Ligand-gated ion channels are critical to brain function. These proteins mediate rapid signaling from one neuron to the next, by opening ion selective pores in the surface membrane in response to the binding of neurotransmitter, and thus eliciting ion flows that cause electrical excitation or inhibition. When ligand-gated channels spend too much or too little time open, the brain cannot process information correctly. Furthermore, alterations in ligand-gated channel activity can result in overt neurological disease, including epilepsy and neurodegenerative diseases. It is thus important to understand the molecular mechanisms that allow channels to open properly. The focus of the work described here is on a class of channels gated by extracellular ATP, which are referred to as P2X receptors. Genes encoding seven different P2X receptors are expressed in the mammalian brain. Understanding the role of specific P2X receptors in normal brain function and in brain disease has been impeded by the absence of subunit specific ligands. In particular, the P2X2 subunit is widely expressed in the brain, but its role is unclear. In Goal 1 we will use a novel random mutagenesis strategy to gain information about the structure and function of P2X receptors. This approach should generate a large number of new mutant receptors that could not be identified by any other strategy. In particular, these experiments will provide important new information about the ATP binding site, which will be of tremendous use in developing new, more selective agents. In Goal 2, we will explore the mechanism by which ATP is able to open P2X2 receptor channels in wild type and mutant receptors in detail using single channel recording. These single channel recordings will allow us to test distinct models of receptor activation. Understanding how P2X2 channels open and close is potentially of great significance for understanding brain injury. A great deal of evidence suggests that overactivity of NMDA-class glutamate receptors is a major cause of brain injury. Like NMDA receptors, P2X2 receptors form calcium permeable channels that are modulated by low pH and elevated Zn. However, these modulators inhibit current through NMDA receptors, but potentiate current through P2X2 receptors. Many neurons co-express NMDA and P2X2 receptors. Thus the ratio of P2X2 receptors to NMDA receptors may be a key determinant of whether changes in the brain environment following seizures or ischemia result in hyper or hypoexcitability. This has important ramifications for which neurons are susceptible to damage as a result of these conditions, and for strategies to try to prevent this damage.
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