Abstract
ATP-sensitive K+ channel (KATP) gating is modulated via adenine nucleotide (ATP or ADP) binding to three classes of nucleotide binding site (NBS). The first site is located on the pore-forming Kir subunit of KATP (Kir6.2 in pancreatic β-cells); binding to this NBS is inhibitory. Two stimulatory NBSs are located on the cytoplasmic nucleotide binding domains of the accessory sulfonylurea receptor subunit (SUR1 in β-cells), part of the ABC transporter family. Despite decades of investigation, the details of nucleotide modulation, the affinity and specificity for each NBS, the interactions between the various NBSs, and the mechanism whereby nucleotide binding events are transmitted to the channel pore are largely unknown. To fully understand the nucleotide effects on KATP, we have labelled each NBS (one at a time) with the fluorescent, non-canonical amino acid ANAP and measured nucleotide binding at each site using FRET between ANAP and trinitrophenyl (TNP) nucleotide derivatives in unroofed membrane fragments. This has allowed us to dissect nucleotide binding to each site, evaluate mechanistically mutations expected to affect nucleotide binding (e.g. SUR1-K1384A, Kir6.2-G334D) or channel gating (e.g. Kir6.2-C166S), and investigate the action of clinically important drugs that inhibit or potentiate KATP (sulfonylureas and K+ channel openers). We have also combined this method with patch-clamp electrophysiology (either in separate experiments or simultaneously with patch-clamp fluorometry) to determine the functional consequences of binding at each NBS. This method will not only yield insights into KATP channel activity, but is readily extended to other channels, ABC transporters, and virtually any protein with a suitable fluorescent ligand.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have