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In vitro Methylation Assay to Study Protein Arginine Methylation
Published on: October 5, 2014
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GABAAergic stimulation modulates intracellular protein arginine methylation
Robert B Denman1, Wen Xie2, George Merz3
1Department of Molecular Biology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York, NY 10314, USA.
Neuroscience Letters
|May 6, 2014
Summary
Gamma-aminobutyric acid type A (GABAA) receptor stimulation causes intracellular alkalinization, affecting protein arginine methyltransferase (PRMT) activity. This pH shift impacts methylation pathways, relevant to neurological conditions like fragile X syndrome.
Area of Science:
- Neuroscience
- Cellular Biology
- Biochemistry
Background:
- Cytoplasmic pH changes regulate cellular processes and neuronal activity.
- Intracellular alkalization in neurons can be triggered by specific channels and receptors, including the Na+/H+ antiporter and GABAA receptors.
- Enzyme activity is sensitive to pH shifts, necessitating investigation into pH-modulated cellular functions.
Purpose of the Study:
- To investigate the impact of GABAA receptor-mediated intracellular pH modulation on protein arginine methyltransferase (PRMT) activities.
- To determine if GABAAergic stimulation influences protein arginine methylation.
- To explore potential pH-dependent and pH-independent methylation pathways activated by GABAA receptors.
Main Methods:
- Expression analysis of major benzodiazepine GABAA receptor subtypes in P19 cells and neurons.
- Induction of intracellular alkalinization using diazepam and muscimol, with detection via fluorescence dye.
- Measurement of PRMT activity using methylation assays with specific protein substrates.
- Assessment of protein arginine methylation changes upon GABAAergic stimulation and with antagonist treatment.
- Verification in hippocampal slice preparations from a fragile X syndrome mouse model.
Main Results:
- The major benzodiazepine GABAA receptor subtype is constitutively expressed in P19 cells and neurons.
- Diazepam and muscimol stimulation induced detectable intracellular alkalinization.
- This alkalinization significantly perturbed protein arginine methylation activity.
- GABAA agonist muscimol altered protein arginine methylation, while the antagonist bicuculline did not.
- Altered methylation was observed in fragile X syndrome mouse models.
Conclusions:
- GABAAergic stimulation can lead to intracellular alkalinization, significantly affecting PRMT activity.
- Both pH-dependent and pH-independent methylation pathways can be activated by GABAAergic stimulation.
- These findings suggest a link between GABAAergic signaling, pH regulation, and protein methylation, with implications for neurological disorders.
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