Carboxamido steroids inhibit the opening properties of transient receptor potential ion channels by lipid raft modulation

Abstract

Transient Receptor Potential (TRP) cation channels, like the TRP Vanilloid 1 (TRPV1) and TRP Ankyrin 1 (TRPA1), are expressed on primary sensory neurons. These thermosensor channels play a role in pain processing. We have provided evidence previously that lipid raft disruption influenced the TRP channel activation, and a carboxamido-steroid compound (C1) inhibited TRPV1 activation. Therefore, our aim was to investigate whether this compound exerts its effect through lipid raft disruption and the steroid backbone (C3) or whether altered position of the carboxamido group (C2) influences the inhibitory action by measuring Ca2+ transients on isolated neurons and calcium-uptake on receptor-expressing CHO cells. Membrane cholesterol content was measured by filipin staining and membrane polarization by fluorescence spectroscopy. Both the percentage of responsive cells and the magnitude of the intracellular Ca2+ enhancement evoked by the TRPV1 agonist capsaicin were significantly inhibited after C1 and C2 incubation, but not after C3 administration. C1 was able to reduce other TRP channel activation as well. The compounds induced cholesterol depletion in CHO cells, but only C1 induced changes in membrane polarization. The inhibitory action of the compounds on TRP channel activation develops by lipid raft disruption, and the presence and the position of the carboxamido group is essential.

Antinociceptive Effects of Lipid Raft Disruptors, a Novel Carboxamido-Steroid and Methyl β-Cyclodextrin, in Mice by Inhibiting Transient Receptor Potential Vanilloid 1 and Ankyrin 1 Channel Activation

“…Both compounds had inhibitory action on formalin-evoked acute neurogenic inflammatory nocifensive reactions (paw liftings, lickings, holdings, and shakings) in the second, neurogenic inflammatory phase by 36 and 51%, respectively. These novel in vivo results are well supported by our previous in vitro findings showing that C1 and MCD significantly and concentration-dependently inhibit TRPV1 and TRPA1 receptor activation both on primary sensory neuronal cultures and receptor-expressing cell line (Szőke et al, 2010;Szánti-Pintér et al, 2015;Sághy et al, 2015Sághy et al, , 2018. We have previously proved by filipin staining and fluorescence spectroscopy that C1 similarly to MCD depleted cholesterol from the plasma membrane of sensory neurons, and therefore, they are both considered to be lipid raft disruptors (Sághy et al, 2018).…”

“…Furthermore, we provided the first evidence and the presence and the position of the carboxamido group was important for this action mediated by cholesterol depletion from the plasma membrane. This effect was similar to that of MCD, but in a much lower, 1000-fold concentration (Szánti-Pintér et al, 2015;Sághy et al, 2018).…”

“…TRPA1 is also considered to Abbreviations: C1, carboxamido-steroid compound; CAPS, capsaicin; CRAC, Cholesterol Recognition/interaction Amino acid Consensus; DHEA, dehydroepiandrosterone; DRG, dorsal root ganglion; E2, 17-β estradiol; MCD, methyl β-cyclodextrin; PGE2, prostaglandin E2; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PS, pregnenolone sulfate; RAMEB, random methylated β-cyclodextrins; RTX, resiniferatoxin; TrkA, tropomyosin-related kinase A; TRP, be a promising analgesic target based on experimental and human studies which seem to be free of severe side effects (Romanovsky et al, 2009;Botz et al, 2017). These data clearly suggest the drug developmental potential of TRPV1 and TRPA1 blockade, therefore alternative mechanisms in addition to the direct antagonism have been proposed as promising inhibitors options (Ferrari and Levine, 2015;Sághy et al, 2015Sághy et al, , 2018Lin et al, 2019).…”

Steroids and TRP Channels: A Close Relationship

“…Recently, it was reported that carboxamide steroids (synthetic compounds) inhibit the opening of TRPV1 and TRPA1 and that this negative regulation is through disruption of the lipid rafts [187]. Although this last study does not assess changes in the temperature threshold of activation of these channels, it will be interesting to evaluate this possibility.…”

Determining the target of membrane sterols on voltage-gated potassium channels

“…Most often channel activity is suppressed by cholesterol enrichment or enhanced by cholesterol depletion of the membrane, as was observed for several K + channels 8,[10][11][12][13][14] , voltage-gated Na + channels 15 , as well as volume-regulated anion channels 16 . In some other channels, however, the opposite was reported: depletion of membrane cholesterol inhibited epithelial Na + channels (eNaC) 17 and transient receptor potential (Trp) channels 18,19 . Based on the conclusions of these studies two possible mechanisms have emerged to explain cholesterol effects on channel gating: specific interaction with the channel via a binding site; or an indirect effect through changes in the physical parameters of the membrane (e.g.…”