Assessment of Ion Channel Oligomerization

As the availability of structural information for ion channels increases, reconstitution and functional measurements have become important to developing structural stories. Creative Bioarray has developed strategies such as Poisson dilution and electron paramagnetic resonance (EPR) spectroscopy to help clients determine information about ion channel function, including oligomerization, conformation, and lipid effects on their regulation.

Background

The diversity of ion channel functions is matched by the diversity of structures and oligomer arrangements, with each possible assembly ranging from monomeric FEX channels, to tetrameric K⁺ channels, to heptameric pannexins and beyond. The oligomeric structure of membrane proteins, including ion channels, is fundamental biochemical knowledge. During assembly, the pore radius and the number of pore-lining sidechains are generally altered and ultimately the function of the protein is affected.

Oligomeric structures can sometimes be solved directly from structural information. However, X-ray crystallography still faces significant technical hurdles, resulting in the significant time required to solve each structure. In addition, the structures of many ion channels are resolved in detergents or require truncation of native sequences, manipulations that can accidentally affect the oligomer structure. Therefore, new biochemical and biophysical methods need to be developed to directly determine the oligomeric structure of ion channels.

Fig. 1 Oligomerization assessment by PELDOR (DEER) spectroscopy. (Pliotas, 2017)

Our Services

For reconstituted systems where protein-to-lipid ratios can be precisely varied, we have developed methods to assess ion channel oligomerization.

• Assessment of ion channel conformation and oligomerization by site-directed spin labeling and pulsed-EPR.
  We help clients refine X-ray crystallography and address specific mechanical problems in solution and membrane proteins' native environments by combining site-directed spin labeling with EPR spectroscopy, providing important information for conformational changes and oligomerization of mechanically sensitive (MS) ion channels. Our dedicated research team has made progress in successfully isolating proteins of interest and introducing paramagnetic labels at desired sites. We have established a variety of EPR methods, such as pulsed electron double resonance (PELDOR), to address a variety of specific problems. And we provide clients with detailed technical consultation from the purification of ion channels, spin labeling, reconstitution into lipid mimics to pulsed EPR experiments.
• Determination of oligomeric architecture in ion channel reconstitution by Poisson distribution statistics.
  We have established a technique for directly determining the oligomeric architecture of ion channels, which utilizes statistical incorporation of ion channel oligomers into liposomes according to Poisson distribution, and uses substrate transport as a proxy for liposome occupancy. Through this technology, we help clients determine the number of ion channel subunits assembled into functional pores and determine ion channel oligomerization, providing important information for understanding and predicting how proteins partition into the liposome population.

Advantages

• Superior technology platform
• Flexible experimental scheme
• Constantly update experimental methods
• Real-time consultation and guidance

Creative Bioarray is committed to providing clients with services to evaluate ion channel oligomerization to complement and confirm structural methods. We have extensive experience in the field of ion channel analysis, providing the foundations for efficient services. If you need our technical support and scientific services, please feel free to contact us.

Reference

1. Pliotas, C. Ion channel conformation and oligomerization assessment by site-directed spin labeling and pulsed-EPR//Methods in enzymology. Academic Press, 2017, 594: 203-242.