I. Development of Rapid Conductance-Based Protocols for Measuring Ion Channel Activity; II. Expression, Characterization, and Purification of the ATP-Sensitive, Inwardly-Rectifying K+ Channel, Kir6.2, and Ion Channel-Coupled Receptors

Abstract

Ligand-gated and ligand-modulated ion channel (IC) sensors have received increased attention for their ability to transduce ligand-binding events into a readily measurable electrical signal. Ligand-binding to an IC modulates the ion flux properties of the channel in label-free manner, often with single-molecule sensitivity and selectivity. As a result, ICs are attractive sensing elements in biosensoring platforms, especially for ligands lacking optical (e.g. fluorescent) or electrochemical properties. Despite the growing number of available ligand-gated and ligand-modulated ICs and artificial lipid bilayer platforms for IC reconstitution, significant work remains in defining the analytical performance capabilities of IC sensors. Particularly, few studies have described platforms for making measurements with rapid temporal resolution and high sensitivity. In this work, we describe an artificial lipid bilayer platform which enables rapid measurement of ion channel activity, a key parameter for developing IC sensors suitable for studying biological events, e.g. single cell exocytosis (Chapter 2 and 3). Additionally, we developed expression, purification, and reconstitution protocols for Kir6.2, a model ligand-gated ion channel, for use in sensor development (Chapter 4). The final goal is to reconstitute ion channel-coupled receptors (ICCRs), G protein-coupled receptor-Kir6.2 fusion proteins, into artificial lipid bilayers to detect small molecules and hormones targeting GPCRs. Towards this goal, we characterized the expression and function of two ICCRs, M2-Kir and D2-Kir, in HEK293 cells (Chapter 5).