Trafficking of Channels Measured by EFC

Discoverx Corporation, Fremont CA

Investigators

Abstract

DESCRIPTION (provided by applicant): Ionic conductance channels (ICC) mediate the transport of ions across the cell membrane and are critical for maintaining membrane excitability. ICC consists of multiple subunits that combine to create the unique conductance properties of the channel. Subunits also confer ligand binding domains for neurotransmitters or regions that allow the channels to interact with G proteins and G protein coupled receptors (GP CRs). The majority of ICC are heteromeric, consisting of subunits of different molecular make up. Recent studies have shown that ICC composition can be influenced by subunit trafficking. Approaches to manipulate ion channel subunit transport to the cell membrane could be used to change the subunit composition and therefore the properties of ICC to alter cell physiology and neurotransmission. Furthermore, modulation of channel transport can alter the number of channels expressed in the cell membrane. Thus, ICC transport could be an important target for drug discovery by the pharmaceutical industry to develop therapeutics that can modulate the number of channels expressed on the cell surface as well as to change channel composition to alter the actions of neurotransmitters and drugs. However, no technologies exist today that can measure ICe transport in a manner amenable for drug discovery. DiscoveRx proposes to employ its proprietary enzyme fragment complementation (EFC) technology as a drug discovery platform to measure ICC transport. The technology is based on a sensitive beta-galactosidase (beta- gal) complementation assay and utilizes two genetically engineered fragments of E. coli beta-gal. The larger fragment, Enzyme Acceptor (EA), contains a deletion near the amino terminus, while the smaller fragment, ProLabel, contains the amino-terminal sequence missing from EA. Alone, EA is inactive, but in vitro it spontaneously recombines with ProLabel to form an active enzyme that catalyzes the formation of a fluorescent or chemiluminescent product that can be detected photometrically as a visually amplified response. In proof-of-principle studies, we propose in this grant to develop an assay to measure ICC transport by tagging channels with ProLabel and using our EFC technology to measure surface expression of the channel. For our studies, we will test whether EFC can be used to measure the transport of two different potassium chancels, HERG and GIRK, to the cell membrane. These initial studies will form the foundation of future phase II SBIR studies to develop EFC into a HTS assay for ICC transport for use by bioteclmology and pharmaceutical companies for drug discovery.