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Proton and non-proton activation of ASIC channels

View Article: PubMed Central - PubMed

ABSTRACT

The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH values below 7.0. By contrast, non-proton ligands are able to trigger sustained ASIC currents at physiological pHs. To analyze the functional basis of the ASIC desensitizing and sustained currents, we have used ASIC1a and ASIC2a mutants with a cysteine in the pore vestibule for covalent binding of different sulfhydryl reagents. We found that ASIC1a and ASIC2a exhibit two distinct currents, a proton-induced desensitizing current and a sustained current triggered by sulfhydryl reagents. These currents differ in their pH dependency, their sensitivity to the sulfhydryl reagents, their ionic selectivity and their relative magnitude. We propose a model for ASIC1 and ASIC2 activity where the channels can function in two distinct modes, a desensitizing mode and a sustained mode depending on the activating ligands. The pore vestibule of the channel represents a functional site for binding non-proton ligands to activate ASIC1 and ASIC2 at neutral pH and to prevent channel desensitization.

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Related in: MedlinePlus

Sustained and desensitizing current of the ASIC2a-A427C mutant.A. pH-dependence of the maximal inward current (black), of the desensitizing current (purple) and the sustained current (red) of the ASIC2a-A427C. Each point represents the mean ± SE of 3 to 5 independent measurements. Curve fit of Imax represents the sum of the individual fits obtained for Idesens and Isust. B. pH-dependence of the Isust recorded from oocytes expressing ASIC2a-A427C after pre-incubation with 100 μM of MTSET (red circles, MTSPTrEA (purple squares), MTSBT (blue triangles), or MTSES (black diamonds). Symbols represent means ± SE of 3 to 15 independent measurements. Isust values were normalized for the maximal inward current elicited at pH 4.0; dashed lines represent best fit to current data. C. SSD determined for ASIC2a wt, A427C, and A427C incubated with MTSET; currents elicited by acidic pH 4.0, and measured after incubation at a conditioning pH ranging from 7.8 to 4.0. Symbols represent means ± SEM of 7 to 8 independent measurements. D. Current-voltage relations obtained for Imax, Isust, and Idesens currents of ASIC2a-A427C unmodified by MTS-reagents; reversal potentials were (mean ± SE) 0.3 ± 1.8 mV, -8.7 ± 2.4 mV and 5.8 ± 1.3 mV (p<0.01), respectively (n = 5). Similar values were obtained for ASIC2wt for these three currents, respectively 1.2 ± 4.5 mV, -7.1 ± 4.0 mV and 12.5 ± 4.5 mV (n = 5).
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pone.0175293.g010: Sustained and desensitizing current of the ASIC2a-A427C mutant.A. pH-dependence of the maximal inward current (black), of the desensitizing current (purple) and the sustained current (red) of the ASIC2a-A427C. Each point represents the mean ± SE of 3 to 5 independent measurements. Curve fit of Imax represents the sum of the individual fits obtained for Idesens and Isust. B. pH-dependence of the Isust recorded from oocytes expressing ASIC2a-A427C after pre-incubation with 100 μM of MTSET (red circles, MTSPTrEA (purple squares), MTSBT (blue triangles), or MTSES (black diamonds). Symbols represent means ± SE of 3 to 15 independent measurements. Isust values were normalized for the maximal inward current elicited at pH 4.0; dashed lines represent best fit to current data. C. SSD determined for ASIC2a wt, A427C, and A427C incubated with MTSET; currents elicited by acidic pH 4.0, and measured after incubation at a conditioning pH ranging from 7.8 to 4.0. Symbols represent means ± SEM of 7 to 8 independent measurements. D. Current-voltage relations obtained for Imax, Isust, and Idesens currents of ASIC2a-A427C unmodified by MTS-reagents; reversal potentials were (mean ± SE) 0.3 ± 1.8 mV, -8.7 ± 2.4 mV and 5.8 ± 1.3 mV (p<0.01), respectively (n = 5). Similar values were obtained for ASIC2wt for these three currents, respectively 1.2 ± 4.5 mV, -7.1 ± 4.0 mV and 12.5 ± 4.5 mV (n = 5).

Mentions: Graphs in Fig 10 illustrate the main characteristics of the ASIC2a-A427C currents before and after modification by MTS-reagents. ASIC2a-A427C shows both Isust and Idesens. The Isust of A427C is smaller in magnitude than Idesens at pH 4, and neither current was detected at pH 7.0 and above (Fig 10A). The pH0.5 for activation of Isust was more alkaline than for Idesens, but still remains below neutral pH. After modification by MTSET, MTSPTrEA, or MTSBT, Isust could be detected above pH 7.0 and further increased up to values corresponding to the maximal current of ASIC2a-A427C when the pH drops below 7.0 (Fig 10B). MTSES was without effect on the Isust, but induced a slight alkaline shift of Idesens (Table 3).


Proton and non-proton activation of ASIC channels
Sustained and desensitizing current of the ASIC2a-A427C mutant.A. pH-dependence of the maximal inward current (black), of the desensitizing current (purple) and the sustained current (red) of the ASIC2a-A427C. Each point represents the mean ± SE of 3 to 5 independent measurements. Curve fit of Imax represents the sum of the individual fits obtained for Idesens and Isust. B. pH-dependence of the Isust recorded from oocytes expressing ASIC2a-A427C after pre-incubation with 100 μM of MTSET (red circles, MTSPTrEA (purple squares), MTSBT (blue triangles), or MTSES (black diamonds). Symbols represent means ± SE of 3 to 15 independent measurements. Isust values were normalized for the maximal inward current elicited at pH 4.0; dashed lines represent best fit to current data. C. SSD determined for ASIC2a wt, A427C, and A427C incubated with MTSET; currents elicited by acidic pH 4.0, and measured after incubation at a conditioning pH ranging from 7.8 to 4.0. Symbols represent means ± SEM of 7 to 8 independent measurements. D. Current-voltage relations obtained for Imax, Isust, and Idesens currents of ASIC2a-A427C unmodified by MTS-reagents; reversal potentials were (mean ± SE) 0.3 ± 1.8 mV, -8.7 ± 2.4 mV and 5.8 ± 1.3 mV (p<0.01), respectively (n = 5). Similar values were obtained for ASIC2wt for these three currents, respectively 1.2 ± 4.5 mV, -7.1 ± 4.0 mV and 12.5 ± 4.5 mV (n = 5).
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pone.0175293.g010: Sustained and desensitizing current of the ASIC2a-A427C mutant.A. pH-dependence of the maximal inward current (black), of the desensitizing current (purple) and the sustained current (red) of the ASIC2a-A427C. Each point represents the mean ± SE of 3 to 5 independent measurements. Curve fit of Imax represents the sum of the individual fits obtained for Idesens and Isust. B. pH-dependence of the Isust recorded from oocytes expressing ASIC2a-A427C after pre-incubation with 100 μM of MTSET (red circles, MTSPTrEA (purple squares), MTSBT (blue triangles), or MTSES (black diamonds). Symbols represent means ± SE of 3 to 15 independent measurements. Isust values were normalized for the maximal inward current elicited at pH 4.0; dashed lines represent best fit to current data. C. SSD determined for ASIC2a wt, A427C, and A427C incubated with MTSET; currents elicited by acidic pH 4.0, and measured after incubation at a conditioning pH ranging from 7.8 to 4.0. Symbols represent means ± SEM of 7 to 8 independent measurements. D. Current-voltage relations obtained for Imax, Isust, and Idesens currents of ASIC2a-A427C unmodified by MTS-reagents; reversal potentials were (mean ± SE) 0.3 ± 1.8 mV, -8.7 ± 2.4 mV and 5.8 ± 1.3 mV (p<0.01), respectively (n = 5). Similar values were obtained for ASIC2wt for these three currents, respectively 1.2 ± 4.5 mV, -7.1 ± 4.0 mV and 12.5 ± 4.5 mV (n = 5).
Mentions: Graphs in Fig 10 illustrate the main characteristics of the ASIC2a-A427C currents before and after modification by MTS-reagents. ASIC2a-A427C shows both Isust and Idesens. The Isust of A427C is smaller in magnitude than Idesens at pH 4, and neither current was detected at pH 7.0 and above (Fig 10A). The pH0.5 for activation of Isust was more alkaline than for Idesens, but still remains below neutral pH. After modification by MTSET, MTSPTrEA, or MTSBT, Isust could be detected above pH 7.0 and further increased up to values corresponding to the maximal current of ASIC2a-A427C when the pH drops below 7.0 (Fig 10B). MTSES was without effect on the Isust, but induced a slight alkaline shift of Idesens (Table 3).

View Article: PubMed Central - PubMed

ABSTRACT

The Acid-Sensing Ion Channels (ASIC) exhibit a fast desensitizing current when activated by pH values below 7.0. By contrast, non-proton ligands are able to trigger sustained ASIC currents at physiological pHs. To analyze the functional basis of the ASIC desensitizing and sustained currents, we have used ASIC1a and ASIC2a mutants with a cysteine in the pore vestibule for covalent binding of different sulfhydryl reagents. We found that ASIC1a and ASIC2a exhibit two distinct currents, a proton-induced desensitizing current and a sustained current triggered by sulfhydryl reagents. These currents differ in their pH dependency, their sensitivity to the sulfhydryl reagents, their ionic selectivity and their relative magnitude. We propose a model for ASIC1 and ASIC2 activity where the channels can function in two distinct modes, a desensitizing mode and a sustained mode depending on the activating ligands. The pore vestibule of the channel represents a functional site for binding non-proton ligands to activate ASIC1 and ASIC2 at neutral pH and to prevent channel desensitization.

No MeSH data available.


Related in: MedlinePlus