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. 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. Members of the Transient Receptor Potential (TRP) family are nonselective cation channels that are multisteric receptors activated by the binding of a variety of exogenous ligands and endogenous mediators, as well as temperature changes. Several TRP receptors are located on the sensory neurons and they are key molecules for sensing pain (1.Gees M. Owsianik G. Nilius B. Voets T. TRP channels.Compr. Physiol. 2012; 2: 563-608Crossref PubMed Scopus (112) Google Scholar, 2.Vay L. Gu C. McNaughton P.A. The thermo-TRP ion channel family: properties and therapeutic implications.Br. J. Pharmacol. 2012; 165: 787-801Crossref PubMed Scopus (202) Google Scholar, 3.Szolcsányi J. Sándor Z. Multisteric TRPV1 nocisensor: a target for analgesics.Trends Pharmacol. 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C2 the group in a position significantly the TRPV1 ion channel activation, but C3 the group a inhibitory effect on the of the TRPV1 ion the position of the carboxamido group in the of C1 and C2 and the in the of compounds in the of and in compound C2 a with a of the a is the of the steroid in as was in by E. J. Z. L. R. of and on TRPV1 receptor PubMed Scopus Google C2 a to the effect of the on the a compound (C3) was for the It the group of C1 and but the as a was the the of that the presence of the carboxamido as well as the of the and the position of the carboxamido are important for the of the inhibitory action on TRP ion have as potential of the TRPV1 cation The and temperature or as a and TRPV1 G. M.F. F. R. R.B. et of the steroid as a transient receptor potential vanilloid 1 with Pharmacol. 2012; PubMed Scopus Google Scholar, D. M. a TRPV1 receptor the in in PubMed Scopus Google Scholar). TRPV1 and ion channels have to and inhibitory have in of TRPV1, and for S. S. S. M. A. V. 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It that our carboxamido steroid compound C1 exerts inhibitory on TRP channels located on primary sensory neurons, such as TRPV1, and and the presence and the position of the carboxamido group are important for this We provided the evidence that our steroid compound C1 is able to cholesterol from the plasma membrane and exerts the effect in as in Therefore, one of the of the is that our compound C1 is a to investigate the effect of cholesterol depletion and the lipid raft disruption on channel and is the most compound in this The other is that the the TRP channels and lipid the of ion channels, and this a and The for with allyl-isothiocyanate extracellular Transient Receptor Potential Transient Receptor Potential Ankyrin 1 Transient Receptor Potential Transient Receptor Potential Transient Receptor Potential Transient Receptor Potential Vanilloid 1