The Na+/Ca2+ exchange inhibitor KB-R7943 potently blocks TRPC channels

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Abstract

Na+/Ca2+ exchangers (NCXs) and members of the canonical transient receptor potential (TRPC) channels play an important role in Ca2+ homeostasis in heart and brain. With respect to their overlapping expression and their role as physiological Ca2+ influx pathways a functional discrimination of both mechanisms seems to be necessary. Here, the effect of the reverse-mode NCX inhibitor KB-R7943 was investigated on different TRPC channels heterologously expressed in HEK293 cells. In patch-clamp recordings KB-R7943 potently blocked currents through TRPC3 (IC50 = 0.46 μM), TRPC6 (IC50 = 0.71 μM), and TRPC5 (IC50 = 1.38 μM). 1-Oleoyl-2-acetyl-sn-glycerol-induced Ca2+ entry was nearly completely suppressed by 10 μM KB-R7943 in TRPC6-transfected cells. Thus, KB-R7943 is able to block receptor-operated TRP channels at concentrations which are equal or below those required to inhibit reverse-mode NCX activity. These data further suggest that the protective effects of KB-R7943 in ischemic tissue may, at least partly, be due to inhibition of TRPC channels.

Section snippets

Materials and methods

Cell culture and transfection. HEK293 cells were cultured as previously described [16]. The cDNAs of the following TRP channels were used for experiments: hTRPC3 (GenBank Accession Nos. NM003305), hTRPC6 (NM004621), and mTRPC5 (NM09428). Cells were plated onto 100 mm dishes and transfected with 1–1.5 μg of plasmid DNA using 5 μl of FUGENE 6 transfection reagent (Roche, Indianapolis, IN, USA) and 95 μl of OptiMEM medium (Invitrogen, Groningen, The Netherlands). Two or three days later cells were

Inhibition of currents through TRPC3, TRPC6, and TRPC5

Whole-cell voltage clamp experiments were performed to investigate the effects of KB-R7943 on canonical TRP channels heterologously expressed in HEK293 cells. Stable activation of currents through TRPC3 and TRPC6 channels can be induced after attaining the whole-cell configuration due to infusion of AlF4-, an unspecific activator of G proteins, added to the pipette solution [16], [17]. Non-transfected HEK293 cells showed negligible currents under these conditions [16].

Intracellular AlF4- (30 μM)

Discussion

The present data provide evidence that KB-R7943 potently inhibits the receptor-operated TRP channels TRPC3, TRPC5, and TRPC6. In this respect, our results are in contradiction with a previous report on a functional coupling between TRPC3 and NCX1 [20], where a possible block of TRPC3 channels by KB-R7943 was not found. In the latter study, acute effects of KB-R7943 on TRPC3-mediated currents were examined less extensively. Instead, pretreatment of TRPC3 expressing HEK293 cells with 5 μM KB-R7943

Acknowledgments

The author wishes to thank Gudrun Bethge for technical assistance, Jens Eilers for critical reading of the manuscript and Christian Harteneck for providing cDNA of hTRPC3, hTRPC6, and mTRPC5.

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