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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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pH-dependence of activation of ASIC1a-G430C currents after channel modification by MTS-reagents.Currents recorded in oocytes expressing ASIC1a-G430C and incubated beforehand with MTSET, MTSPTrEA, MTSPT, MTSBT, MTSEA-biotin, MTSEA, MTSES (100 μM) during 10 min. at pH 7.8. Currents are elicited by acidic pH changes ranging from 7.8 to 5.5. Black circles and black solid lines represent the total inward current, pink circles and lines represent the desensitizing current (Idesens), red circles and lines the sustained current (Isust). The lines represent the best non-linear fit for the pH-dependence of current activation. For comparison, the dashed line represents the pH dependence of activation of ASIC1-G430C without pre-incubation with MTS-reagents obtained from data shown in S2 Fig. Each current value was normalized for the maximal total inward current elicited at pH 5.5. Each symbol represents the mean ± SE of 10 to 53 measurements.
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pone.0175293.g003: pH-dependence of activation of ASIC1a-G430C currents after channel modification by MTS-reagents.Currents recorded in oocytes expressing ASIC1a-G430C and incubated beforehand with MTSET, MTSPTrEA, MTSPT, MTSBT, MTSEA-biotin, MTSEA, MTSES (100 μM) during 10 min. at pH 7.8. Currents are elicited by acidic pH changes ranging from 7.8 to 5.5. Black circles and black solid lines represent the total inward current, pink circles and lines represent the desensitizing current (Idesens), red circles and lines the sustained current (Isust). The lines represent the best non-linear fit for the pH-dependence of current activation. For comparison, the dashed line represents the pH dependence of activation of ASIC1-G430C without pre-incubation with MTS-reagents obtained from data shown in S2 Fig. Each current value was normalized for the maximal total inward current elicited at pH 5.5. Each symbol represents the mean ± SE of 10 to 53 measurements.

Mentions: In Fig 3 we show the analysis of the total current elicited after modification of ASIC1a-G430C by the different MTS-reagents at different pHs. The maximal ASIC1a current (Imax) elicited by protons is represented as the sum of a transient desensitizing current (Idesens) and a sustained current (Isust). After modification by MTSET, an alkaline shift of the pH-dependency (pH0.5) of ASIC1a-G430C activation was observed for both Idesens and Isust currents; the Isust levels off at around pH 7.0, plateauing at 10% of Imax, the major fraction of this current accounting for Idesens. A similar analysis was performed for the ASIC1a-G430C currents after modification by MTSPTrEA: compared to MTSET, the alkaline shift of pH0.5 of Isust activation was more pronounced, whereas the pH0.5 values of Idesens were comparable for the two MTS-reagents; after modification with MTSPTrEA, Isust represented up to 45% of the Imax at pHs for maximal activation (see also Table 1). The pH0.5 for activation of the ASIC1a-G430C currents was measured after modification by MTSPT and MTSBT and show values for Isust between those obtained for MTSET and MTSPTrEA, as for the relative magnitude of Isust (Table 1). The effect of MTSEA-biotin on both the pH0.5 and the magnitude of Isust was comparable to that of MTSBT and MTSPT. Modification of ASIC1a-G430C with MTSEA had only minimal effects on Isust and modification by MTSES did not induce any detectable Isust. MTSES, like the other MTS-reagents, shifted the pH0.5 of the Idesens to a similar extent. The values for pH0.5 obtained for both Idesens and Isust are summarized in Table 1: the pH0.5 for Idesens, of the non-modified ASIC1a-G430C was 6.55 ±0.02, similar to ASIC1a wt, but after modification by the different MTS reagents including MTSES, the pH0.5 for Idesens was on average 6.89 ± 0.08 (mean ± SD). The average pH0.5 for Isust, when detectable, varied among the different MTS-reagents and was on average around 7.29 ± 0.11 (mean ± SD). The fraction of Imax carried by the Isust also varied greatly among the MTS used for G430C modification and ranged from zero for the MTSES to around 0.45 for the MTSPTrEA.


Proton and non-proton activation of ASIC channels
pH-dependence of activation of ASIC1a-G430C currents after channel modification by MTS-reagents.Currents recorded in oocytes expressing ASIC1a-G430C and incubated beforehand with MTSET, MTSPTrEA, MTSPT, MTSBT, MTSEA-biotin, MTSEA, MTSES (100 μM) during 10 min. at pH 7.8. Currents are elicited by acidic pH changes ranging from 7.8 to 5.5. Black circles and black solid lines represent the total inward current, pink circles and lines represent the desensitizing current (Idesens), red circles and lines the sustained current (Isust). The lines represent the best non-linear fit for the pH-dependence of current activation. For comparison, the dashed line represents the pH dependence of activation of ASIC1-G430C without pre-incubation with MTS-reagents obtained from data shown in S2 Fig. Each current value was normalized for the maximal total inward current elicited at pH 5.5. Each symbol represents the mean ± SE of 10 to 53 measurements.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5383329&req=5

pone.0175293.g003: pH-dependence of activation of ASIC1a-G430C currents after channel modification by MTS-reagents.Currents recorded in oocytes expressing ASIC1a-G430C and incubated beforehand with MTSET, MTSPTrEA, MTSPT, MTSBT, MTSEA-biotin, MTSEA, MTSES (100 μM) during 10 min. at pH 7.8. Currents are elicited by acidic pH changes ranging from 7.8 to 5.5. Black circles and black solid lines represent the total inward current, pink circles and lines represent the desensitizing current (Idesens), red circles and lines the sustained current (Isust). The lines represent the best non-linear fit for the pH-dependence of current activation. For comparison, the dashed line represents the pH dependence of activation of ASIC1-G430C without pre-incubation with MTS-reagents obtained from data shown in S2 Fig. Each current value was normalized for the maximal total inward current elicited at pH 5.5. Each symbol represents the mean ± SE of 10 to 53 measurements.
Mentions: In Fig 3 we show the analysis of the total current elicited after modification of ASIC1a-G430C by the different MTS-reagents at different pHs. The maximal ASIC1a current (Imax) elicited by protons is represented as the sum of a transient desensitizing current (Idesens) and a sustained current (Isust). After modification by MTSET, an alkaline shift of the pH-dependency (pH0.5) of ASIC1a-G430C activation was observed for both Idesens and Isust currents; the Isust levels off at around pH 7.0, plateauing at 10% of Imax, the major fraction of this current accounting for Idesens. A similar analysis was performed for the ASIC1a-G430C currents after modification by MTSPTrEA: compared to MTSET, the alkaline shift of pH0.5 of Isust activation was more pronounced, whereas the pH0.5 values of Idesens were comparable for the two MTS-reagents; after modification with MTSPTrEA, Isust represented up to 45% of the Imax at pHs for maximal activation (see also Table 1). The pH0.5 for activation of the ASIC1a-G430C currents was measured after modification by MTSPT and MTSBT and show values for Isust between those obtained for MTSET and MTSPTrEA, as for the relative magnitude of Isust (Table 1). The effect of MTSEA-biotin on both the pH0.5 and the magnitude of Isust was comparable to that of MTSBT and MTSPT. Modification of ASIC1a-G430C with MTSEA had only minimal effects on Isust and modification by MTSES did not induce any detectable Isust. MTSES, like the other MTS-reagents, shifted the pH0.5 of the Idesens to a similar extent. The values for pH0.5 obtained for both Idesens and Isust are summarized in Table 1: the pH0.5 for Idesens, of the non-modified ASIC1a-G430C was 6.55 ±0.02, similar to ASIC1a wt, but after modification by the different MTS reagents including MTSES, the pH0.5 for Idesens was on average 6.89 ± 0.08 (mean ± SD). The average pH0.5 for Isust, when detectable, varied among the different MTS-reagents and was on average around 7.29 ± 0.11 (mean ± SD). The fraction of Imax carried by the Isust also varied greatly among the MTS used for G430C modification and ranged from zero for the MTSES to around 0.45 for the MTSPTrEA.

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