepared from mesenteric arteries of male Sprague-Dawley rats (100-150 g) (41). Mouse cortical protoplasmic type-1 astrocytes were prepared from fetuses on embryonic day 18 or 19 (42). ASMCs and astrocytes were plated onto 25-mm coverslips and were grown in primary culture for 4-6 and 7-9 days, respectively.…”

Section: Methodsmentioning

confidence: 99%

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

Lee¹,

Song²,

Nakai

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Accumulating evidence indicates that plasma membrane (PM) microdomains and the subjacent ''junctional'' sarcoplasmic͞endo-plasmic reticulum (jS͞ER) 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 jS͞ER may represent cross-talk between the PM and jS͞ER. The Ca 2؉ sensor protein, GCaMP2, was targeted to different PM domains by constructing genes for fusion proteins with either the ␣1 or ␣2 isoform of the Na ؉ pump catalytic (␣) subunit. These fusion proteins were expressed in primary cultured mouse brain astrocytes and arterial smooth muscle cells. Immunocytochemistry demonstrated that ␣2(f)GCaMP2, like native Na ؉ pumps with ␣2-subunits, sorted to PM domains that colocalized with subjacent S͞ER; ␣1(f)GCaMP2, like Na ؉ pumps with ␣1-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 S͞ER unloading with cyclopiazonic acid (in Ca 2؉ -free medium). When cytosolic Ca 2؉ diffusion was markedly restricted with EGTA, however, only ␣2(f)GCaMP2 detected the local, storeoperated Ca 2؉ channel-mediated Ca 2؉ entry signal. Thus, ␣1 Na ؉ pumps are apparently excluded from the PM microdomains occupied by ␣2 Na 2؉ pumps. The jS͞ER 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.arterial smooth muscle ͉ astrocytes ͉ Na ϩ pump ͉ PLasmERosome ͉ store-operated Ca 2ϩ entry

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Section: Methodsmentioning

confidence: 99%

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

Lee¹,

Song²,

Nakai

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Standard PCR methods were used for genotyping genomic DNA from fetus tails. Cortical protoplasmic type-1 astrocytes were prepared from WT and KO mouse fetuses on days 18 or 19 (E18 -19) as described (14,26). The cells were plated onto poly-L-lysine-coated 25-mm glass coverslips (ϳ50,000 cells/coverslip) for immunocytochemistry and Ca 2ϩ imaging, or onto 10-cm Petri dishes for immunoblots.…”

Section: Methodsmentioning

confidence: 99%

An N-terminal Sequence Targets and Tethers Na+ Pump α2 Subunits to Specialized Plasma Membrane Microdomains

Song

Lee

Kinsey

et al. 2006

Journal of Biological Chemistry

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Sodium pumps (␣␤ dimers) with the ␣1 isoform of the catalytic (␣) subunit are expressed in all cells. Additionally, most cells express Na ؉ pumps with a second ␣ isoform. For example, astrocytes and arterial myocytes also express Na ؉ pumps with the ␣2 isoform. The ␣2 pumps localize to plasma membrane (PM) microdomains overlying "junctional" sarco-/endoplasmic reticulum (S/ER), but the ␣1 pumps are more uniformly distributed. To study ␣2 targeting, we expressed ␣1/␣2 and ␣2/␣1 chimeras and 1-90 and 1-120 amino acid N-terminal peptides in primary cultured mouse astrocytes. Immunocytochemistry revealed that ␣2/␣1 (but not ␣1/␣2) chimeras markedly reduced native ␣2 (i.e. were "dominant negatives"). N-terminal (1-120 and 1-90 amino acids) ␣2 (and ␣3), but not ␣1 peptides also targeted to the PM-S/ER junctions and were dominant negative for native ␣2 in astrocytes and arterial myocytes. Thus ␣2 and ␣3 have the same targeting sequence. Ca 2؉ (fura-2) signals in astrocytes expressing the 1-90 ␣2 peptide were comparable to signals in cells from ␣2 null mutants (i.e. functionally dominant negative): 1 M ATP-evoked Ca 2؉ transients were augmented, and 100 nM ouabain-induced amplification was abolished. Amino acid substitutions in the 1-120 ␣1 and ␣2 constructs, and in full-length ␣1, revealed that Leu-27 and Ala-35 are essential for targeting/tethering the constructs to PM-S/ER junctions.The sodium pump (Na ϩ , K ϩ -ATPase) plays a critical role in maintaining a low cytosolic Na ϩ concentration ([Na ϩ ] CYT ) and a large Na ϩ electrochemical gradient across the plasma membrane (PM) 3 in nearly all animal cells (1, 2). This Na ϩ gradient not only enables the generation of Na ϩ -dependent action potentials in excitable cells, but it also drives many solute transport processes, including the Na ϩ /Ca 2ϩ exchanger that modulates Ca 2ϩ signaling. Functional Na ϩ pumps are ␣␤ dimers (2-4). The catalytic (␣) subunits contain the Na ϩ , K ϩ , ATP, and cardiotonic steroid binding sites (2-4). The ␣ subunits (molecular mass of Ϸ110 kDa) have 10 membrane-spanning helices (M1-M10), cytoplasmic N-and C-terminal segments, and a large cytoplasmic loop between M4 and M5 ( Fig. 1A) (2). The ␣ subunit tertiary structure has been deduced by homology modeling (5) based on the known structure of the sarco-/endoplasmic reticulum (S/ER) Ca 2ϩ pump, SERCA (see Fig. 1B) (6). There are four isoforms of the ␣ subunit, ␣1-␣4 (2). Most cells express two different ␣ isoforms, ␣1 and either ␣2 or ␣3, but sperm express ␣1 and ␣4 (7). For example, astrocytes express ␣1 and ␣2, and most neurons express ␣1 and ␣3 (8 -10). In rodents, ␣1 has an unusually low affinity for ouabain, whereas the high ouabain affinity binding sites in ␣2 and ␣3 have been retained through evolution (2, 11). Na ϩ pumps with ␣1 subunits distribute differently from those with ␣2 subunits in astrocytes or ␣3 in neurons (9, 13), and they apparently serve different functions (14). In particular, Na ϩ pumps with ␣2 or ␣3 subunits cluster, along with the Na ϩ /Ca 2ϩ exchanger, in PM micr...

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“…NCX is largely distributed close to the sarcoplasmic reticulum / endoplasmic reticulum (ER) Ca 2+ stores in smooth muscle (18, 19) and astrocytes (20). In addition, Golovina et al (21) reported that Ca 2+ entry across plasmalemma via NCX increases the Ca 2+ level of ER stores in astrocytes, and Chernaya et al (22) reported that Ca 2+ release from intracellular stores induces regulatory activation of NCX activity in transfected Chinese hamster ovary cells expressing NCX. These findings suggest that NCX plays a role in regulation of the intracellular Ca 2+ stores in the ER.…”

Section: No-induced Cell Toxicitymentioning

confidence: 99%

Topics on the Na+/Ca2+ Exchanger: Responses of Na+/Ca2+ Exchanger to Interferon-γ and Nitric Oxide in Cultured Microglia

et al. 2006

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Abstract. The Na + / Ca 2+ exchanger (NCX) plays a role in regulation of intracellular Ca 2+ levels, but little is known about the functional role of NCX in microglia. To clarify the role of NCX in microglia, we studied the responses of NCX to pathological conditions such as interferon-γ or nitric oxide (NO) exposure. Treatment with interferon-γ caused a biphasic increase in NCX activity. The delayed increase in NCX activity was accompanied by increases in the mRNA and protein levels. Pharmacological studies show that protein kinase C and tyrosine kinase are involved in the transient and delayed increases in NCX activity, and the extracellular signalregulated protein kinase is involved in the delayed increase in NCX activity. On the other hand, NO causes apoptotic cell death in cultured microglia. We observed, using the specific NCX inhibitor SEA0400, that NO activates NCX activity and NCX is involved in NO-induced depletion of Ca 2+ in the endoplasmic reticulum (ER), leading to ER stress. These results suggest that NCX is involved in the regulation of Ca 2+ levels in the ER. The responses of NCX to interferon-γ and NO implies that NCX plays a key role in microglial function.

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Modulation of two functionally distinct Ca2+ stores in astrocytes: Role of the plasmalemmal Na/Ca exchanger

Cited by 76 publications

References 42 publications

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

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

An N-terminal Sequence Targets and Tethers Na+ Pump α2 Subunits to Specialized Plasma Membrane Microdomains

Topics on the Na+/Ca2+ Exchanger: Responses of Na+/Ca2+ Exchanger to Interferon-γ and Nitric Oxide in Cultured Microglia

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Modulation of two functionally distinct Ca2+ stores in astrocytes: Role of the plasmalemmal Na/Ca exchanger

Cited by 76 publications

References 42 publications

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

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

An N-terminal Sequence Targets and Tethers Na+ Pump α2 Subunits to Specialized Plasma Membrane Microdomains

Topics on the Na+/Ca2+ Exchanger: Responses of Na+/Ca2+ Exchanger to Interferon-γ and Nitric Oxide in Cultured Microglia

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

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