7] Identification of ion channel-associated proteins using the yeast two-hybrid system

Abstract

Publisher Summary A wide variety of ion channels are involved in generating and regulating the electrical behavior of a neuronal cell. Typically, these ion channels are not randomly distributed on the cell surface but rather localized at specific subcellular sites. Good examples include ligand-gated ion channels (iono- tropic neurotransmitter receptors) at postsynaptic sites and voltage-gated sodium channels at nodes of Ranvier. Presumably, the targeted localization of these ion channels depends on protein-protein interactions between ion channel subunits and the cytoskeleton. In addition it is likely that ion channels interact with intracellular proteins that are involved in their modulation—for example, protein kinases such as the tyrosine kinase Src or protein kinase C 3 –or that are involved in downstream signaling mechanisms activated by the ion channel. Thus, knowledge about intracellular proteins associated with ion channels is critical to our understanding of signal transduction by ion channels and of mechanisms of ion channels clustering at specific subcellular sites. Currently, few such proteins have been identified. This chapter describes the yeast two-hybrid assay that has been used to identify proteins binding to Shaker-type channels and NMDA receptor subunits. It also discusses some general and specific considerations about using ion channel proteins in the two-hybrid system.