The function of BK channels extracted and purified within SMALPs

Area of Science:

• Biophysics
• Molecular Biology
• Membrane Protein Biochemistry

Background:

• Human BK channels are large, voltage- and Ca2+-activated K+ channels crucial for physiological functions.
• Their function is modulated by accessory subunits (β and γ).
• Efficient purification and functional reconstitution of these large channels remain challenging.

Purpose of the Study:

• To evaluate styrene maleic acid (SMA) for solubilizing and purifying human BK channels.
• To assess the functional reconstitution of purified BK channels in planar lipid bilayers (PLBs).
• To investigate the impact of β1 subunits on channel activity after purification.

Main Methods:

• Solubilization and purification of human BK channels (α and α+β1 subunits) from HEK cells using SMA polymer and nickel affinity chromatography.
• Native SMA-PAGE analysis to determine subunit assembly.
• Reconstitution of purified SMA lipid particles (SMALPs) into PLBs for single-channel recording.

Main Results:

• SMA successfully extracted and purified tetrameric α-subunit BK channels and heteromeric α+β1 complexes.
• Reconstituted channels in PLBs exhibited high conductance (≈260 pS).
• Co-purified β1 subunits increased channel open probability but restricted voltage-dependent gating.

Conclusions:

• SMA is an effective tool for purifying large, complex human ion channels from low-expression sources.
• SMALP-reconstituted BK channels can display functional activity, including voltage-dependent gating.
• SMA appears to reduce the voltage-dependent gating of BK channels.