Richard P. Lifton, Department of Genetics, Yale University
Intracellular chloride levels, [Cl-]i, define whether the neurotransmitter GABA is excitatory or inhibitory. This fundamental property is essential to the neural circuitry of the mammalian brain. We aim to reveal how phosphorylation of the WNK family of signaling kinases directly controls [Cl-]i in neurons. In addition, we aim to identify novel proteins (genes) that control the physiological set point of [Cl-]i in neurons; an essential component of brain function where major players (protein) have yet to be defined. These studies aim to identify fundamental molecular mechanisms that modulate the normal brain physiology that is ultimately altered by drugs of addiction. WNK kinases co-expressed in mammalian cells with their known targets, proteins that control [Cl-]i levels in neurons, will serve as a model system to identify phosphoproteins that modulate neuronal [Cl-]i. The complexity of this system requires novel applications of phosphoproteomic technology. Preliminary studies with WNK kinases and K-Cl co-transporters have identified 14 phosphorylation sites. Investigating dynamics in this complex system will require SILAC approaches, coupled to targeted phosphoproteomics. Targeting phosphorylation dynamics with SILAC coupled to titanium dioxide based proteomic technologies will pinpoint phosphorylation dynamics that modulate [Cl-]i in response to alterations via physiologic stimuli or site directed mutagenesis of target proteins. The results of the study will define novel signaling mechanisms in the mammalian brain and thus contribute to the over all goals of the NIDA Center. These studies are expected to serve as a template for current and future investigations by other members of the NIDA Center. Achieving these goals will serve to fill a knowledge gap in understanding the molecular mechanisms in the brain that establish the normal physiology that is altered by drugs of addiction.