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Conformational changes associated with proton-dependent gating of ASIC1a
Christopher J Passero1, Sora Okumura1, Marcelo D Carattino1
1Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
The Journal of Biological Chemistry
|October 28, 2009
Summary
Acid-sensing ion channels (ASICs) are crucial for neuronal function. This study reveals that the pre-TM2 region of ASIC1a undergoes conformational changes during proton-gated channel activation and recovery from desensitization.
Area of Science:
- Neuroscience
- Biophysics
- Molecular Biology
Background:
- Acid-sensing ion channels (ASICs) are proton-gated ion channels critical for neuronal signaling.
- The precise structural mechanisms underlying ASIC gating, particularly the role of specific domains like the pre-TM2 region, remain incompletely understood.
Purpose of the Study:
- To investigate the role of the pre-TM2 region in the activation and desensitization of mouse ASIC1a.
- To elucidate the conformational dynamics of the pre-TM2 region during proton-dependent gating.
Main Methods:
- Voltage clamp fluorometry using Alexa Fluor 488 C5-maleimide labeling of cysteine-substituted ASIC1a channels (E425C).
- Assessment of fluorescence changes correlated with channel gating.
- Substituted-cysteine-accessibility (SCA) method to probe conformational states using methanethiosulfonate bromide derivatives.
Main Results:
- Extracellular acidification induced fluorescence changes in E425C channels, correlating with channel activation.
- Mutations disrupting channel inactivation abolished the fluorescence response, indicating pore opening is linked to pre-TM2 rearrangement.
- SCA revealed state-dependent accessibility changes in the pre-TM2 region (residues K421, K422, Y424, E425) between resting and desensitized states.
Conclusions:
- The pre-TM2 region of ASIC1a undergoes dynamic conformational rearrangements during proton-dependent gating.
- Pore opening is associated with a conformational shift in the pre-TM2 region.
- These findings provide insights into the molecular mechanisms of ASIC channel function and proton sensing.
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