Riluzole activates TRPC5 channels independently of PLC activity
Version of Record online: 10 DEC 2013
© 2013 The British Pharmacological Society
British Journal of Pharmacology
Volume 171, Issue 1, pages 158–170, January 2014
How to Cite
Richter, J. M., Schaefer, M. and Hill, K. (2014), Riluzole activates TRPC5 channels independently of PLC activity. British Journal of Pharmacology, 171: 158–170. doi: 10.1111/bph.12436
- Issue online: 10 DEC 2013
- Version of Record online: 10 DEC 2013
- Accepted manuscript online: 4 OCT 2013 04:05AM EST
- Manuscript Accepted: 15 SEP 2013
- Manuscript Revised: 5 SEP 2013
- Manuscript Received: 5 JUN 2013
Figure S1 U73343, the inactive analogue of U73122, does not interfere with the riluzole-mediated TRPC5 activation. Whole cell patch clamp recording of T-RExTRPC5 (Tet+) cells pretreated with U73343 (5 μM). (A) Representative whole cell recording. Data were extracted from voltage ramps and represent current densities at +100 mV (upper trace) and −100 mV (lower trace) in response to Amix, riluzole (50 μM), and 2-APB (75 μM). (B) I/V curves for basal (1), Amix (2)-, and riluzole (3)-induced currents. (C) Statistical analysis of several experiments such as in A (*P < 0.05, n.s.: no significant difference). Data represent means and SEM of 5 independent experiments.
Figure S2 Riluzole activates TRPC5 in cell-attached patches. Examples of cell-attached recordings on tetracycline induced T-RExTRPC5 (Tet+; upper trace) and non-induced T-RExTRPC5 (Tet-; lower trace) cells at −80 mV. Pipette and bath solution consist of standard extracellular buffer, the pipette solution was additionally supplemented with 100 μM riluzole or with 100 μM riluzole together with 75 μM 2-APB as indicated. (Right-hand figure) The NPo was averaged for 30s intervals and represent means and SEM of several independent experiments. Numbers of individual experiments are stated in the figure.
Figure S3 Gene expression of TRPC1, TRPC5 and GAPDH in U87 cells, which were transiently transfected with TRPC5-specific siRNA (100 pmol, si C5) or unspecific scrambled siRNA (100 pmol, si sc). Untreated U87 cells served as a control. Total RNA was isolated by using RNeasy Mini Kit purchased from Qiagen. The RNA (1 μg) was reverse transcribed to obtain cDNA by using M-MuLV Reverse Transcriptase according to the manufacturer's instructions (NEB: Ipswich, MA, USA). The PCRs were carried out on PTC-200 DNA Engine Cycler (Bio-Rad: Hercules, CA, USA) by using GoTaq DNA Polymerase Kit obtained from Promega (Madison, WI, USA). The following primers were used: TRPC1 forward primer: AGTGACGAGCCTCTTGACAAAC, reverse primer: GGGCTTGCGTCGGTAAC. TRPC5 forward primer: AACTCCCTCTACCTGGCAACTA, reverse primer: GGATATGAGACGCAACGAACTT. GAPDH forward primer: CTCATTTCCTGGTATGAC, reverse primer: GAGCACAGGGTACTTTAT. The cDNA was amplified for 35 cycles (TRPC1, TRPC5) or 22 cycles (GAPDH) and PCR products (5 μL) were subjected to 1% agarose gel electrophoresis.
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