Isoform specificity of P2X(2) purinergic receptor C-terminus binding to tubulin

Adenosine triphosphate (ATP) and other nucleotides can be released in the central and peripheral nervous systems and act as neurotransmitters/ neuromodulators. They can activate G-protein coupled receptors and ligand-gated ion channels, which are present throughout the central nervous system (CNS). P2X(2) is one of seven known ion channels gated by ATP, and is characterized by having two transmembrane domains, a large extracellular loop and intracellular N- and C-termini. Recently, work from several laboratories has shown that neurotransmitter receptors can interact with other proteins thereby changing their functional attributes. More specifically, it was demonstrated that P2X(2) binds beta-tubulin. Our goal was to investigate this interaction, by comparing P2X(2) with a naturally occurring splicing variant named P2X(2b). These isoforms differ in their C-terminal regions which contain the proposed P-tubulin-binding domain. Indeed we were able to demonstrate that only the long variant P2X(2) binds beta-tubulin I in various biochemical assays. In addition, this interaction can be direct since it also occurred when the P2X(2) C-terminus was exposed to purified brain tubulin. When expressed in heterologous cells, P2X(2) interacted with beta-tubulin I while present on the outer membrane, as demonstrated by biotinylation of surface proteins. Therefore, the present data strongly support a functional interaction between an ATP-gated channel and the cytoskeleton. Moreover, we show a biochemical difference between the splicing alternatives that might account for novel distinct functional roles. (C) 2007 Elsevier Ltd. All rights reserved.