Conformational Fingerprints Underlying Thermal Modulation of PAC/TMEM206 Gating

Area of Science:

• Biophysics
• Molecular Biology
• Ion Channel Physiology

Background:

• The proton-activated chloride channel (PAC/TMEM206) is crucial in acid-induced cell injury during pathological conditions like ischemia and tumor acidosis.
• PAC exhibits sensitivity to both protons and temperature, but the structural basis for this dual modulation remains elusive.

Purpose of the Study:

• To elucidate the structural mechanisms underlying temperature-dependent modulation of the proton-activated chloride channel (PAC).
• To identify specific residues and regions within PAC that respond to thermal and proton stimuli.

Main Methods:

• Site-specific incorporation of the fluorescent amino acid ANAP (4-azido-2,3-difluorophenylalanine) into PAC.
• Automated patch-clamp electrophysiology to measure proton-evoked currents.
• Molecular dynamics simulations and ANAP spectral analysis to probe local environmental changes.

Main Results:

• ANAP reporters revealed distinct, yet overlapping, structural environments modulated by heating and acidification.
• Specific residues (R93, Y111, F196, R237, F282) showed significant temperature-dependent changes.
• Proton-dependent changes were prominent at residues R93, Y111, H130, and R237.
• Temperature-sensitive reporters localized to an intersubunit region, supported by dynamic correlation analysis.

Conclusions:

• Protonation likely establishes an activation-permissive state, while heating reconfigures intersubunit coupling.
• Structural correlates for PAC thermal modulation have been identified, providing insights into dual gating mechanisms.
• The findings propose a model for integrated proton and thermal sensing in PAC, generating testable predictions.