Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold

Catalina-Hernández, Èric; López-Martín, Mario; Masnou-Sánchez, David; Martins, Marco; Lorenz-Fonfria, Victor A.; Jiménez-Altayó, Francesc; Hellmich, Ute A.; Inada, Hitoshi; Alcaraz, Antonio; Furutani, Yuji; Nonell-Canals, Alfons; Vázquez-Ibar, Jose Luis; Domene, Carmen; Gaudet, Rachelle; Perálvarez-Marín, Alex

doi:10.1016/j.csbj.2023.12.028

Cited by 2 publications

(2 citation statements)

References 41 publications

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“…EV2A–O ). In agreement with a recent study on TRPV2 (Catalina-Hernandez et al, 2024 ), we also observed an activation and sensitization of rTRPV2 by benzoic acid (Fig EV2P–S ). However, the non-deprotonatable β-hydroxybutyric acid failed to activate rTRPV2 (Appendix Fig.…”

Section: Resultssupporting

confidence: 93%

“…Although HOAc-sensitivity was almost abolished in the rTRPV1-H521A mutant, further binding sites may be involved. To this end, a recent study suggested that activation of TRPV2 by probenecid and benzoic acid derivatives may be due to an interaction with the proposed binding sites of the TRPV2-inhibitor piperlongumine, including the residue Arg539 (Catalina-Hernandez et al, 2024 ; Conde et al, 2021 ). While 2-APB-sensitivity of rTRPV2 only partially depends on His521, a stronger reduction of the 2-APB-sensitivity was observed in the double mutant rTRPV2-H521A/R539K (Pumroy et al, 2022 ).…”

Section: Discussionmentioning

confidence: 99%

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Functional and structural insights into activation of TRPV2 by weak acids

Haug,

Pumroy,

Sridhar

et al. 2024

EMBO J

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Transient receptor potential (TRP) ion channels are involved in the surveillance or regulation of the acid-base balance. Here, we demonstrate that weak carbonic acids, including acetic acid, lactic acid, and CO2 activate and sensitize TRPV2 through a mechanism requiring permeation through the cell membrane. TRPV2 channels in cell-free inside-out patches maintain weak acid-sensitivity, but protons applied on either side of the membrane do not induce channel activation or sensitization. The involvement of proton modulation sites for weak acid-sensitivity was supported by the identification of titratable extracellular (Glu495, Glu561) and intracellular (His521) residues on a cryo-EM structure of rat TRPV2 (rTRPV2) treated with acetic acid. Molecular dynamics simulations as well as patch clamp experiments on mutant rTRPV2 constructs confirmed that these residues are critical for weak acid-sensitivity. We also demonstrate that the pore residue Glu609 dictates an inhibition of weak acid-induced currents by extracellular calcium. Finally, TRPV2-expression in HEK293 cells is associated with an increased weak acid-induced cytotoxicity. Together, our data provide new insights into weak acids as endogenous modulators of TRPV2.

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Section: Resultssupporting

confidence: 93%

Section: Discussionmentioning

confidence: 99%

Functional and structural insights into activation of TRPV2 by weak acids

Haug,

Pumroy,

Sridhar

et al. 2024

EMBO J

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Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry

Werin,

Hansson Wennersten,

Olsson

et al. 2024

Microb Cell Fact

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Background The wasabi receptor, also known as the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel, is a potential target for development of repellents for insects, like the pine weevil (Hylobius abietis) feeding on conifer seedlings and causing damage in forestry. Heterologous expression of TRPA1 from pine weevil in the yeast Pichia pastoris can potentially provide protein for structural and functional studies. Here we take advantage of the Green Fluorescent Protein (GFP) tag to examine the various steps of heterologous expression, to get more insight in clone selection, expression and isolation of the intact purified protein. Results The sequence of HaTRPA1 is reported and GFP-tagged constructs were made of the full-length protein and a truncated version (Δ1-708 HaTRPA1), lacking the N-terminal ankyrin repeat domain. Clones were screened on GFP expression plates, induced in small liquid cultures and in fed-batch cultures, and evaluated by flow cytometry and fluorescence microscopy. The screening on plates successfully identifies low-expression clones, but fails to predict the ranking of the best performing clones in small-scale liquid cultures. The two constructs differ in their cellular localization. Δ1-708 HaTRPA1 is found in a ring at the perimeter of cell, whereas HaTRPA1 is forming highly fluorescent speckles in interior regions of the cell. The pattern is consistent in different clones of the same construct and persists in fed-batch culture. The expression of Δ1-708 HaTRPA1 decreases the viability more than HaTRPA1, and in fed-batch culture it is clear that intact cells first express Δ1-708 HaTRPA1 and then become damaged. Purifications show that both constructs suffer from degradation of the expressed protein, but especially the HaTRPA1 construct. Conclusions The GFP tag makes it possible to follow expression by flow cytometry and fluorescence microscopy. Analyses of localization, cell viability and expression show that the former two parameters are specific for each of the two evaluated constructs, whereas the relative expression of the constructs varies with the cultivation method. High expression is not all that matters, so taking damaged cells into account, something that may be linked to protein degradation, is important when picking the most suitable construct, clone, and expression scheme.

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Experimental and computational biophysics to identify vasodilator drugs targeted at TRPV2 using agonists based on the probenecid scaffold

Cited by 2 publications

References 41 publications

Functional and structural insights into activation of TRPV2 by weak acids

Functional and structural insights into activation of TRPV2 by weak acids

Evaluation of heterologous expression in Pichia pastoris of Pine Weevil TRPA1 by GFP and flow cytometry

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