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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.

No MeSH data available.


Activation of ASIC2a-A427C by MTS-reagents at neutral pH.Oocytes expressing ASIC2a-A427C were subjected to a protocol similar as in Fig 5A. After an initial current pulse elicited at pH 4.0, ASIC2a-A427C was activated at pH 7.0 by 100 μM of MTSET (A), MTSES (B), or MTSPTrEA (C). The recording was terminated by a final pH pulse at pH 4.0. Blue line corresponds to pH 7.8, the red line to acidic pH 7.0 or 4.0, dashed line to the addition of 300 μM amiloride. D. pH-dependence of Isust normalized for the maximal current elicited at pH 4.0 elicited by MTSET (n = 4–13) or MTSPTrEA (n = 6–10).
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pone.0175293.g011: Activation of ASIC2a-A427C by MTS-reagents at neutral pH.Oocytes expressing ASIC2a-A427C were subjected to a protocol similar as in Fig 5A. After an initial current pulse elicited at pH 4.0, ASIC2a-A427C was activated at pH 7.0 by 100 μM of MTSET (A), MTSES (B), or MTSPTrEA (C). The recording was terminated by a final pH pulse at pH 4.0. Blue line corresponds to pH 7.8, the red line to acidic pH 7.0 or 4.0, dashed line to the addition of 300 μM amiloride. D. pH-dependence of Isust normalized for the maximal current elicited at pH 4.0 elicited by MTSET (n = 4–13) or MTSPTrEA (n = 6–10).

Mentions: Finally, the representative tracings on Fig 11 show that, as for ASIC1a-G430C, ASIC2a-A427C can be activated at physiological pH by MTSET (A) or MTSPTrEA (C), but not by MTSES (B). After a first pulse to pH 4.0, the pH was set at 7.0, and the addition of MTSET triggered an amiloride-sensitive current with slow activation kinetics. After returning to pH 7.8, removal of the amiloride shows the persistence of an ASIC2a current that can be further stimulated at pH 4.0. MTSES was without effect on channel activation or desensitization except for an increase in the magnitude of the peak current elicited at pH 4.0 (Fig 11B, right panel). Furthermore, the Isust elicited by MSTET or MTSPTrEA was insensitive to pH between 7.8 and 7.0, the magnitude of Isust being larger for MTSPTrEA than for MTSET (Fig 11D). In this pH range, the Isust elicited by MTSET and MTSPTrEA closely matches the current elicited by protons on ASIC2a-A427C that has been previously modified by the same MTS-reagents (Fig 10).


Proton and non-proton activation of ASIC channels
Activation of ASIC2a-A427C by MTS-reagents at neutral pH.Oocytes expressing ASIC2a-A427C were subjected to a protocol similar as in Fig 5A. After an initial current pulse elicited at pH 4.0, ASIC2a-A427C was activated at pH 7.0 by 100 μM of MTSET (A), MTSES (B), or MTSPTrEA (C). The recording was terminated by a final pH pulse at pH 4.0. Blue line corresponds to pH 7.8, the red line to acidic pH 7.0 or 4.0, dashed line to the addition of 300 μM amiloride. D. pH-dependence of Isust normalized for the maximal current elicited at pH 4.0 elicited by MTSET (n = 4–13) or MTSPTrEA (n = 6–10).
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pone.0175293.g011: Activation of ASIC2a-A427C by MTS-reagents at neutral pH.Oocytes expressing ASIC2a-A427C were subjected to a protocol similar as in Fig 5A. After an initial current pulse elicited at pH 4.0, ASIC2a-A427C was activated at pH 7.0 by 100 μM of MTSET (A), MTSES (B), or MTSPTrEA (C). The recording was terminated by a final pH pulse at pH 4.0. Blue line corresponds to pH 7.8, the red line to acidic pH 7.0 or 4.0, dashed line to the addition of 300 μM amiloride. D. pH-dependence of Isust normalized for the maximal current elicited at pH 4.0 elicited by MTSET (n = 4–13) or MTSPTrEA (n = 6–10).
Mentions: Finally, the representative tracings on Fig 11 show that, as for ASIC1a-G430C, ASIC2a-A427C can be activated at physiological pH by MTSET (A) or MTSPTrEA (C), but not by MTSES (B). After a first pulse to pH 4.0, the pH was set at 7.0, and the addition of MTSET triggered an amiloride-sensitive current with slow activation kinetics. After returning to pH 7.8, removal of the amiloride shows the persistence of an ASIC2a current that can be further stimulated at pH 4.0. MTSES was without effect on channel activation or desensitization except for an increase in the magnitude of the peak current elicited at pH 4.0 (Fig 11B, right panel). Furthermore, the Isust elicited by MSTET or MTSPTrEA was insensitive to pH between 7.8 and 7.0, the magnitude of Isust being larger for MTSPTrEA than for MTSET (Fig 11D). In this pH range, the Isust elicited by MTSET and MTSPTrEA closely matches the current elicited by protons on ASIC2a-A427C that has been previously modified by the same MTS-reagents (Fig 10).

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.