SUPPLEMENTARY INFORMATION. The Calcium-activated Chloride Channel Anoctamin 1 acts as a Heat. Sensor in Nociceptive Neurons

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1 SUPPLEMENTARY INFORMATION The Calcium-activated Chloride Channel Anoctamin 1 acts as a Heat Sensor in Nociceptive Neurons Hawon Cho, Young Duk Yang, Jesun Lee, Byeongjoon Lee, Tahnbee Kim Yongwoo Jang, Seung Keun Back, Heung Sik Na, Brian D Harfe, Fan Wang, Ramin Raouf, John N Wood, and Uhtaek Oh. 1

2 Supplementary Figure 1. Pharmacological and biophysical properties of heat-induced ANO1 currents. 2

3 (a) Left, Heat evoked inward currents in 0 Ca 2+ condition in ANO1/HEK cells (E hold = 60 mv). The pipette solution contained 10 mm of BAPTA. Right, a summary of heat-induced currents in the absence of intracellular Ca 2+. Numbers in parenthesis are experiment numbers. (b) Current-voltage relationship of heat-evoked currents in ANO1/HEK cells. Voltage ramps of 350 ms duration were applied from 100 to +100 mv. The pipette and bath solutions contained 140 mm NMDG-Cl. (c) Relative anion permeabilities of heat-evoked currents in ANO1- expressing cells. The pipette solution contained 140 mm NMDG-Cl. Bath solutions contained equimolar amounts of NaF and NaCl, NaBr, NaI or NaNO 3. (d) Mefloquine inhibited heat-evoked currents in ANO1/HEK cells. Mefloquine (5 µm) was applied 5 min before the second heat stimulus. (e) A summary of the effects of mefloquine on heat evoked currents inano1/hek 293T cells. *** p < versus the untreated control (I 0 ) (Student s unpaired t-test 2 tailed). 3

4 Supplementary Figure 2. The activation of ANO1 by heat in inside-out patches. (a) Application of heat to isolated an inside-out patch failed to evoke currents in non-transfected HEK 293T (CTL) cells (upper panel) whereas it evoked robust inward currents in a patch isolated from an ANO1-HEK cell (ANO1). (b) Summary of single-channel currents of ANO1 activated by heat (n = 5, Student s unpaired t-test 2 tailed, **p<0.001 versus the CTL). 4

5 Supplementary Figure 3. Synergistic effects of heat and Ca 2+ on ANO1 currents. (a) Sub-threshold heat (40 o C) markedly augmented ANO1 current activated by supra-maximal Ca 2+ (10 µm). (b) Summary of ANO1 currents by Ca 2+ and heat (n = 6, Student s unpaired t- test 2 tailed, **p<0.01). 5

6 Supplementary Figure 4. Mefloquine blocked heat-evoked currents in DRG neurons from Trpv1 -/- mice (a), but not in TRPV1/HEK cells (b). Right, Summaries of the effects of 100 µm mefloquine on heat-evoked currents in DRG neurons isolated from Trpv1 / mice. Mefloquine (10 µm) was applied 5 min before the second heat stimulus. *** p < versus control, Student s unpaired t-test 2-tailed. 6

7 Supplementary Figure 5. Comparison of the transcript levels of ANO1 and ANO2 in DRG cells. Mean relative mrna levels of ANO1 and ANO2 were estimated with RTQ- PCR normalized with GAPDH. Each reaction was performed in triplicate. ANO1 relative mrna level was approximately 10 fold greater than ANO2. ***p<0.001 versus ANO1 (Student s unpaired t-test 2 tailed) 7

8 Supplementary Figure 6. ANO1 antagonist failed to reduce hyperalgesic or allodynic responses of rats to mechanical stimuli. Carrageenan was injected into right hindpaws to induce inflammation. Three hours later, hindpaws were stimulated with von Frey hairs (the von Frey test) (a) or with a blunt plastic needle (the Randall Selitto test) (b). Mefloquine (MFQ, 40 mg/kg, i.p.) was administered one hour before the test. ***p<0.001 versus CTL (right paw). Statistical analyses were performed using one way ANOVA followed Tukey s post-hoc test. 8

9 Supplementary Figure 7. Heat-evoked Cl currents in DRG neurons isolated from scrambled sirna-treated (a) and from ANO1 sirna-treated mice (b). (c) Summary of heat-evoked currents in DRGs from scrambled sirna- or ANO1 sirna-treated mice. **p<0.01 versus scrambled sirna (Student s unpaired t-test 2 tailed) 9

10 Supplementary Figure 8. Balance, motor or locomotive behaviors of control and ANO1 CKO mice. (a) Body weights of CTL and CKO mice were not different (p < 0.357, Student s unpaired t-test 2 tailed,). (b) Rotarod test was performed to examine difference of balance and motor coordination in CTL and ANO1 CKO mice. The latency to fall was not different in CTL and ANO1 CKO mice (p < 0.626, Student s unpaired t-test 2 tailed) (c) In open field test, CTL and CKO ANO1 mice didn t show difference in the total distance traveled (p <0.789, Student s unpaired t-test 2 tailed). 10

11 Supplementary Figure 9. Reverse-transcriptase (RT)-PCR analysis of ano1, trpm3 and stim1 in ANO1-transfected and GFP-transfected HEK293T cells. 11