mOrange2, a Genetically Encoded, pH Sensitive Fluorescent Protein, is an Alternative to BCECF-AM to Measure Intracellular pH to Determine NHE3 and DRA Activity.
Intracellular pH; mORANGE2; NHE3; DRA
Journal
Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology
ISSN: 1421-9778
Titre abrégé: Cell Physiol Biochem
Pays: Germany
ID NLM: 9113221
Informations de publication
Date de publication:
26 Jan 2022
26 Jan 2022
Historique:
accepted:
08
01
2022
entrez:
25
1
2022
pubmed:
26
1
2022
medline:
8
2
2022
Statut:
ppublish
Résumé
NHE3 (Na The use of a genetically modified fluorescent protein, mOrange2 was explored as an intracellular pH sensor protein to measure exchange activity of NHE3 and DRA. The model used was FRT cells stably expressing NHE3 or DRA with intracellular pH measured by changes of mOrange2 fluorescence intensity. Intracellular pH was monitored using a) Isolated single clones of FRT/mOrange2/HA-NHE3 cells studied in a confocal microscope with time-lapse live cell imaging under basal conditions and when NHE3 was inhibited by exposure to forskolin and stimulated by dexamethasone, b) coverslip grown FRT/mOrange2 cells expressing NHE3 or DRA using a computerized fluorometer with a perfused cuvette with standardization of the mOrange2 absorption and emission signal using K A similar rate of intracellular alkalization by Na This study demonstrates that mOrange2 protein can be an effective alternate to BCECF-AM in measuring intracellular pH (preferred setting Ex520nm, Em 563nm) as affected by NHE3 and DRA activity, with the advantage, compared to AM ester dyes, that genetic expression can provide uniform expression of the pH sensor.
Sections du résumé
BACKGROUND/AIMS
OBJECTIVE
NHE3 (Na
METHODS
METHODS
The use of a genetically modified fluorescent protein, mOrange2 was explored as an intracellular pH sensor protein to measure exchange activity of NHE3 and DRA. The model used was FRT cells stably expressing NHE3 or DRA with intracellular pH measured by changes of mOrange2 fluorescence intensity. Intracellular pH was monitored using a) Isolated single clones of FRT/mOrange2/HA-NHE3 cells studied in a confocal microscope with time-lapse live cell imaging under basal conditions and when NHE3 was inhibited by exposure to forskolin and stimulated by dexamethasone, b) coverslip grown FRT/mOrange2 cells expressing NHE3 or DRA using a computerized fluorometer with a perfused cuvette with standardization of the mOrange2 absorption and emission signal using K
RESULTS
RESULTS
A similar rate of intracellular alkalization by Na
CONCLUSION
CONCLUSIONS
This study demonstrates that mOrange2 protein can be an effective alternate to BCECF-AM in measuring intracellular pH (preferred setting Ex520nm, Em 563nm) as affected by NHE3 and DRA activity, with the advantage, compared to AM ester dyes, that genetic expression can provide uniform expression of the pH sensor.
Substances chimiques
Antiporters
0
Fluoresceins
0
Luminescent Proteins
0
Slc26a3 protein, rat
0
Slc9a3 protein, rat
0
Sodium-Hydrogen Exchanger 3
0
Sulfate Transporters
0
fluorescent protein 583
0
2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein
85138-49-4
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
39-49Subventions
Organisme : NIH NIDDK
ID : RO1-DK26523, RO1-DK61765, PO1-DK64388, P30-DK89502
Pays : United States
Organisme : NIH NIAID
ID : P01-AI125181
Pays : United States
Organisme : NIH SIG
ID : 1S10OD025244
Pays : United States
Informations de copyright
© Copyright by the Author(s). Published by Cell Physiol Biochem Press.
Déclaration de conflit d'intérêts
The authors declare that no conflict of interests exists.