20-Hydroxyeicosatetraenoic Acid (20-HETE) Activates Mouse TRPC6 Channels Expressed in HEK293 Cells

Abstract

In the present study, we show that the eicosanoid compound, 20-hydroxyeicosatetraenoic acid (20-HETE), an important arachidonic acid metabolite, activates mouse TRPC6 in a stable, overexpressing HEK293 cell line, Hek-t6.11. Application of 20-HETE rapidly induced an inward, non-selective current in whole-cell recordings, which was inhibited by N-methyl-D-glucamine, 1.8 mM Ca 2؉ , and 100 M Gd 3؉ but remained unaffected by flufenamate and indomethacin. The current-voltage relationship obtained at low concentrations of 20-HETE (1-10 M) demonstrated slight inward rectification, whereas the highest concentration of 20-HETE tested (30 M) showed outward rectification, as shown previously for these channels using 100 M 1-oleoyl-2-acetylsn-glycerol. Dose-response curves indicate that 20-HETE activated TRPC6 channels with an EC 50 ‫؍‬ 0.8 M. Single channel analysis using inside-out patches revealed that 20-HETE increased open probability of mouse TRPC6 channels ϳ3-fold, and this was in a membrane-delimited fashion. Interestingly, 20-HETE did not provoke changes in intracellular Ca 2؉ concentrations. Thus, we have identified an arachidonic acid metabolite, 20-HETE, as a novel activator for a TRP family member, TRPC6.Cationic channels thought to be responsible for capacitative Ca 2ϩ entry, as originally defined by Putney (1, 2), have been identified in the plasmalemmal membrane of a spectrum of cells (3, 4). Capacitative Ca 2ϩ entry, through store-operated channels (SOCs) 1 is triggered by the emptying of intracellular Ca 2ϩ stores by a variety of maneuvers (4 -6). SOCs differ in their ionic selectivity, conductance, and sensitivity to inorganic and organic blockers. The best characterized SOC is the calcium release-activated calcium channel, a highly selective Ca 2ϩ ion channel first described in mast cells (7) and T lymphocytes (8), whereas non-selective cationic channels activated by store depletion have also been described (9). The molecular identity of store-operated channels has not yet been firmly established despite accumulating evidence that they could be members of the TRPC family (10 -12). The transient receptor potential (dTRP) channel, found in Drosophila eye, is a storeoperated Ca 2ϩ channel, whereas the trp-like (dTRPL) protein functions as a constitutively activated, non-selective cation channel (13-15). In mammals, seven dTRP homologs, TRPC1-TRPC7 (16 -18), have been found. Overexpression studies have demonstrated that these channels show important differences in a number of properties including their mode of activation, unitary conductance, and selectivity, which may or may not be a result of the expression system used (for reviews, see Refs. 4, 17, and 19). Moreover, the formation of heteromeric TRPC channels has been reported, whose characteristics were quite distinct from those recorded for the individual, homomeric channels; such behavior was shown for coassembly of TRPC1/ TRPC3 (20) and TRPC1/TRPC5 (21). A number of studies have reported that some members of the TRPC family can be activated by produ...

5-Oxo-ETE regulates tone of guinea pig airway smooth muscle via activation of Ca2+pools and Rho-kinase pathway

“…This result contrasts with the data recently reported to explain the inotropic effects and mode of action of 20-HETE on ASM cells, which are known to express various lipid-gated nonselective cationic TRPC channel isoforms (2,6,31). Among those, TRPC3 and -6 are recognized as direct modulators of resting smooth muscle tone (1,2,6). In contrast to Gd 3ϩ , nifedipine relaxes 48.1% the tension induced by 5-oxo-ETE, suggesting that L-type Ca 2ϩ channels (14) are directly involved in the control of ASM tone triggered by this eicosanoid.…”

“…In bronchi, Ca 2ϩ entry could also result from the activation of the capacitative Ca 2ϩ entry after depletion of intracellular Ca 2ϩ stores, as reported previously (6). Nevertheless, the tonic response induced by 5-oxo-ETE was poorly sensitive to 100 M Gd 3ϩ , a concentration known to block both capacitative and noncapacitative Ca 2ϩ entry (1,4,6), suggesting that this process contributes only marginally to the response to 5-oxo-ETE. This result contrasts with the data recently reported to explain the inotropic effects and mode of action of 20-HETE on ASM cells, which are known to express various lipid-gated nonselective cationic TRPC channel isoforms (2,6,31).…”

Mechanisms of Low-Glucose Sensitivity in Carotid Body Glomus Cells

“…These channels appear to belong to the TRPC3/6 subtypes, highly expressed in CB glomus cells (30) and having a pharmacology compatible with that observed for the background Na ϩ current (33,34). Heterologously expressed recombinant TRPC6 channels exhibit single-channel events quite similar to those observed in glomus cells after activation by low glucose or 1-oleoyl-2-acetyl-snglycerol (39). Interestingly, TRPC3 channels have been shown to be directly involved in Ca 2ϩ signaling, since Na ϩ influx is coupled with reversal activation of the Na ϩ /Ca 2ϩ exchanger, thus leading to the rise of cytosolic Ca 2ϩ (40).…”

Functional characterization and physiological relevance of the TRPC3/6/7 subfamily of cation channels

“…TRPC6 was the first ion channel identified that is activated by DAG in a membrane-delimited fashion, independently of protein kinases C. The exact location of a putative binding site for diacylglycerols in the TRPC6 protein is still elusive because an OAG-insensitive splice variant of TRPC6, (TRPC6B; Zhang and Saffen 2001) characterized by means of fluorometry turned out to be activated by DAG when analyzed by electrophysiological methods (Jung et al 2003). More recently, TRPC6 has also been reported to be sensitive to the arachidonic acid metabolite 20-hydroxyeicosatetraenoic acid (Basora et al 2003). In contrast to the scenario with TRPC6 which is a tightly receptor-regulated store-independent cation channel, TRPC3 and TRPC7 display considerable basal activity (see Fig.…”