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Drofenine: A 2-APB Analogue with Greater Selectivity for Human TRPV3.

Deering-Rice CE, Mitchell VK, Romero EG, Abdel Aziz MH, Ryskamp DA, Križaj D, Gopal VR, Reilly CA - Pharmacol Res Perspect (2014)

Bottom Line: The antispasmodic agent drofenine was identified as a new TRPV3 agonist.Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin.Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Utah, 30 S. 2000 E., Room 201 Skaggs Hall, Salt Lake City, UT 84112, USA.

ABSTRACT
Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology, as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 over-expressing HEK-293 cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

No MeSH data available.


Related in: MedlinePlus

Drofenine induces inward currents in TRPV3-overexpressing HEK-293 cells. (A) Example traces of whole-cell recordings at a negative holding potential. 250 μmol/L, 500 μmol/L, and 1 mmol/L drofenine increased the change in inward current (ΔI). (B–D) Dose-dependent responses were characterized by comparisons between basal current and the peak response. (B) n = 6, P = 0.197. (C) n = 5, P = 0.00151. (D) n = 8, P = 0.00574. Data are represented as mean ± SEM and asterisks indicate a significant difference between the baseline current and the peak response detected using a one-tailed, paired t-test.
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fig03: Drofenine induces inward currents in TRPV3-overexpressing HEK-293 cells. (A) Example traces of whole-cell recordings at a negative holding potential. 250 μmol/L, 500 μmol/L, and 1 mmol/L drofenine increased the change in inward current (ΔI). (B–D) Dose-dependent responses were characterized by comparisons between basal current and the peak response. (B) n = 6, P = 0.197. (C) n = 5, P = 0.00151. (D) n = 8, P = 0.00574. Data are represented as mean ± SEM and asterisks indicate a significant difference between the baseline current and the peak response detected using a one-tailed, paired t-test.

Mentions: Whole-cell recordings were made using borosilicate pipettes (3–5 MΩ). Coverslips were coated with concanavalin A as described previously (Ryskamp et al. 2011) and rinsed with phosphate buffer solution. Suspended TRPV3-overexpressing HEK-293 cells were plated on these coverslips in supplemented DMEM:F12 and used within 24 h. Throughout experiments, cells were perfused with external saline containing 145 mmol/L NaCl, 5 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L HEPES, 10 mmol/L glucose, and 2 mmol/L CaCl2 (pH 7.4, 320 mOsm). Drofenine was bath applied via perfusion (250 and 500 μmol/L) or injection (1 mmol/L). The internal pipette solution consisted of 133 mmol/L CsCl, 10 mmol/L HEPES, 5 mmol/L ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mmol/L CaCl2, and 1 mmol/L MgCl2 (pH of 7.3 set with CsOH) (Bae et al. 2011). Pipette and membrane capacitances were automatically compensated and current recordings were Bessel-filtered at 10 kHz using an EPC-10 amplifier (Patchmaster; HEKA Instruments Inc., Bellmore, NY). Cells were voltage clamped at −60 or −70 mV to measure inward currents. The holding potential was adjusted to approximately normalize the baseline current from cell to cell. For each drofenine concentration, we only used cells that were recorded at the same holding potential. Fresh cells were used for each recording to prevent variability due to sensitization or desensitization. Data were collected at 1.0 kHz and analyzed with IGOR Pro 6 software (WaveMetrics Inc., Lake Oswego, OR). Every 100th data point was plotted in example traces ( Fig. 3A).


Drofenine: A 2-APB Analogue with Greater Selectivity for Human TRPV3.

Deering-Rice CE, Mitchell VK, Romero EG, Abdel Aziz MH, Ryskamp DA, Križaj D, Gopal VR, Reilly CA - Pharmacol Res Perspect (2014)

Drofenine induces inward currents in TRPV3-overexpressing HEK-293 cells. (A) Example traces of whole-cell recordings at a negative holding potential. 250 μmol/L, 500 μmol/L, and 1 mmol/L drofenine increased the change in inward current (ΔI). (B–D) Dose-dependent responses were characterized by comparisons between basal current and the peak response. (B) n = 6, P = 0.197. (C) n = 5, P = 0.00151. (D) n = 8, P = 0.00574. Data are represented as mean ± SEM and asterisks indicate a significant difference between the baseline current and the peak response detected using a one-tailed, paired t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4115637&req=5

fig03: Drofenine induces inward currents in TRPV3-overexpressing HEK-293 cells. (A) Example traces of whole-cell recordings at a negative holding potential. 250 μmol/L, 500 μmol/L, and 1 mmol/L drofenine increased the change in inward current (ΔI). (B–D) Dose-dependent responses were characterized by comparisons between basal current and the peak response. (B) n = 6, P = 0.197. (C) n = 5, P = 0.00151. (D) n = 8, P = 0.00574. Data are represented as mean ± SEM and asterisks indicate a significant difference between the baseline current and the peak response detected using a one-tailed, paired t-test.
Mentions: Whole-cell recordings were made using borosilicate pipettes (3–5 MΩ). Coverslips were coated with concanavalin A as described previously (Ryskamp et al. 2011) and rinsed with phosphate buffer solution. Suspended TRPV3-overexpressing HEK-293 cells were plated on these coverslips in supplemented DMEM:F12 and used within 24 h. Throughout experiments, cells were perfused with external saline containing 145 mmol/L NaCl, 5 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L HEPES, 10 mmol/L glucose, and 2 mmol/L CaCl2 (pH 7.4, 320 mOsm). Drofenine was bath applied via perfusion (250 and 500 μmol/L) or injection (1 mmol/L). The internal pipette solution consisted of 133 mmol/L CsCl, 10 mmol/L HEPES, 5 mmol/L ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mmol/L CaCl2, and 1 mmol/L MgCl2 (pH of 7.3 set with CsOH) (Bae et al. 2011). Pipette and membrane capacitances were automatically compensated and current recordings were Bessel-filtered at 10 kHz using an EPC-10 amplifier (Patchmaster; HEKA Instruments Inc., Bellmore, NY). Cells were voltage clamped at −60 or −70 mV to measure inward currents. The holding potential was adjusted to approximately normalize the baseline current from cell to cell. For each drofenine concentration, we only used cells that were recorded at the same holding potential. Fresh cells were used for each recording to prevent variability due to sensitization or desensitization. Data were collected at 1.0 kHz and analyzed with IGOR Pro 6 software (WaveMetrics Inc., Lake Oswego, OR). Every 100th data point was plotted in example traces ( Fig. 3A).

Bottom Line: The antispasmodic agent drofenine was identified as a new TRPV3 agonist.Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin.Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Utah, 30 S. 2000 E., Room 201 Skaggs Hall, Salt Lake City, UT 84112, USA.

ABSTRACT
Transient receptor potential vanilloid-3 (TRPV3) is a member of the TRPV subfamily of TRP ion channels. The physiological functions of TRPV3 are not fully understood, in part due to a lack of selective agonists and antagonists that could both facilitate the elucidation of roles for TRPV3 in mammalian physiology, as well as potentially serve as therapeutic agents to modulate conditions for which altered TRPV3 function has been implicated. In this study, the Microsource Spectrum Collection was screened for TRPV3 agonists and antagonists using alterations in calcium flux in TRPV3 over-expressing HEK-293 cells. The antispasmodic agent drofenine was identified as a new TRPV3 agonist. Drofenine exhibited similar potency to the known TRPV3 agonists 2-aminoethoxydiphenylboronate (2-APB) and carvacrol in HEK-293 cells, but greater selectivity for TRPV3 based on a lack of activation of TRPA1, V1, V2, V4, or M8. Multiple inhibitors were also identified, but all of the compounds were either inactive or not specific. Drofenine activated TRPV3 via interactions with the residue, H426, which is required for TRPV3 activation by 2-APB. Drofenine was a more potent agonist of TRPV3 and more cytotoxic than either carvacrol or 2-APB in human keratinocytes and its effect on TRPV3 in HaCaT cells was further demonstrated using the antagonist icilin. Due to the lack of specificity of existing TRPV3 modulators and the expression of multiple TRP channels in cells/tissue, drofenine may be a valuable probe for elucidating TRPV3 functions in complex biological systems. Identification of TRPV3 as a target for drofenine may also suggest a mechanism by which drofenine acts as a therapeutic agent.

No MeSH data available.


Related in: MedlinePlus