LRET Determination of Molecular Distances during pH Gating of the Mammalian Inward Rectifier Kir1.1b

Area of Science:

• Biophysics
• Ion Channel Physiology
• Molecular Biology

Background:

• The gating mechanism of the inward rectifier potassium channel Kir1.1, particularly the role of its C-terminal domain (CTD) in response to intracellular pH, is not fully understood.
• Previous studies using crystal structures and single-molecule fluorescence resonance energy transfer suggested CTD contraction or rigid body rotation during Kir channel gating.

Purpose of the Study:

• To investigate the conformational changes of the Kir1.1b CTD during pH-dependent gating.
• To clarify the motion of the CTD relative to the helix bundle crossing (HBC) gate during channel opening and closing.

Main Methods:

• Utilized lanthanide-based resonance energy transfer (La-RET) on single-Cys dimeric constructs of Kir1.1b incorporated into anionic liposomes with PIP2.
• Measured state-dependent distances between paired Cys residues on diagonally opposite subunits under closed (pH 6) and open (pH 8) conditions.
• Verified channel functionality and pH dependence using electrophysiological experiments.

Main Results:

• La-RET measurements showed no significant expansion or contraction of the CTD during Kir1.1b gating.
• The helix bundle crossing (HBC) gate widened substantially (8.8 ± 4 Å) upon opening (from 6.3 ± 2 Å to 15.1 ± 6 Å).
• Observed gating distances are consistent with a model involving rigid body rotation of the CTD.

Conclusions:

• The CTD of Kir1.1b does not undergo conformational changes like contraction or expansion during pH-induced gating.
• Channel opening is correlated with the rigid body rotation of the CTD around the permeation axis.
• This rotation facilitates the opening of the HBC gate, enabling the passage of hydrated potassium ions.