Inward rectifier potassium channel Kir 2.3 is inhibited by internal sulfhydryl modification

Area of Science:

• Molecular biology
• Neuroscience
• Ion channel biophysics

Background:

• The inward rectifier potassium channel Kir 2.3 plays a crucial role in neuronal excitability.
• Understanding the structural organization of ion channels is essential for elucidating their function.
• Identifying residues accessible to the aqueous environment provides insights into channel pore structure.

Purpose of the Study:

• To map the regions of the Kir 2.3 channel that interact with the aqueous environment.
• To identify specific cysteine residues involved in channel gating and pore accessibility.

Main Methods:

• Utilized sulfhydryl reagent sensitivity to probe channel structure.
• Employed N-ethylmaleimide (NEM) to identify reactive cysteine residues.
• Generated chimeric constructs and performed site-directed mutagenesis to pinpoint specific residues (Cys28 and Cys50).

Main Results:

• Kir 2.3 currents were inhibited by NEM, unlike Kir 2.1.
• Cysteine residues Cys28 and Cys50 were identified as the sites of NEM modification.
• Cys28 and Cys50 were accessible to intracellularly applied MTSET but not extracellularly applied pCMPS.

Conclusions:

• Cys28 and Cys50 are located in a cytoplasmically accessible aqueous region of the Kir 2.3 channel.
• These findings suggest the N-terminus of Kir 2.3 is part of the internal vestibule or modulates gating.
• This study provides critical structural information about the Kir 2.3 potassium channel pore.