The function of BK channels extracted and purified within SMALPs

Abstract

Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of β and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and β1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA–PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of β1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260 pS), as expected. The open probability was increased in the presence of co-purified β1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.

High-Resolution Proteomics Unravel a Native Functional Complex of Cav1.3, SK3, and Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels in Midbrain Dopaminergic Neurons

“…Cross-linking coupled with mass spectrometry (XL-MS) based on the covalent binding of proximal amino acid residues of protein partners [ 111 , 112 ] will help to obtain a fine description of the complex topography of these protein interactions. This approach could be complemented with native gel electrophoresis analysis [ 113 , 114 ]. Then, it would be possible to identify a strategy to dissociate the complex in order to determine its influence on SNc DA neuron pacemaking activity and the vulnerability of this neuronal type in pathological conditions.…”