A synthetic method to assay polycystin channel biophysics

Abstract

Ion channels are biological transistors that control flux of ions across membranes to regulate electrical transmission and cell signaling. They are found in all eukaryotic cell membranes and their conductive states are frequently disrupted in human diseases. Organelle ion channels are among the most resistant to functional and pharmacological interrogation. Traditional channel protein reconstitution methods rely upon exogenous expression and/or purification from endogenous cellular sources which are frequently contaminated by resident channels. Here we describe a fully synthetic method to assay the functional properties of polycystin subfamily of transient receptor potential (TRP) channels that natively traffic to primary cilia and endoplasmic reticulum organelles. Using this method, we characterize their membrane integration, orientation and conductance while comparing these results to their endogenous channel properties. Outcomes define a novel synthetic approach that can be applied broadly to investigate other channels resistant to biophysical analysis and pharmacological characterization.