An activity-based protein profiling probe for the nicotinic acetylcholine receptor

Area of Science:

• Biochemistry
• Neuroscience
• Chemical Biology

Background:

• Activity-based protein profiling (ABPP) is a powerful technique for studying enzyme function.
• ABPP has not been widely applied to ion channels, limiting the characterization of their physiological roles.
• Nicotinic acetylcholine receptors (nAChRs) are crucial ion channels involved in neurotransmission.

Purpose of the Study:

• To develop and validate a state-dependent photoaffinity probe for ion channel directed ABPP.
• To demonstrate the feasibility of using ABPP for studying ion channel conformations and functions.
• To investigate the potential of activity-based probes in understanding nAChR physiology, particularly desensitization.

Main Methods:

• Synthesis of a novel photoaffinity probe, BPyneTEA, incorporating nAChR binding, photolabeling, and click chemistry moieties.
• Single-molecule current measurements to assess probe interaction with different nAChR states (closed and open).
• Cell-based photolabeling experiments to evaluate probe selectivity for closed versus desensitized nAChR states.

Main Results:

• BPyneTEA effectively blocks both closed and open (nondesensitized) conformations of nAChRs with comparable kinetics.
• In living cells, BPyneTEA demonstrates selective photolabeling of the closed nAChR state over the desensitized state.
• The probe's design and reactivity suggest broad applicability for ion channel ABPP.

Conclusions:

• BPyneTEA serves as a promising probe for activity-based protein profiling of nondesensitized nAChRs.
• This approach may offer new insights into the molecular mechanisms of ion channel desensitization.
• Ion channel pores represent a viable and broadly applicable target for future ABPP development.