Probing a potassium channel pore with an engineered protonatable site

Area of Science:

• Molecular biology
• Biophysics
• Ion channel function

Background:

• Intracellular cations like Mg2+ and polyamines block outward K+ conduction in inward rectifier K+ channels.
• Residue 171 in the second transmembrane (M2) segment of the ROMK1 channel is critical for the affinity of this blockade.

Purpose of the Study:

• To investigate the mechanism by which residue 171 mediates ion blockade.
• To determine the location and interaction of residue 171 within the ion conduction pore.

Main Methods:

• Site-directed mutagenesis of the ROMK1 channel, introducing a proton acceptor (histidine) at residue 171.
• Electrophysiological analysis to assess ion conduction and blockade.

Main Results:

• The side chain of residue 171 is positioned within the ion conduction pore.
• Residue 171 is located approximately halfway across the transmembrane voltage drop.
• The side chain of residue 171 directly interacts electrostatically with blocking ions.

Conclusions:

• Residue 171 plays a key role in the physical obstruction of ion flow through the ROMK1 channel pore.
• The location and electrostatic interaction of residue 171's side chain are crucial for cation blockade.
• Understanding residue 171's function provides insights into the gating mechanisms of inward rectifier K+ channels.