Determinants of TRPV4 Activity following Selective Activation by Small Molecule Agonist GSK1016790A

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 Ca2+ influx in HeLa-TRPV4 cells, but not in control transfected cells, which can be inhibited by ruthenium red and Ca2+-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 Ca2+-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.

Function of Transient Receptor Potential Cation Channel Subfamily V Member 4 (TRPV4) as a Mechanical Transducer in Flow-sensitive Segments of Renal Collecting Duct System

“…The GSK101-induced time course of [Ca 2ϩ ] i was relatively slow, typically reaching a peak value within 1-6 min. The results are consistent with our earlier characterization of GSK101 in TRPV4-transfected HeLa cells and in the M-1 collecting duct cells which endogenously express TRPV4 (19,25). Comparison of the responses of individual cells from a single split-opened CCD demonstrates variable responses from cell to cell that are likely reflective of the individual properties and TRPV4 expression levels of different cells from a single tubule (Fig.…”

“…To verify the TRPV4 specificity of our antibody for TRPV4, in separate studies we have demonstrated that our antibody (directed at the C terminus, Alomone Laboratory anti-TRPV4) recognized an appropriate double band (ϳ100 kDa) for TRPV4 in TRPV4-transfected HEK cells, but not in nontransfected HEK cells (19). Immunocytochemistry and functional assays confirmed identification of TRPV4 only in the TRPV4-expressing cells.…”

“…Using fluorescence ratiometric imaging of the Ca 2ϩ -sensitive probe, fura-2, [Ca 2ϩ ] i was monitored and presented as the ratio F 340 /F 380 (or [Ca 2ϩ ] i in summary figures) as described previously (3,4). To activate TRPV4 selectively, we took advantage of the TRPV4 agonist, GSK101, to demonstrate specific activation of TRPV4 (19,24). As shown in Fig.…”

“…Measurement of Intracellular Calcium-Ratiometric fluorescence imaging of fura-2 was used to monitor intracellular calcium levels, [Ca 2ϩ rsqb] i , in split-opened tubule preparations using standard procedures as done extensively before (4,17,19). Briefly, tubule fragments on coverslip chips were loaded with fura-2/AM (2 M) for 40 min, washed, placed on the coverslip bottom of an open perfusion chamber (0.5 ml), and imaged with a InCa Imaging Work station (Intracellular Imaging, Inc.) at 37°C using a 20ϫ Nikon Super Fluor objective.…”

Angiotensin II Increases Activity of the Epithelial Na+ Channel (ENaC) in Distal Nephron Additively to Aldosterone

“…The Fura-2 fluorescence intensity ratio was determined by excitation (an average for ϳ300 ms) at 340 nm and 380 nm and calculating the ratio of the emission intensities at 511 nm in the usual manner every 5 s. We observed no significant Fura-2 bleaching and minimal Fura-2 leakage at both wavelengths during the experiments. The changes in the ratio were converted to intracellular Ca 2ϩ concentrations using the calibration methods as we have done before (34,35). Experimental traces from individual cells were inspected visually prior to acceptance.…”