Reliable measurement of free Ca

Autor: Ana M, Rossi, Colin W, Taylor
Rok vydání: 2020
Předmět:
IP3
inositol 1
4
5-trisphosphate
[Ca2+]m
medium free [Ca2+]
KD
equilibrium dissociation constant
CLM-H
CLM supplemented with HEDTA
Endoplasmic Reticulum
IP3R
IP3 receptor
Antibodies
Fluorescence
Article
Inositol 1
4
5-trisphosphate receptor
Cell Line
ER
endoplasmic reticulum
Animals
Humans
GECI
genetically-encoded Ca2+ indicator
Ca2+ signal
RFU
relative fluorescence unit
AM
acetoxymethyl
ComputingMethodologies_COMPUTERGRAPHICS
CLM
cytosol-like medium
Aniline Compounds
Esterification
QAb
quenching antibody
CPA
cyclopiazonic acid
DT40-IP3R1
DT40 cells expressing rat type 1 IP3R
G-CEPIA1er
green-CEPIA indicator targeted to ER lumen
h
Hill coefficient
EC50
half-maximally effective concentration
Ca2+ indicator
Xanthenes
SERCA
sarcoplasmic/endoplasmic reticulum Ca2+-ATPase
Calcium
Acetoxymethyl ester
Chickens
Zdroj: Cell Calcium
ISSN: 1532-1991
Popis: Graphical abstract
Highlights • Mag-Fluo-4 loaded into ER by incubation of cells with Mag-Fluo-4 AM reliably reports ER free [Ca2+]. • However, the Ca2+ affinity of Mag-Fluo-4 determined in vitro appears too high to allow measurements of ER luminal [Ca2+]. • We use an antibody (QAb) to quench the fluorescence of leaked indicator. • Using QAb, we show that indicator within the ER has low affinity and sensitivity across a wide range of [Ca2+]. • Incomplete de-esterification of compartmentalized indicator allows it to effectively report ER free [Ca2+].
Synthetic Ca2+ indicators are widely used to report changes in free [Ca2+], usually in the cytosol but also within organelles. Mag-Fluo-4, loaded into the endoplasmic reticulum (ER) by incubating cells with Mag-Fluo-4 AM, has been used to measure changes in free [Ca2+] within the ER, where the free [Ca2+] is estimated to be between 100 μM and 1 mM. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free [Ca2+] within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for Ca2+ measured in vitro (KDCa ∼22 μM). Using an antibody to quench the fluorescence of indicator that leaked from the ER, we established that the affinity of Mag-Fluo-4 within the ER is much lower (KDCa ∼1 mM) than that measured in vitro. We show that partially de-esterified Mag-Fluo-4 has reduced affinity for Ca2+, suggesting that incomplete de-esterification of Mag-Fluo-4 AM within the ER provides indicators with affinities for Ca2+ that are both appropriate for the ER lumen and capable of reporting a wide range of free [Ca2+].
Databáze: OpenAIRE
Externí odkaz: