Pharmacological modulation of diacylglycerol-sensitive TRPC3/6/7 channels

Abstract

Members of the classic type of transient receptor potential channels (TRPC) represent important molecules involved in hormonal signal transduction. TRPC3/6/7 channels are of particular interest as they are components of phospholipase C driven signalling pathways. Upon receptor-activation, G-protein-mediated stimulation of phospholipase C results in breakdown of phosphatidylinositides leading to increased intracellular diacylglycerol and inositol-trisphosphate levels. Diacylglycerol activates protein kinase C, but more interestingly diacylglycerol directly activates TRPC2/3/6/7 channels. Molecular cloning, expression and characterization of TRP channels enabled reassignment of traditional inhibitors of receptor-dependent calcium entry such as SKF-96365 and 2-APB as blockers of TRPC3/6/7 and several members of non-classic TRP channels. Furthermore, several enzyme inhibitors have also been identified as TRP channel blockers, such as ACA, a phospholipase A(2) inhibitor, and W-7, a calmodulin antagonist. Finally, the naturally occurring secondary plant compound hyperforin has been identified as TRPC6-selective drug, providing an exciting proof of concept that it is possible to generate TRPC-selective channel modulators. The description of Pyr3 as the first TRPC3-selective inhibitor shows that not only nature but also man is able to generate TRP-selective modulators. The review summarizes the data on pharmacological modification of TRPC3/6/7. Sheds lights on the current knowledge and historical development of pharmacological modulators of TRPC3/6/7. Our analysis indicates that Pyr3 and hyperforin provide promising core structures for the development of new, skeletive and more potent modulators of TRPC3/6/7 activity.

Fig. (1)

Signalling cascade leading to TRPC3/6/7 activation. Hormonal stimulation of G-protein coupled receptor (GPCR) activates phospholipase C isoforms (PLC β) via a G-protein dependent mechanism. Phospholipase C activity results in the breakdown of phosphatidylinositides (PIP₂) leading to the formation of inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG). Inositol 1,4,5-trisphosphate as ligand of inositol 1,4,5-trisphosphate receptors located at the endoplasmic reticulum induce calcium release from intracellular stores, whereas DAG activates mammalian TRPC2, TRPC3, TRPC6 and TRPC7 channels. In contrast to the mammalian signalling cascade, Drosophila TRPL and TRPγ are activated by poly-unsaturated fatty acids (PUFA) generated by phospholipase A₂ (PLA₂) from subsequent degradation of diacylglycerols.

Fig. (2)

Chemical structures of broad range TRP channel blocker. SKF-96365: 1-{β[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride; 2-APB: 2-aminoethoxydiphenyl borate; ACA: N-(p-amylcinnamoyl)anthranilic acid; ML-9: [1-(5-Chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride]; W-7: N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride.

Fig. (3)

Concentration-response relationship of ML-9 and W-7 related compounds on hyperforin-induced, TRPC6-mediated fluorescence changes. A) The chemical structures of the tested compound is shown. ML-9: 1-(5-Chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine; W-7: N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide; W-5: N-(6-Aminohexyl)-1-naphthalenesulfonamide; W-7 isomer: N-(6-Aminohexyl)-5-chloro-2-naphthalenesulfonamide; J-8: (N-8-Aminooctyl)-5-iodo-1-naphthalenesulfonamide; W-7 decyl: N-(6-Aminodecyl)-5-chloro-1-naphthalenesulphonamide. B) Data from a representative experiment show the effect of ML-9, W-7, W-7 decyl, W-7 isomer, W-5 and J-8 on calcium entry in TRPC6-expressing cells upon hyperforin stimulation. Calcium entry was measured using FLIPR^Tetra and data analysed as described in Jörs et al. [5]. The data were calculated from one experiment of at least three experiments performed in quadruplicates per concentration and TRP channel. The concentration-response curves determined by calcium imaging showed a quite distinct inhibition profile. The IC₅₀ values of ML-9, W-7, W-7 isomer, W-7 decyl W-5, J-8 were 36 µM, 26 µM, 35 µM, 29 µM, 20 µM, 85 µM, respectively.

Fig. (4)

Concentration-dependent inhibition of TRPC6-, TRPM2-, TRPM3-, TRPV4- and TRPγ-mediated calcium entry by W-7. Fluorescence of TRPC6-, TRPM2-, TRPM3-, TRPV4- and TRPγ-expressing, fluo-4-loaded cells in were stimulated with hyperforin (10 µM), hydrogen peroxide (5 mM), pregnenolone sulphate (35 µM), 4α-phorbol-didecanoate (5 µM) and eicosatretranoic acid (20 µM), respectively. Calcium entry was measured using FLIPR^Tetra and data analysed as described in Jörs et al. [5]. The data were calculated from one experiment of at least three experiments performed in quadruplicates per concentration and TRP channel. The concentration-response curves determined by calcium imaging showed a quite distinct inhibition profile. The IC₅₀ values to block TRPC6, TRPM2, TRPM3, TRPV4, Drosophila TRPγ were 28 µM, 26 µM, 15 µM, 65 µM, 5 µM, respectively.

Fig. (5)

Mechanistic model of the hyperforin-dependent antidepressive effect. Within the neurotransmitter circuit of release and uptake, hyperforin and classic, synthetic antidepressants attack different targets resulting in reduced presysnaptic uptake of neurotransmitter and increase concentration of neurotransmitter in the synaptic cleft. Whereas classic synthetic antidepressants directly block the neurotransmitter transporters, hyperforin activates TRPC6 channel in proximity of the neurotransmitter transporters. TRPC6 is a non-selective cation channel enabling the permeation of sodium and calcium. Sodium entry mediated by TRPC6 activation reduced the sodium gradient across the plasma membrane. As the activity of the neurotransmitter transporters depends on the electrochemical sodium gradient, TRPC6 activity indirectly inhibits neurotransmitter uptake. On the other hand, TRPC6-mediated calcium entry may trigger calcium-dependent differentiation processes responsible for changes in neuronal plasticity.

Fig. (6)

Comparison of the chemical structure of Hyp1 and Pyr-PP.