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Determinants of TRPV4 activity following selective activation by small molecule agonist GSK1016790A.

Jin M, Wu Z, Chen L, Jaimes J, Collins D, Walters ET, O'Neil RG - PLoS ONE (2011)

Bottom Line: Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4).Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells.TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas, United States of America.

ABSTRACT
TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, non-noxious heat and mechanical stress and some small molecule agonists (e.g. phorbol ester 4α-PDD). GSK1016790A (GSK101) is a recently discovered specific small molecule agonist of TRPV4. Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4). GSK101 (10 nM) causes a TRPV4 specific Ca(2+) influx in HeLa-TRPV4 cells, but not in control transfected cells, which can be inhibited by ruthenium red and Ca(2+)-free medium more significantly at the early stage of the activation rather than the late stage, reflecting apparent partial desensitization. Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells. Patch clamp analysis also revealed an early partial desensitization of the channel which was Ca(2+)-independent. FRET analysis of TRPV4 subunit assembly demonstrated that the GSK101-induced TRPV4 channel activation/desensitization was not due to alterations in homotetrameric channel formation on the plasma membrane. It is concluded that GSK101 specifically activates TRPV4 channels, leading to a rapid partial desensitization and downregulation of the channel expression on the plasma membrane. TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation.

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Dose-dependent activation of Ca2+ influx under GSK101 stimulation.HeLa-TRPV4 cells were stimulated with GSK101 at 1, 3, 5, 10, 20 and 100 nM. Delta [Ca2+]i was determined by calcium imaging and the dose-response curve was fitted by a sigmoidal dose-response function using SigmaPlot 10.0. The calculated EC50 = 3.3 nM.
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pone-0016713-g001: Dose-dependent activation of Ca2+ influx under GSK101 stimulation.HeLa-TRPV4 cells were stimulated with GSK101 at 1, 3, 5, 10, 20 and 100 nM. Delta [Ca2+]i was determined by calcium imaging and the dose-response curve was fitted by a sigmoidal dose-response function using SigmaPlot 10.0. The calculated EC50 = 3.3 nM.

Mentions: In HeLa-TRPV4 cells, Ca2+ influx was stimulated by GSK101 in a dose-dependent manner as shown in Figure 1. The GSK101 - Ca2+ influx relationship could be fitted by asigmoidal dose-responsefunction, which yielded an EC50 of 3.3 nM for GSK101 stimulation. GSK101 at 10 nM displayed a near maximum stimulation and was, therefore, used as the preferred concentration to activate TRPV4 channels in this study.


Determinants of TRPV4 activity following selective activation by small molecule agonist GSK1016790A.

Jin M, Wu Z, Chen L, Jaimes J, Collins D, Walters ET, O'Neil RG - PLoS ONE (2011)

Dose-dependent activation of Ca2+ influx under GSK101 stimulation.HeLa-TRPV4 cells were stimulated with GSK101 at 1, 3, 5, 10, 20 and 100 nM. Delta [Ca2+]i was determined by calcium imaging and the dose-response curve was fitted by a sigmoidal dose-response function using SigmaPlot 10.0. The calculated EC50 = 3.3 nM.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3038856&req=5

pone-0016713-g001: Dose-dependent activation of Ca2+ influx under GSK101 stimulation.HeLa-TRPV4 cells were stimulated with GSK101 at 1, 3, 5, 10, 20 and 100 nM. Delta [Ca2+]i was determined by calcium imaging and the dose-response curve was fitted by a sigmoidal dose-response function using SigmaPlot 10.0. The calculated EC50 = 3.3 nM.
Mentions: In HeLa-TRPV4 cells, Ca2+ influx was stimulated by GSK101 in a dose-dependent manner as shown in Figure 1. The GSK101 - Ca2+ influx relationship could be fitted by asigmoidal dose-responsefunction, which yielded an EC50 of 3.3 nM for GSK101 stimulation. GSK101 at 10 nM displayed a near maximum stimulation and was, therefore, used as the preferred concentration to activate TRPV4 channels in this study.

Bottom Line: Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4).Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells.TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, Texas, United States of America.

ABSTRACT
TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, non-noxious heat and mechanical stress and some small molecule agonists (e.g. phorbol ester 4α-PDD). GSK1016790A (GSK101) is a recently discovered specific small molecule agonist of TRPV4. Its effects on physical determinants of TRPV4 activity were evaluated in HeLa cells transiently transfected with TRPV4 (HeLa-TRPV4). GSK101 (10 nM) causes a TRPV4 specific Ca(2+) influx in HeLa-TRPV4 cells, but not in control transfected cells, which can be inhibited by ruthenium red and Ca(2+)-free medium more significantly at the early stage of the activation rather than the late stage, reflecting apparent partial desensitization. Western blot analysis showed that GSK101 activation did not induce an increase in TRPV4 expression at the plasma membrane, but caused an immediate and sustained downregulation of TRPV4 on the plasma membrane in HeLa-TRPV4 cells. Patch clamp analysis also revealed an early partial desensitization of the channel which was Ca(2+)-independent. FRET analysis of TRPV4 subunit assembly demonstrated that the GSK101-induced TRPV4 channel activation/desensitization was not due to alterations in homotetrameric channel formation on the plasma membrane. It is concluded that GSK101 specifically activates TRPV4 channels, leading to a rapid partial desensitization and downregulation of the channel expression on the plasma membrane. TRPV4 subunit assembly appears to occur during trafficking from the ER/Golgi to the plasma membrane and is not altered by agonist stimulation.

Show MeSH
Related in: MedlinePlus