The goal of this research is to understand how the subunits of ATP sensitive potassium channels (KATP) interact by utilizing known disease-causing mutations as starting points for biochemical and electrophysiological analysis. KATP channels are expressed in and responsible for beta-cell physiology. Their conductance state is regulated by intracellular nucleotide levels, and thus is an important link between cellular metabolic status and cell excitability. KATP channels are composed of two types of subunits: Kir6.2 and SUR1, But how these subunits communicate with each other-their specific inter-subunit amino acid interactions, and how these interactions specify KATP biogenesis and activity-is not known. High- resolution crystal structures do not exist for either subunit or for the KATP complex; therefore, functional experiments are needed to answer these important questions. I will perform a detailed study of two residues within SUR1, E128 and R74. These residues are associated with congenital hyperinsulinism due to trafficking defects, but their activity profiles mimic neonatal diabetes mutations. Both residues are members of a select group, mutation of which leads to trafficking defects that can be 'rescued' by sulfonylurea (SU) treatment. Based on the fact that mutation of either residue leads to KATP biogenesis and activity defects, I hypothesize that E128 and R74 contribute to SUR1-Kir6.2 interactions. Specific Aim 1 addresses how these two residues facilitate proper KATP biogenesis using biochemical experiments. I will determine how systematic mutagenesis of these residues effect SUR1-Kir6.2 associations (co-immunoprecipitation) and trafficking to the plasma membrane (western blot & chemiluminescence). Further, the influence of SU on both processes will be measured. Specific Aim 2 utilizes the same mutant SUR1 subunits and considers how they affect KATP channel activity, both ATP and SU sensitivities. Finally, in Specific Aim 3,1 will identify the residues of Kir6.2 that interact with SUR1 E128 and R74 by a systematic mutagenesis scan of Kir6.2. Verification of SUR1-Kir6.2 interacting pairs will be achieved by study of double-mutant electrophysiological properties and the ability of prospective pairs to form disulphide-bridges following cysteine-replacement and oxidation. This work is of particular public health importance because drugs widely used in diabetes treatment, sulfonylureas, have recently been found to change an important regulator of insulin secretion, the KATP ion channel. Studying how these drugs work has the potential for discovery of new treatments for a different disease, congenital hyperinsulinism. In addition, this work will further our understanding of the basic biology of the KATP channel. [unreadable] [unreadable] [unreadable]
NIH ExPorter
NIH ExPorter