Molecular determinants of ATP binding to P2X2 ion channels.

Abstract

ATP, in addition to its myriad roles in intracellular processes, is an extracellular signaling molecule. ATP serves as a neurotransmitter by binding to and opening ion channels known as P2X receptors. P2X receptors assemble as trimers of subunits. It is unclear if ATP binding occurs between or within subunits and if three ATP are required to bind the receptor before the channel opens. To address these questions, we adopted two approaches, both based on introducing mutations that disrupt ATP binding at one or two of the three ATP binding sites of P2X2 receptors. Some of these mutations (K69A and K308A) were sufficiently severe that homotrimers bearing them were non-responsive to ATP. First, we characterized in <italic>Xenopus laevis</italic> oocytes several concatamers in which the open reading frames for three subunits were joined together to form a single coding unit with binding site mutations introduced at various positions. We obtained evidence that these concatamers could form a subpopulation of cross-assembly side-products, which limited the conclusions we could draw. Second, we co-expressed subunits encoding ATP binding mutants either together or with subunits which respond normally to ATP. From these experiments we concluded that receptors with one wild type and two mutant binding sites could respond to ATP and that binding is between receptor subunits. In a final set of experiments, we used two cysteine mutants, one at a critical ATP binding residue (K69C) and another in a neighboring but non-binding residue (I67C). K69C did not give rise to detectable ATP responses, but treatment with Alexa Fluor 546 C₅-maleimide (AM546), a thiol-reactive drug, caused the channels to be constitutively open, and to respond to the allosteric modulators zinc and acidic pH in the absence of exogenous ATP. Therefore, this mutant could serve as a specific biosensor of physiological zinc and pH. In contrast, AM546 caused the responses of I67C channels to decline to less than 10% of their initial amplitude. Additional experiments provided evidence that ATP binding was abolished at these receptors, therefore this mutant will be useful in concatamer and co-expression based experiments to explore the requirement for ATP binding in receptor activation.