Probing ion-channel pores one proton at a time

Area of Science:

• Biochemistry
• Structural Biology
• Neuroscience

Background:

• Membrane proteins utilize ionizable residues for critical structure and function.
• Determining the ionization states of these residues under physiological conditions is experimentally challenging.

Purpose of the Study:

• To investigate the impact of the local microenvironment on the proton affinity of ionizable residues.
• To characterize the residue-specific microenvironment within the transmembrane pore of the nicotinic acetylcholine receptor.

Main Methods:

• Engineered specific ionizable residues (lysine, histidine, arginine) along the M2 transmembrane alpha-helix.
• Utilized electrophysiology to detect single proton binding-unbinding events as cation current fluctuations.
• Performed kinetic analysis of fluctuations to determine position-dependent proton transfer rates and calculate pK(a) values.

Main Results:

• Developed a residue-by-residue energetic description of the microenvironment around the M2 helices in the open-channel state.
• Quantified the excess free energy required for protonation of engineered residues relative to bulk water.
• Found minimal M2 helix rotation in the open-channel conformation compared to closed states.

Conclusions:

• The study provides a novel, high-resolution map of the microenvironment influencing ionizable residues in membrane proteins.
• The findings challenge the proposed significant M2 helix rotation as a primary gating mechanism in Cys-loop receptors.
• This methodology offers a powerful approach to study membrane protein electrostatics and dynamics.