Supplementary Figure 1

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1 Supplementry Figure 1 c d Wistr SHR Wistr AF-353 SHR AF-353 n = 6 n = 6 n = 28 n = 3 n = 12 n = 12 Supplementry Figure 1 Neurophysiologicl properties of petrosl chemoreceptive neurones in Wistr nd SH rts. Control memrne potentils were mesured from physiologiclly chrcterized chemoreceptive petrosl neurones t high smpling frequency nd compred etween rt strins efore () nd fter AF-353 ppliction to the CB ((); 15 nl 2 mm, n = 1 or 11). The more depolrized potentils in SHR neurones (P <.1) ws olished fter P2X3 receptor lockde. (c): There ws no difference in memrne input resistnce of petrosl chemoreceptive neurones etween Wistr nd SH rts. (d): No difference ws found in petrosl memrne cpcitnce etween Wistr nd SHR for oth chemoreceptive nd non-chemoreceptive petrosl neurones. Nture Medicine: doi:1.138/nm.4173

2 2 mv 2 mv Supplementry Figure 2 WISTAR SHR WISTAR AF-353 SHR AF-353 c Tyrosine Hydoxylse e.5 s -57 mv -52 mv -61 mv -6 mv 1nA d Post-Synptic Density-95 f 1.5 s NCN -57mV Supplementry Figure 2. Chemoreceptive petrosl neurone chrcteristics in Wistr nd SH rts. (), (), The firing responses of chemoreceptive petrosl cells to injected current were more excitle in SH rts nd this ws reduced to levels seen in Wistr rts y locking P2X3 receptors within the crotid ody (not petrosl gngli) with AF-353 microinfusion. (c), In chemoreceptive petrosl cells there ws no difference in tyrosine hydroxylse (TH) mrna etween rt strins. (d), Post-synptic density protein 95 (PSD-95) mrna ws upregulted in chemoreceptive petrosl cells of SH rts indictive of more synptic contcts. (e), There ws no difference in P2x2 or P2x3 receptor mrna in non-chemoreceptive (unidentified) petrosl neurones etween SH nd Wistr rts. (f), There ws no rt strin difference in mrna of P2x2 or P2x3 receptors in identified chemoreceptive neurones locted in the nucleus trctus solitrii. Nture Medicine: doi:1.138/nm.4173

3 Supplementry Figure 3 Wistr P2X3 TH P2X3/TH Supplementry Figure 3 P2X3 receptor nd tyrosine hydroxylse (TH) immunofluorescence in the crotid ody of rts. Note the P2X3 receptor expression in TH positive cells (glomus cells) nd fires were more evident in the SH rts () compred with Wistr rts (). Scle rs 5 mm. SHR P2X3 TH P2X3/TH Nture Medicine: doi:1.138/nm.4173

4 2 mv Supplementry Figure 4 25 WISTAR SHR Firing freq (Hz) 2 s -53 mv -57 mv α,β-methyl ATP (1 µm),-atp α,β-methyl ATP (1 µm) Supplementry Figure 4 Chemoreceptive petrosl neurone sensitivity to --methylene ATP. In Wistr nd SH rts The stle nlogue of ATP (-methylene ATP evoked greter firing responses () nd memrne depolristions () in chemoreceptive petrosl neurones from SH (n = 13) thn Wistr rts (n = 1). All dt from the in situ rterilly perfused preprtion. Unpired t-test. AUC, re under the curve. *, P <.5. Concentrtion of ATP (mm) Nture Medicine: doi:1.138/nm.4173

5 Supplementry Figure 5 Concentrtion of AF-219 (ng/ml) mg/kg/h 4 mg/kg/h 8 mg/kg/h Oserved (1 mg/kg/h) Oserved (8 mg/kg/h) Oserved (8 mg/kg/h) Time (h) AF-219 Oservtion time points Supplementry Figure 5 Phrmcokinetic profile of AF-219 in rt plsm. The phrmcokinetic dt re indicted y symols for the three doses of AF219 (colour coded) infused i.v. into conscious rts (n = 7). Blood smples were tken t 6 nd 12 minutes nd highlighted y shded lue res. Solid lines represent modeled phrmcokinetic response to AF-219 t corresponding doses. Two rts were excluded from lood smpling due to technicl issues with the venous line. Nture Medicine: doi:1.138/nm.4173

6 Supplementry Figure S B P (m m H g ) V e h ic le 1 m g /k g 4 m g /k g 8 m g /k g B s e lin e A F In fu s io n R e c o v e ry D B P (m m H g ) B s e lin e A F In fu s io n R e c o v e ry H e r t r te ( p m ) B s e lin e A F In fu s io n R e c o v e ry Supplementry Figure 6 Crdiovsculr nd respirtory response dynmics during infusion of AF-219 in SH rts. Time profile of the chnge in systolic lood pressure (SBP); distolic lood pressure (DBP), hert rte nd respirtory rte with Vehicle (grey), 1mg/kg/h (lck); 4mg/kg/h (lue); 8 mg/kg/h (red) i.v. infusion of AF-219 in the SH rt (n = 7). Dt re corrected for vehicle effect. * P <.5, one-wy ANOVA with Dunnett s test. Dt points were excluded if rts overtly strted moving during the infusion period. R e s p ir to r y r te ( p m ) B s e lin e A F In fu s io n R e c o v e ry Nture Medicine: doi:1.138/nm.4173

7 Supplementry Figure S B P (m m H g ) S H R S H R /C B R W S T B s e lin e A F In fu s io n R e c o v e ry D B P (m m H g ) B s e lin e A F In fu s io n R e c o v e ry H e r t r te ( p m ) B s e lin e A F In fu s io n R e c o v e ry B s e lin e A F In fu s io n R e c o v e ry Supplementry Figure 7 Dependence on crotid ody for the crdiovsculr nd respirtory responses following AF-219 infusion into SH rts. Time profile of the reduction in systolic lood pressure (SBP); distolic lood pressure (DBP), hert rte nd respirtory rte with 8 mg/kg/h i.v. infusion of AF-219 in the SH rts (n = 7) efore nd fter selective crotid ody resection (SHR/CBR). Note sence of response in the normotensive Wistr rt (n = 7). Dt re corrected for vehicle effect, *, P <.5, one-wy ANOVA with Dunnett s test. Dt points were excluded if rts overtly strted moving during the infusion period. R e s p ir to r y r te ( p m ) Nture Medicine: doi:1.138/nm.4173

8 Supplementry Figure 8 Bsl Reflex Reflex Supplementry Figure 8 Bsl nd chemoreflex evoked sympthetic ctivity responses fter TNP-ATP (1 µm) microinfused into the crotid ody of Wistr nd SH rts. TNP-ATP is high ffinity, nonselective P2X receptor ntgonist tht inhiits P2X 1, P2X 3 nd heteromeric P2X 2/3 with 1- fold selectivity over P2X 2, P2X 4 nd P2X 7 receptors tht is structurlly distinct to AF-353. These dt support the selectivity of AF-353 in locking P2X3 homomeric nd P2X2/3 heterotrimeric receptors rther thn non-selective off-trget effects. The two reflex r chrts relte to the sympthetic ctivity tht is inspirtory or expirtory modulted.. Nture Medicine: doi:1.138/nm.4173

9 L F :H F (H R ) H F (H R ) Supplementry Figure A F In fu s io n S H R 8 m g /k g S H R C B R 8 m g /k g R e c o v e ry c sbrg (ms/mmhg) AF-219 Infusion Recovery Time (min) A F In fu s io n R e c o v e ry Supplementry Figure 9 Spectrl nlysis of the hert rte nd spontneous crdic roreflex during infusion of AF-219. We found no chnge in the high frequency spectr () nor chnge in the low to high frequency rtio (LF:HF; ()) following lockde of P2X3 receptors with AF-219 (8 mg/kg/h; n = 7). However, there ws significnt improvement in the spontneous roreceptor reflex gin of hert rte (c). * P <.5, ** P <.1. One-wy ANOVA with Dunnett s test. Nture Medicine: doi:1.138/nm.4173

10 Supplementry Figure 1 Hum CB P2X3 P2X3 + Peptide Rt CB P2X3 P2X3 + Peptide c Rt CB; Immunocytochemistry Wistr TH, P2X3/Peptide SHR TH, P2X3/Peptide 66kD 44kD Supplementry Figure 1 P2X3 receptor ntiody specificity testing for western lot nd immunohistochemistry. Antiody specificity ws determined y western lotting on humn (Hum; ()) nd rt () crotid odies (CB). Memrnes were incuted with nti-p2x3 ntiody in the sence (left lne) or presence (right lne) of the locking peptide. The sence of the nd on the western lot performed in the presence of the locking peptide reveled tht the P2X3 receptor migrtes t 44 kd in humn nd t 66 kd in rt. (c), the specificity of the P2X3 receptor ntiody for immunocytochemistry ws tested on CBs from Wistr nd SHR using the locking peptide. No P2X3 receptor immunofluorescence ws detected. Glomus cells were identified using tyrosine hydroxylse (TH) immunocytochemistry. Scle rs 1 mm. Nture Medicine: doi:1.138/nm.4173

11 Supplementry Figure 11 P2X3/DAPI P2X3/DAPI/TH Supplementry Figure 11 P2X3 receptor immunofluorescence within the humn crotid ody. () shows P2X3 receptor expression in presumed glomus cells. These dt re from different cdver to those shown in Fig 6. Scle r 25 mm. () Superimposed P2X3, DAPI nd tyrosine hydroxylse (TH) immnunofluorescence stining of xons within the CB (exmples rrowed) tht were immunopositive for P2X3 receptors nd TH nd pper yellow. These fires could e petrosl fferents or sympthetic efferents, lthough the ltter is unlikely. Scle r 5 mm. Nture Medicine: doi:1.138/nm.4173

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