ATP-sensitive potassium channels (KATP channels) in pancreatic beta-cells regulate insulin secretion in response to changes in glucose metabolism. KATP channels are composed of 2 subunits: an inward rectifier (KIR6.2) which forms the pore and a sulfonylurea receptor, SUR1, that regulates channel activity giving sensitivity to MgADP, sulfonylureas (SUs) and potassium channel openers (KCOs). Some forms of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) are caused by mutations in KATP channel subunits that result in loss of channel activity. KATP channels set the membrane potential and loss of activity leads to depolarization that allows voltage-gated Ca2+ channels to open, increasing the Ca2+ levels enough to release of insulin. KATP channels are the targets for SUs used to treat non-insulin dependent diabetes mellitus because they close KATP channels and cause insulin secretion. KCOs inhibit insulin secretion by opening KATP channels and are used to treat PHHI and hyperinsulinemic states. Both of these drugs bind to SUR1, the regulatory subunit, which is an ATP binding cassette protein with two nucleotide binding folds (NBFs) and an ABC signature sequence in each NBF. The long-term objectives of this proposal are to understand the structure and regulation of KATP channels. Studies on the ATP binding and hydrolytic properties of SUR1 are critical for understanding how these channels are regulated by nucleotides, how novel compounds can be developed to regulate cellular electrical activity, and they should provide insight into diabetes and other disorders of glucose metabolism. These studies will also help us understand the physiological role of KATP channels in the neuro-endocrine system. My objective is to perform a structure-function analysis of SUR1, specifically I will pursue the following aims: (1) Determine the domains of SUR1 and K IR6.2 that are in close proximity to 1251-azidoglibenclamide, a derivative of a commonly used hypoglycemic agent that binds tightly to SUR1, covalently photolabeling it and K IR6.2; (2) Interchange the signature sequences of SUR1 to determine if they are required for ATP binding, hydrolysis, and regulation of channel activity; (3) Pursue my early observation that there is an interaction between the N-terminus of K IR6.2 and the C-terminus of SUR1. This will be done using a two-hybrid system approach and GST-""""""""pull-downs""""""""; (4) Attempt to express and purify SUR1 using the yeast S. cerevisiae in order to develop assays to measure its ATPase activity directly.
| Vila-Carriles, Wanda H; Zhao, Guiling; Bryan, Joseph (2007) Defining a binding pocket for sulfonylureas in ATP-sensitive potassium channels. FASEB J 21:18-25 |
