Probing structure and function of ion channels using limited proteolysis and microfluidics

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Ion channel dysfunction is linked to numerous diseases, yet developing targeted pharmaceuticals is challenging due to limited structure-function data.
• Obtaining correlated structural and functional information for membrane proteins like ion channels has been a significant hurdle in drug development.

Purpose of the Study:

• To develop a novel microfluidic method for correlating ion channel structure with function.
• To identify specific regions of the TRPV1 channel that influence its electrophysiological properties and response to agonists.

Main Methods:

• A microfluidic system was employed to expose TRPV1 channels in proteoliposomes or excised patches to limited trypsin proteolysis.
• Cleaved peptides were identified using mass spectrometry (MS), and electrophysiological properties were measured via patch clamp recordings.
• Structure-function relationships were assessed by correlating functional changes with the removal of specific structural elements.

Main Results:

• The study successfully pinpointed regions of TRPV1 whose removal altered channel properties, including current amplitude, single-channel conductance, and EC50 for capsaicin.
• The method allowed for the functional assessment of various peptide regions within the TRPV1 channel.

Conclusions:

• This microfluidic approach provides a rapid "shotgun" method for chemical truncation of membrane proteins, enabling functional analysis of peptide regions.
• The findings offer a new strategy for understanding ion channel structure-function relationships and could accelerate the development of novel therapeutics.