SUMMARY Mutations that render the cystic fibrosis transmembrane conductance regulator (CFTR) defective in function lead to cystic fibrosis, a devastating multisystem disease affecting tens of thousands of people worldwide. Drug discovery efforts by Vertex, Inc. (Cambridge, Mass. USA) have yielded clinically efficacious drug combinations, establishing CFTR as a therapeutically accessible target. Thus far all of the successfully tested therapies include Ivacaftor, which as a “potentiator,” rather than an activator of CFTR relies at least to some degree on the phosphorylation state of CFTR, which is subject to dynamic hormonal regulation in vivo. In addition, accumulating evidence suggests that Ivacaftor works through an ATP-independent mechanism, meaning that the canonical route by which stable CFTR openings are achieved, namely ATP-driven dimerization of the intracellular binding domains, is not exploited by Ivacaftor. By aiming to better understand both phospho regulation and ATP binding in CFTR, the two aims of this proposal are expected to support future efforts to develop mechanism-based therapies that increase CFTR function. Two scientific aims in my proposal describe the means to achieve these goals. The first of these aims will use a powerful method we have developed whereby the phosphorylation state of a specific site in the CFTR channel is controlled by a brief (<1 second) flash of light. This will allow me to observe the intrinsic phosphorylation rates of the channel, and the functional consequence, in real time, in a cellular environment. Given that phosphoregulation of ion channels is well-described in the lung and heart, and often defective in cardiovascular disease, this training and the anticipated ensuing discoveries will likely lead directly to additional opportunities on other ion channel proteins with ties to human health. The second aim will examine the interaction chemistry that is utilized between nucleotide binding domains (NBD) and ATP, their regulatory target. NBDs are ancient domains (billions of years old) that are found throughout biology, thus advancing their mode of action will simultaneously impact multiple areas. I will use structural biology and advanced spectroscopic methods to examine the mechanism of how the soluble NBDs from the CFTR channel bind to their regulatory target, ATP. The likely common output from these combined efforts will be the publication of multiple high value papers and the advanced training in modern techniques for the study of ion channel proteins. Additionally, CFTR’s evolution allows it to serve as a model for both phospho-regulation of ion channels (in common with many other clinically relevant channels in the lung and heart) and for ATP-based activation of other ABC transporters which play important roles in lung physiology. Accordingly, execution of this proposal will establish a platform to ask similarly important questions relating to the regulation of other membrane proteins. As a tr...