Single acetylcholine receptor channel currents recorded at high hydrostatic pressures

Area of Science:

• Biophysics
• Neuroscience
• Molecular Biology

Background:

• Patch-clamp electrophysiology is a crucial technique for studying ion channel function.
• Understanding the physical forces, such as hydrostatic pressure, influencing ion channel gating is essential for comprehending cellular signaling.

Purpose of the Study:

• To describe a novel technique for patch-clamp experiments under high hydrostatic pressure (up to 60 MPa).
• To investigate the pressure dependence of acetylcholine receptor (AChR) channel properties.
• To determine the activation volumes associated with AChR channel gating and ion translocation.

Main Methods:

• Development of a method to transfer patch-clamp recording configurations into a high-pressure vessel.
• Recording single-channel currents from excised outside-out membrane patches of cultured rat muscle cells expressing AChRs.
• Application of hydrostatic oil pressure ranging from 0.1 to 60 MPa.

Main Results:

• Open channel conductance of AChRs remained largely unchanged (within 2%) across the pressure range, indicating minimal volume changes during ion translocation.
• At high acetylcholine concentrations, pressurization to 40 MPa increased mean open and closed times, yielding activation volumes of approximately 59 A3 and 139 A3, respectively.
• A net volume increase of 80 A3 was calculated for the transition from the agonist-free to the open state, potentially involving agonist binding.

Conclusions:

• The described high-pressure patch-clamp technique is effective for studying pressure-dependent ion channel behavior.
• AChR channel gating involves significant volume changes, suggesting conformational rearrangements.
• The calculated activation volumes for AChR gating are comparable to those of other voltage-gated ion channels, implying conserved gating mechanisms in ion channel proteins.