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Figure 1. Effects of peptide mixes on ASIC1a current amplitude and desensitization. (a) Top, representative current traces of ASIC1a expressing oocytes conditioned with pH 7.25 (grey bars) and activated with pH 6.5 (black bars). Peptide mixes 1–13 (M1-M13) were present in the conditioning solution as indicated by the blue bars with a concentration of 20 μM per individual peptide. Bottom, scatter plot showing ratios of the peak currents after pre-application of peptides (I) to the mean of the two flanking control peak currents (Icontrol). Bars show the mean and error bars the SD. (b) Scatter plot showing ratios of time constants of desensitization after pre-application of peptides (τ) to the time constants of desensitization of the two flanking control activations (τcontrol). *p < 0.05, **p < 0.01, ***p < 0.001 (paired Student’s t-test).
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Figure 2. Dynorphin A(1–13) and dynorphin A(1–17) increase ASIC1a current amplitude. Top, representative current trace of an ASIC1a expressing oocyte conditioned with pH 7.25 (grey bars) and activated with pH 6.5 (black bars). Peptide mixes 7′ and 9′ (M7′ and M9′), dynorphin A(1–13) (DynA(1–13)) or dynorphin A(1–17) (DynA(1–17)) were present in the conditioning solution as indicated by the blue bars with a concentration of 20 μM per individual peptide. Mix 7′ contained all peptides of mix 7 except for dynorphin A(1–13), mix 9′ contained all peptides of mix 9 except for dynorphin A(1–17) (See Table 1). Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD. **p < 0.01, ***p < 0.001 (paired Student’s t-test).
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Figure 3. Effect of peptide mixes on ASIC1b current amplitude and desensitization. (a) Top, representative current traces of ASIC1b expressing oocytes conditioned with pH 7.1 (grey bars) and activated with pH 5.9 (black bars). Peptide mixes 1–13 (M1-M13) were present in the conditioning solution as indicated by the blue bars with a concentration of 20 μM per individual peptide. Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD. (b) Scatter plot showing ratios of τ to τcontrol. *p < 0.05, **p < 0.01 (paired Student’s t-test).
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Figure 4. YFMRFamide decreases peak currents and slows desensitization of ASIC1b. (a) Top, representative current trace of an ASIC1b expressing oocyte conditioned with pH 7.1 (grey bars) and activated with pH 5.9 (black bars). Individual peptides of mix 2.1 (M2.1) were applied as indicated by the blue bars with a concentration of 20 μM each: YFMRFamide (YFMRFa), neuromedin N (NM-N), NT-2 (fragment analogue of neurotensin) (NT), angiotensin IV (AT-4) and ORG2766 (ORG). Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD. (b) Scatter plot showing ratios of τ to τcontrol. **p < 0.01, ***p < 0.001 (paired Student’s t-test).
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Figure 5. Effect of peptide mixes on ASIC3 current amplitude and desensitization. (a) Top, representative current traces of ASIC3 expressing oocytes conditioned with pH 7.0 (grey bars) and activated with pH 6.5 (black bars). Peptide mixes 1–13 (M1-M13) were present in the conditioning solution as indicated by the blue bars with a concentration of 20 μM per individual peptide. Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD. (b) Scatter plot showing ratios of τ to τ control. *p < 0.05, **p < 0.01, ***p < 0.001 (paired Student’s t-test).
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Figure 6. Endomorphin-1 slows the desensitization of ASIC3. (a) Top, representative current trace of an ASIC3 expressing oocyte conditioned with pH 7.0 (grey bars) and activated with pH 6.5 (black bars). Individual peptides of mix 1.2 (M1.2) were applied as indicated by the blue bars with a concentration of 20 μM each: endomorphin-1 (EM-1), leu-enkephalin (L-Enk), met-enkephalin (M-Enk), long proCART(55–59) (CART) and angiotensin I/II(1–5) (AT1–5). Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD. (b) Scatter plot showing ratios of τ to τcontrol. **p < 0.01 (paired Student’s t-test).
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Figure 7. YFMRFamide slows the desensitization of ASIC3. (a) Top, representative current trace of an ASIC3 expressing oocyte conditioned with pH 7.0 (grey bars) and activated with pH 6.5 (black bars). Peptide mix 2′ (M2′) and YFMRFamide (YFMRFa) were present in the conditioning solution as indicated by the blue bars with a concentration of 20 μM per individual peptide. Mix 2′ contained all peptides of mix 2 except for YFMRFa. (See Table 1). Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD. (b) Scatter plot showing ratios of τ to τcontrol. (c) Top, representative current trace of an ASIC3 expressing oocyte conditioned with pH 7.4 (green bars) and activated with pH 6.5 (black bars). YFMRFamide (YFMRFa), RPRFamide (RPRFa) and FMRFamide (FMRFa) were present in the conditioning solution as indicated by the blue bars with a concentration of 20 μM per individual peptide. Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD. **p < 0.01 (paired Student’s t-test). (d) Scatter plot showing ratios of τ to τcontrol. *p < 0.05, **p < 0.01, ***p < 0.001 (paired Student’s t-test followed by Bonferroni correction).
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Figure 8. There is no direct agonist for ASIC4 and BASIC among the 109 neuropeptides. (a,b) Top, representative current traces of ASIC4 (a) or BASIC (b) expressing oocytes activated with divalent free solution (div free; red bars). Controls (ctrl; black bars) before each peptide mix (M; blue bars) contained the solvent in its respective concentration. Peptides were applied at pH 7.4. Bottom, bar graphs showing the mean current amplitudes; error bars show the SD. *p < 0.05 (paired Student’s t-test followed by Bonferroni correction).
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Supplementary Figure 1. Effect of submixes on ASIC1a current amplitude. Top,
representative current trace of an ASIC1a expressing oocyte conditioned with pH 7.25 (grey bars) and activated with pH 6.5 (black bars). Peptide mixes (M) 5.1, 5.2, 6.1, 6.2, 10.1 and 10.2 were present in the conditioning solution as indicated by the blue bars with a concentration of 20 µM per individual peptide. Bottom, scatter plot showing ratios of I to I control. Bars show the mean and error bars the SD. *p < 0.05, **p < 0.01 (paired Student’s t-test).
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Supplementary Figure 2. Effect of submixes on ASIC1b current amplitude. Top,
representative current trace of an ASIC1b expressing oocyte conditioned with pH 7.1 (grey bars) and activated with pH 5.9 (black bars). Peptide mixes (M) 2.1, 2.2, 6.1 and 6.2 were present in the conditioning solution as indicated by the blue bars with a concentration of 20 µM per individual peptide. Bottom, scatter plot showing ratios of I to I control. Bars show the mean and error bars the SD. *p < 0.05 (paired Student’s t-test).
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Supplementary Figure 3. Effect of submixes on ASIC3 current amplitude and
desensitization. (a) Top, representative current traces of ASIC3 expressing oocytes conditioned with pH 7.0 (grey bars) and activated with pH 6.5 (black bars). Peptide mixes (M) 1.1, 1.2, 8.1, 8.2, 9.1, 9.2, 6.1 or 6.2 were present in the conditioning solution as indicated by the blue bars with a concentration of 20 µM per individual peptide. Bottom, scatter plot showing ratios of I to I control. Bars show the mean and error bars the SD. (b) Scatter plot showing ratios of t to t control. *p < 0.05, ***p < 0.001 (paired Student’s t-test).
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Supplementary Figure 4. Effect of submixes on ASIC3 current amplitude. Top,
representative current trace of an ASIC3 expressing oocyte conditioned with pH 7.0 (grey bars) and activated with pH 6.5 (black bars). Peptide mixes 2.1’ and 2.2’(M2.1’, M2.2’) were present in the conditioning solution as indicated by the blue bars with a concentration of 20 µM per individual peptide. Bottom, scatter plot showing ratios of I to Icontrol. Bars show the mean and error bars the SD.
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