Katp: dynamics of a channel-enzyme complex

This $385,191 Project Grant awarded by the National Institute of General Medical Sciences (NIGMS) Biomedical Research and Research Training Program (CFDA 93.859) supports research to study the structure, function, and regulation of ATP-sensitive potassium (KATP) channels. The project aims to develop novel spectroscopic tools to simultaneously probe KATP channel structure and function, with the goal of understanding how ligand binding and long-range communication between subunits in the multi-protein KATP complex regulate channel activity. Key activities include measuring site-specific nucleotide binding using Förster resonance energy transfer (FRET) techniques, assessing the enzymatic activity of the sulfonylurea receptor (SUR) subunit, and probing the structural basis of coupling between the KATP channel subunits. This research will yield new insights into the physiological role of KATP channels and their dysfunction in certain forms of diabetes. The award is held by The Trustees of Trinity College, a private non-profit educational institution in Connecticut, and the project will be conducted at their facilities.

Abstract

ATP-sensitive k+ channels (katp) couple a cell's metabolic state to its electrical excitability. This process is crucial in pancreatic β-cells where katp couples glucose uptake to insulin secretion. In the heart, katp plays a protective role, opening during hypoxia. The role of these channels is so vital that katp dysfunction is associated with diseases of insulin secretion, with severe mutations causing treatment-resistant syndromes with accompanying neurological deficits. To study this critical channel, we developed spectroscopic tools that allow US to simultaneously probe katp structure and function. This is an ideal approach for understanding katp's physiological role and probing long-standing questions of biochemistry. How is the input of energy from ligand binding coupled to changes in protein function? How can one mechanistically separate ligand binding from protein activation? How is long-range communication achieved between subunits in a multi-protein complex? Katp comprises four pore-forming inward-rectifier k+ channel subunits (KIR), each associated with a sulfonylurea receptor (SUR). The connection between katp and metabolism is established by ATP/ADP binding to three classes of nucleotide binding site (NBS). Binding to KIR shuts katp; binding to two sites on SUR opens katp. Thus, channel activity is a function of the occupancy of each NBS and their relative energetic contribution to gating. We have taken a novel approach to understanding the role of each NBS in katp gating: measuring site-specific binding using förster resonance energy transfer (fret) between fluorescent ATP derivatives and channels tagged with fluorescent, non-canonical amino acids. Simultaneous recordings of binding and ionic current allow US to model each NBS in terms of its nucleotide affinity and energetic influence on gating. This experimental/analytical framework will be applied to examine the difference between heart and pancreatic katp subtypes and the consequences of katp mutations associated with diabetes. We will assess the enzymatic activity of SUR (an atpase) with mutations in putative catalytic amino acids and novel fluorescent non-hydrolyzable ATP derivatives synthesized in house. Finally, we will probe the structural basis of coupling between the KIR and SUR subunits using an enhanced fret technique in which colored metal ions are used as short-distance fluorescence quenchers. These combined approaches will yield a new appreciation for the physiological role of katp and its dysfunction in certain forms of diabetes. Novel insights into the energetics and conformational dynamics of katp will provide a template for understanding the behavior of other protein complexes.