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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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Effects of GSK101 on whole-cell membrane current (patch clamp) in HeLa-TRPV4 cells.A–C: HeLa-TRPV4 cells in Ca2+ containing media. A. A representative trace of GSK101-induced whole cell current showing a rapid increase at 1 min stimulation followed by channel desensitization (holding potential at −90 mV). B. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) displaying a peak activation at 1 min followed by a partial desensitization of the channels. The time course for desensitization was fit to a single exponential equation and yielded a time constant (τ) for decay of 0.8±0.1 minutes.C. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation, using a voltage ramp protocol from −100 to 100 mV over 200 msec. D–E: HeLa-TRPV4 cells in Ca2+ free media (Na+ containing media). D. A representative trace of GSK101-induced whole cell current showing a similar pattern of channel activation as shown in A, but at a larger magnitude in Ca2+ free media. E. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) in Ca2+ free media. The time course for desensitization was fit to a single exponential equation witha τ for decay of 3.8±1.8 minutes (NS from Ca2+-containing media in B). F. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation in Ca2+ free media, using a voltage ramp protocol from −100 to 100 mV over 200 msec.
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pone-0016713-g004: Effects of GSK101 on whole-cell membrane current (patch clamp) in HeLa-TRPV4 cells.A–C: HeLa-TRPV4 cells in Ca2+ containing media. A. A representative trace of GSK101-induced whole cell current showing a rapid increase at 1 min stimulation followed by channel desensitization (holding potential at −90 mV). B. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) displaying a peak activation at 1 min followed by a partial desensitization of the channels. The time course for desensitization was fit to a single exponential equation and yielded a time constant (τ) for decay of 0.8±0.1 minutes.C. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation, using a voltage ramp protocol from −100 to 100 mV over 200 msec. D–E: HeLa-TRPV4 cells in Ca2+ free media (Na+ containing media). D. A representative trace of GSK101-induced whole cell current showing a similar pattern of channel activation as shown in A, but at a larger magnitude in Ca2+ free media. E. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) in Ca2+ free media. The time course for desensitization was fit to a single exponential equation witha τ for decay of 3.8±1.8 minutes (NS from Ca2+-containing media in B). F. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation in Ca2+ free media, using a voltage ramp protocol from −100 to 100 mV over 200 msec.

Mentions: Although calcium imaging detected significant Ca2+ influx in response to GSK101 in HeLa-TRPV4 cells, it was necessary to determine the specific role of TRPV4 channels in regulating intracellular Ca2+ levels. The whole cell patch clamp technique was utilized to study the channel currents under GSK101 stimulation using either Ca2+-containing or Ca2+-free media in HeLa-TRPV4 cells. Whole-cell recordings in Ca2+-containing media are shown in Figure 4A–C. A representative current trace following GSK101 stimulation is shown in Figure 4A. At a holding potential of −90 mV, the GSK101-induced current occurredimmediately, reached a peak current at 1 min after activation, and relaxed to a pseudosteady state for the remainder of the recording (20 min). A similar time course was observed for four separate cells, with each demonstrating an early rapid activation, a peak near 1 min, followed by an apparent partial desensitization of the channel (Figure 4B). To further characterize the GSK101-induced current, a whole-cell current-voltage relation was generated using a voltage ramp protocol (−100 mV to +100 mV) over 200 msec duration. The IV-plot shows the GSK101-induced inward and outward currents, including the peak current at 1 min and the expected decay at the 5-min time pointin HeLa-TRPV4 cells. The characteristic outward rectification for TRPV4-currents at positive voltages is readily apparent (Figure 4C). Figure 4D–F shows the whole cell recording obtained in Ca2+-free media where Na+ is the dominant charge carry for this non-selective cation channel. The GSK101-induced currents in Ca2+-free media displayed similar patterns. This demonstrates that in the absence of Ca2+ in the media, Na+ readily permeated the TRPV4 channels under GSK101 stimulation causing even larger transmembrane currents. Similar current-voltage relationship under GSK101 stimulation was observed by others in TRPV4-expressing HEK-293 cells and human aortic endothelial cells [15], [16]. These electrophysiological findings indicate that the Ca2+ influx detected by calcium imaging during the first 3 minutes in HeLa-TRPV4 cells was likely primarily through TRPV4 channels. Moreover, the desensitization of TRPV4 under prolonged GSK101 stimulation did not appear to be calcium-dependent since it still occurred in calcium-free media.


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)

Effects of GSK101 on whole-cell membrane current (patch clamp) in HeLa-TRPV4 cells.A–C: HeLa-TRPV4 cells in Ca2+ containing media. A. A representative trace of GSK101-induced whole cell current showing a rapid increase at 1 min stimulation followed by channel desensitization (holding potential at −90 mV). B. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) displaying a peak activation at 1 min followed by a partial desensitization of the channels. The time course for desensitization was fit to a single exponential equation and yielded a time constant (τ) for decay of 0.8±0.1 minutes.C. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation, using a voltage ramp protocol from −100 to 100 mV over 200 msec. D–E: HeLa-TRPV4 cells in Ca2+ free media (Na+ containing media). D. A representative trace of GSK101-induced whole cell current showing a similar pattern of channel activation as shown in A, but at a larger magnitude in Ca2+ free media. E. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) in Ca2+ free media. The time course for desensitization was fit to a single exponential equation witha τ for decay of 3.8±1.8 minutes (NS from Ca2+-containing media in B). F. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation in Ca2+ free media, using a voltage ramp protocol from −100 to 100 mV over 200 msec.
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getmorefigures.php?uid=PMC3038856&req=5

pone-0016713-g004: Effects of GSK101 on whole-cell membrane current (patch clamp) in HeLa-TRPV4 cells.A–C: HeLa-TRPV4 cells in Ca2+ containing media. A. A representative trace of GSK101-induced whole cell current showing a rapid increase at 1 min stimulation followed by channel desensitization (holding potential at −90 mV). B. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) displaying a peak activation at 1 min followed by a partial desensitization of the channels. The time course for desensitization was fit to a single exponential equation and yielded a time constant (τ) for decay of 0.8±0.1 minutes.C. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation, using a voltage ramp protocol from −100 to 100 mV over 200 msec. D–E: HeLa-TRPV4 cells in Ca2+ free media (Na+ containing media). D. A representative trace of GSK101-induced whole cell current showing a similar pattern of channel activation as shown in A, but at a larger magnitude in Ca2+ free media. E. Summary data showing the time course of GSK101-induced membrane current (Mean ± SE, n = 4) in Ca2+ free media. The time course for desensitization was fit to a single exponential equation witha τ for decay of 3.8±1.8 minutes (NS from Ca2+-containing media in B). F. Current-voltage plot of a HeLa-TRPV4 cell under GSK101 stimulation in Ca2+ free media, using a voltage ramp protocol from −100 to 100 mV over 200 msec.
Mentions: Although calcium imaging detected significant Ca2+ influx in response to GSK101 in HeLa-TRPV4 cells, it was necessary to determine the specific role of TRPV4 channels in regulating intracellular Ca2+ levels. The whole cell patch clamp technique was utilized to study the channel currents under GSK101 stimulation using either Ca2+-containing or Ca2+-free media in HeLa-TRPV4 cells. Whole-cell recordings in Ca2+-containing media are shown in Figure 4A–C. A representative current trace following GSK101 stimulation is shown in Figure 4A. At a holding potential of −90 mV, the GSK101-induced current occurredimmediately, reached a peak current at 1 min after activation, and relaxed to a pseudosteady state for the remainder of the recording (20 min). A similar time course was observed for four separate cells, with each demonstrating an early rapid activation, a peak near 1 min, followed by an apparent partial desensitization of the channel (Figure 4B). To further characterize the GSK101-induced current, a whole-cell current-voltage relation was generated using a voltage ramp protocol (−100 mV to +100 mV) over 200 msec duration. The IV-plot shows the GSK101-induced inward and outward currents, including the peak current at 1 min and the expected decay at the 5-min time pointin HeLa-TRPV4 cells. The characteristic outward rectification for TRPV4-currents at positive voltages is readily apparent (Figure 4C). Figure 4D–F shows the whole cell recording obtained in Ca2+-free media where Na+ is the dominant charge carry for this non-selective cation channel. The GSK101-induced currents in Ca2+-free media displayed similar patterns. This demonstrates that in the absence of Ca2+ in the media, Na+ readily permeated the TRPV4 channels under GSK101 stimulation causing even larger transmembrane currents. Similar current-voltage relationship under GSK101 stimulation was observed by others in TRPV4-expressing HEK-293 cells and human aortic endothelial cells [15], [16]. These electrophysiological findings indicate that the Ca2+ influx detected by calcium imaging during the first 3 minutes in HeLa-TRPV4 cells was likely primarily through TRPV4 channels. Moreover, the desensitization of TRPV4 under prolonged GSK101 stimulation did not appear to be calcium-dependent since it still occurred in calcium-free media.

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