Targeting α7nAChR for Neurotherapeutics: Mutagenesis and Electrophysiological Studies in Xenopus Oocytes
Irina Kim Cavdar1, Quynh Hoa Do1, Anne Laurie Pierre1, Michaela Jansen1
1Biophysiology Department, Texas Tech Health Science Center
Objective:
Identify and structurally characterize the RIC-3 a7 nAChR interface for future structure-based drug discovery efforts.
Background:

Ion channels have been implicated in numerous neurological conditions, including epilepsy, migraines, and addiction. Existing therapies targeting these channels frequently result in undesirable side effects, highlighting the need for more precise and effective treatments. Neuronal nicotinic acetylcholine receptor (nAChR) is a key ion channel targeted for treating neurological and neuropsychiatric diseases. Notably, the α7 nAChR subtype has been implicated in schizophrenia and Alzheimer's disease. Within the central nervous system, α7 nAChRs primarily exhibit modulatory functions, distinct from other nAChRs that directly mediate neurotransmission. Resistance to Inhibitors of Cholinesterase (RIC-3), regulates the surface expression of functional nAChRs on neuronal membranes, which is altered in various diseases. For developing potent and efficacious drugs with reduced side effects, the intracellular domain (ICD) of α7 nAChR, particularly its protein-protein interaction with RIC-3, presents a promising therapeutic target.


Design/Methods:

We employed a pull-down assay using synthetic peptides of the α7 nAChR ICD to identify the segment mediating interaction with RIC-3. To investigate RIC-3's modulatory role in functional α7 nAChR expression, we injected corresponding cRNAs into Xenopus laevis oocytes and performed two-electrode voltage-clamp (TEVC) recordings. Current amplitudes were acquired and analyzed using a TEV-200 amplifier, a Digidata 1440A data interface, and pClamp 10.4 software.


Results:
Our study identifies the L1-MX segment of α7 nAChR as sufficient to interact with RIC-3.
Conclusions:
This provides the basis for future detailed mechanistic and structural studies aimed at developing compounds to mimic or interfere with this protein-protein interaction that is crucial for determining the amount of functional α7 nAChR on the plasma membrane. Our findings underscore the significance of the intracellular domain in ion channel function, paving the way for targeted therapies for various neurological disorders involving ion channels.
10.1212/WNL.0000000000211554
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