|
Figure 1. The inhibition by PcTx1 is pH dependent. ASIC1a currents were repeatedly activated by pH 5.0. They were completely inhibited by application of the P. cambridgei venom (1:20,000 dilution) for 60 s at pH 7.4. In contrast, the venom did not inhibit the current, when it was applied at pH 7.9. Only a linear current rundown can be observed that is usual for ASIC1a and does not depend on the presence of the venom.
|
|
Figure 2. PcTx1 caused a robust shift of the steady-state desensitization curve of ASIC1a to lower H+ concentrations. (A) Apparent affinity of synthetic PcTx1 to ASIC1a. Left, an example of ASIC1a currents inhibited by 20 nM PcTx1 (applied for 125 s in the conditioning period). Currents were elicited by a pH drop from pH 7.4 to pH 6. Right, dose–response relationship for inhibition of ASIC1a currents by PcTx1. The first test current after PcTx1 application was normalized to the test current just before the toxin application. The line represents a fit to the Hill equation (IC50 = 3.7 nM, Hill coefficient = 1.65). Each data point represents mean ± SEM (n = 5–7). (B) Representative traces of ASIC1a currents evoked by application of pH 6.0 with varying conditioning pH as indicated. Top, without PcTx1, bottom, with PcTx1 (30 nM). Conditioning pH was applied for 120 s. Holding potential was −70 mV. (C) H+ dependence of steady-state desensitization of ASIC1a currents in the absence (open circles, n = 8) or presence (filled squares, n = 5) of 30 nM PcTx1.
|
|
Figure 3. Kinetics of inhibition by PcTx1. (A, i) ASIC1a current traces illustrating inhibition by PcTx1. PcTx1 (30 nM) was applied for 50 and 100 s, respectively. (A, ii) Current peak amplitudes (normalized to the current amplitude before applying the toxin) are plotted against the corresponding time period of PcTx1 application. Each data point represents the mean ± SEM of 5–7 individual measurements. The time constant for onset of inhibition, obtained by fitting the mean data to a single exponential function, was 52 s. (B, i) Representative current traces of ASIC1a channels recovering from inhibition by PcTx1 (30 nM, applied for 150 s). ASIC1a channels that had recovered from inhibition were assessed every 70 s by application of pH 6. The conditioning period between channel activation was 60 s, allowing ASIC1a to fully recover from low pH–induced desensitization. (B, ii) Current peak amplitudes (normalized to the maximal current amplitude after toxin washout) are plotted against the corresponding time after washout. Each data point represents the mean ± SEM of 10 individual measurements. The time constant for recovery from inhibition was 125 ± 56 s.
|
|
Figure 4. PcTx1 increased the apparent affinity for H+ of ASIC1a. (A) Representative current traces elicited by pH ranging from 7.1 to 6.0 with conditioning pH 7.9 (applied for 70 s). Top, without PcTx1, bottom, with PcTx1 (30 nM). (B) H+ dependence of ASIC1a activation in the absence (n = 7, open circles) or in the presence (n = 7, filled squares) of 30 nM PcTx1. PcTx1 significantly (P < 0.01) shifted the pH activation curve to lower H+ concentrations.
|
|
Figure 5. Ca2+ dependence of the shift of the steady-state desensitization curve. (A) Representative traces of ASIC1a currents evoked by application of pH 6.0 with varying conditioning pH as indicated. Top, without PcTx1, bottom, with PcTx1 (30 nM). Conditioning solution contained 0.1 mM Ca2+ (left) or 10 mM Ca2+/1.0 mM Mg2+ (right) and was applied for 120 s. Solution of pH 6.0 always contained 1.8 mM Ca2+/1.0 mM Mg2+. (B) H+ dependence of steady-state desensitization of ASIC1a currents in the absence (open circles, 0.1 mM Ca2+, n = 7; open squares, 10 mM Ca2+/1.0 mM Mg2+, n = 8) or presence (filled circles, 0.1 mM Ca2+, n = 6; filled squares, 10 mM Ca2+/1.0 mM Mg2+, n = 5) of 30 nM PcTx1. At low Ca2+ concentrations, PcTx1 caused a more robust shift (by 0.42 pH units) of the steady-state desensitization curve than at high Ca2+ concentrations (by 0.16 pH units). Note that Ca2+ itself also shifts the steady-state desensitization curves.
|
|
Figure 6. Ca2+ inhibits the binding of PcTx1 to ASIC1a channels. (A) 30 nM PcTx1 was applied together with either 0.1 mM Ca2+ (top) or 10 mM Ca2+/1.0 mM Mg2+ (bottom), in the conditioning solution (pH 8.0). Test solution always contained 1.8 mM Ca2+/1.0 mM Mg2+, pH 6.7. (B) The potentiation of the ASIC1a current, due to increased affinity to H+, is expressed as the ratio of peak currents after and before application of PcTx1. White bar, 0.1 Ca2+ (n = 6); black bar, 10 mM Ca2+/1.0 mM Mg2+ (n = 6). High Ca2+ significantly (P < 0.01) reduced the potentiation of ASIC1a currents by PcTx1.
|
|
Figure 7. Ca2+ speeds recovery from inhibition by PcTx1. (A) Representative traces of ASIC1a currents recovering from the inhibition by PcTx1 (30 nM). Protocol as in Fig. 3 B, but with 0.1 mM Ca2+ and pH 7.55 (top) or 10 mM Ca2+/1.0 mM Mg2+ and pH 7.06 (bottom) during the conditioning period. The different pH values were chosen to account for the influence of Ca2+ on the speed of recovery from desensitization. (B) Current peak amplitudes (normalized to the maximal current amplitude) are plotted against the corresponding time after toxin washout. The time constants, obtained by fitting the data to a mono-exponential function, were 441 ± 141 s (0.1 Ca, open circles, n = 7), 125 ± 56 s (1.8 Ca, open squares, n = 10), and 118 ± 38 s (10 Ca, filled diamonds, n = 6). Data for 1.8 Ca2+ are from Fig. 3.
|
|
Figure 8. PcTx1 directly opens ASIC1a at certain conditions. (A) At pH 7.35, 100 nM PcTx1 elicited transient inward currents when coapplied with low Ca2+ concentrations (0.1 mM, left, n = 5). These PcTx1-elicited currents could be blocked by 100 μM amiloride (right, n = 4). (B) When coapplied with pH 7.1, 100 nM PcTx1 elicited transient inward currents (left, n = 5), which could be blocked by 100 μM amiloride (right, n = 3).
|
|
Figure .
|
