Nitric Oxide Lacks Direct Effect on TRPC5 Channels but Suppresses Endogenous TRPC5-Containing

SUPPLEMENTARY INFORMATION

Nitric oxide lacks direct effect on TRPC5 channels but suppresses endogenous TRPC5-containing channels in endothelial cells

†1,2Wong, C.O., †3,4Sukumar, P., *3,4Beech, D.J. & *1,2Yao, X.

1School of Biomedical Sciences, 2Li Ka Shing Institute of Health Sciences, The ChineseUniversity of Hong Kong, Hong Kong, China. 3Multidisciplinary Cardiovascular Research Centre and 4Institute of Membrane & Systems Biology, Faculty of BiologicalSciencesUniversity of Leeds, Leeds, LS2 9JT, UK.

*Authors for correspondence: Prof X Yao (School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, China) or Prof DJ Beech (Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, England, UK).

†These authors contributed equally.

Supplementary figure legends

Suppl. Fig. 1 TRPC5expression in TRPC5-overexpressing HEK293 cells.

aRepresentative immunoblot showing increased TRPC5expression in mTRPC5-overexpressing HEK293 cells. These cells were stably transfected with vector or mTRPC5. bRepresentative immunoblot comparing TRPC5 protein level in Tet-induced (Tet+) and not induced (Tet-) HEK293 cells. These cells were stably transfected with tetracycline-induciblehuman TRPC5. The anti--actin antibody was used to confirm equal loading of lanes. Masses of protein markers are indicated on the left. n=3.

Suppl. Fig. 2Effect of SNAP in a minority of HEK293 cells that were transfected with vectoror mTRPC5.

Time courses of single-cell [Ca2+]i changesin some batches of cells that showed responses to SNAP (300 M).Each trace represents the mean ± SEM data of 20 cells.Suchresponseswere observed in 5 out of 23 batches of mTRPC5-transfected (b)and 4 out of 32 batches of vector-transfected (a)cells

Suppl. Fig. 3 Complete original data sets for SNAP studies on HEK 293 cells conditionally induced (Tet+) or non-induced (Tet-, control) to express human TRPC5.

Time-series graphs indicating multi-well Ca2+ measurements andshowing results for addition of SNAP(at the concentrations indicated inµM), DMSO (0.2 %) or Gd3+ (100µM). Five independent test/control (Tet+/Tet-) experiments are shown (a&b, c&d, e&f, g&h, i&j). Each is a repeat of the type of experiment shown in Fig 1d&e of the main paper and a&b are the same data set as Fig 1d&e but shown in full. All data points represent the mean±SEM for ≥ 3wells.

Suppl. Fig. 4 Selectivity of T5E3 antibody for TRPC5-mediated Ca2+ influx.

a-bInhibitory effect ofT5E3 onLaCl3 (La3+, 100 µM)and carbachol (CCh, 100 µM)-induced [Ca2+]i rise in TRPC5-overexpressingHEK293 cells.La3+ and carbachol are two known activator of TRPC5.a-bRepresentative time courses of [Ca2+]i changes with (b) or without (a) 4 µg/ml T5E3 in the bath solution. c-d Ineffectiveness ofT5E3 onOAG (100 µM)-induced [Ca2+]i rise in TRPC3-overexpressingHEK293 cells. OAG is a known activator of TRPC3. c-dRepresentative time courses of [Ca2+]i changes with 4 µg/ml preimmune IgG (c) or T5E3 (d) in the bath solution. (a-d) Each trace in represents the mean ± SEM of 20 cells in a representative experiment.Total number of independent experiments: 3-4.

Suppl. Fig. 5Data generated using mTRPC5 and SNAP provided by Yoshida et al (2006).

Single-cell Ca2+-imaging data obtained using mouse TRPC5kindly provided by Y. Mori and using the transient transfection protocolfrom Yoshida et al 2006 [Nat Chem Biol 2:596-607]. In brief, transiently transfected cells were trypsinized after 16 hours and seeded and cultured on coverslip for 24 hours before recordings.The bath solution wasHBS and [Ca2+]iwas detected bysingle-cell Ca2+-imaging system (see main manuscript). Arrows indicate the time points when chemicals were added. Each trace represents the mean ± SEM from20 cellsin onerepresentative experiment (typical of 4-6 independent experiments). SNAP (provided by Yoshida et al), 300 µM; carbachol (CCh), 100 µM; LaCl3 (La3+), 100 µM; 2-APB, 75 µM. Note that mTRPC5-transfected cells showed a prolonged [Ca2+]irise in response to CCh and La3+, and this [Ca2+]i rise could be inhibited by 2-APB, a TRPC5 inhibitor. In contrast, the [Ca2+]i rise to CCh in vector-transfected cells is transient. Both types of cells failed to respond to SNAP.

Suppl. Fig. 6 Involvement of TRPC5 in ATP-stimulated whole-cell currents in BAECs.

aCurrent-voltage relationships (I-Vs) of ATP (100 µM)-stimulated whole-cell current in BAECs, calculated by subtracting the basal currents (before ATP-application) from that at 1 minute after ATP-application. The cells were pre-incubated with T5E3 (T5E3) or pre-immune IgG (pre-immune) for 30 min.b Percentage increase in whole-cell currents at -80 mV in response toATPstimulation.Mean ± SEM (n = 4;*, p<0.05).Whole-cell currents of single BAEC were recorded by using an EPC7patch clamp amplifier (HEKA) controlled by Pulse software (HEKA). The patch pipette contained in mM: 115 Cs-aspartate, 10 EGTA, 2 MgCl2, 5 Na2ATP, 0.1 NaGTP, 10 Hepes, 5.7 CaCl2(pH 7.2 with CsOH). The extracellular solution was HBS.A 0.5-s ramp protocol from –100 mV to +100 mV was applied at a frequency of 0.1 Hz from a holding potential of 0 mV. A liquid junction potential of +15 mV was corrected after the recordings.