Applications for Mass Spectrometry in the Study of Ion Channel Structure and Function

Ion channels are intrinsic membrane proteins that form gated ion permeable pores across biological membranes. Depending on the type, ion channels exhibit sensitivities to a diverse range of stimuli including changes in membrane potential, binding by diffusible ligands, changes in temperature and direct mechanical force. The purpose of these proteins is to facilitate the passive diffusion of ions down their respective electrochemical gradients into and out of the cell, and between intracellular compartments. In doing so, ion channels can affect transmembrane potentials and regulate the intracellular homeostasis of the important second messenger, Ca²⁺, modulating a multitude of cell signaling systems in the process. The ion channels of the plasma membrane are of particular clinical interest due to their regulation of cell excitability and cytosolic Ca²⁺ levels, and the fact that they are particularly amenable to manipulation by exogenously applied drugs and toxins. A critical step in improving the pharmacopeia of chemicals available that influence the activity of ion channels is understanding how their three-dimensional structure relates to their function. Historically, elucidation of the structure of membrane proteins has been slow relative to that for soluble proteins, due to limitations inherent in the most widely used methods, in particular X-ray crystallography. Over the course of the last decade, starting with significant advances in X-ray crystallography followed by the more recent, and profound, surge in the use of single particle cryo-electron microscopy (cryo-EM), a slew of high resolution ion channel structures have been resolved. Overshadowed during this period have been the equally marked advances in mass spectrometry, pushing this method to the fore as an important complimentary approach to studying the structure and function of ion channels. In addition to revealing the subtle conformational changes in ion channel structure that accompany gating and permeation, mass spectrometry is already being used effectively for identifying tissue-specific posttranslational modifications and mRNA splice variants. Furthermore, the use of mass spectrometry for high throughput proteomics analysis, which has proven so successful for soluble proteins, is already providing valuable insight into the functional interactions of ion channels within the context of the macromolecular signaling complexes that they inhabit in vivo. In this chapter, the potential for mass spectrometry as a complementary approach to the study of ion channel structure and function will be reviewed with examples of its application.