The Selectivity Filter Is Involved in the U-Type Inactivation Process of Kv2.1 and Kv3.1 Channels.

Area of Science:

• Molecular Biology
• Biophysics
• Ion Channel Physiology

Background:

• Voltage-gated potassium (Kv) channels regulate cellular excitability through ion flux.
• Kv channels exhibit diverse inactivation mechanisms, including N-, C-, and U-types.
• C-type inactivation involves conformational changes in the selectivity filter (SF), suggesting allosteric coupling between the activation gate and SF.

Purpose of the Study:

• To investigate the role of the second threonine in the SF signature sequence in U-type inactivation.
• To elucidate the contribution of the SF to the gating mechanisms of hKv2.1 and hKv3.1 channels.

Main Methods:

• Site-directed mutagenesis was used to substitute the second threonine with alanine in hKv2.1 and hKv3.1 channels.
• Electrophysiological recordings (macroscopic currents) were performed to assess channel gating and inactivation properties.
• The effect of extracellular potassium concentration on macroscopic current amplitude was analyzed.

Main Results:

• Mutations hKv2.1-T377A and hKv3.1-T400A rendered the channels resistant to U-type inactivation.
• Mutant channels displayed non-inactivating currents, remaining fully conductive during prolonged depolarizations.
• Increased extracellular K+ concentration enhanced macroscopic current amplitude in mutant channels, mirroring findings in C-type inactivation mutants.

Conclusions:

• The second threonine of the SF signature sequence plays a critical role in U-type inactivation gating for hKv2.1 and hKv3.1 channels.
• These findings support the allosteric coupling model between the activation gate and the SF in Kv channel function.
• The study highlights the conserved importance of this residue across different Kv channel subtypes and inactivation mechanisms.