Multiple Nucleotide-Dependent Conformations of a Mycobacterial RCK Domain

Abstract

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 K ATP , 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 K ATP (sulfonylureas and K þ 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 K ATP channel activity, but is readily extended to other channels, ABC transporters, and virtually any protein with a suitable fluorescent ligand. 543-Pos Single Molecule FRET Reveals Lipid Induced Conformational Changes inCytoplasmic Domain of Kir2.1 Anionic phospholipids are common regulators of ion channel activity, including Kir2.1, an inward rectifying potassium channel crucial for setting and maintaining resting membrane potential in many tissues, which is activated specifically by PIP2 and phosphatidyl glycerol (PG). Crystal structures indicate that a simple upward translocation of the cytoplasmic Kir domain is induced by PIP2 and PG binding. However, recent computational and single-molecule FRET experiments show more complex intra-domain movements and rotation in the Kir domain of prokaryotic KirBac1.1 upon PIP2 binding. Single molecule FRET measurements on eukaryotic human Kir2.1 indicate similar complex motions that are not detected in static crystal structures. Our measurements indicate dynamic conformational 'breathing' throughout the Kir domain, suggesting greater flexibility in the intracellular domain of ion channels than may be apparent in structural studies. Specifically, our data indicate a PIP2 dependent shifts in average single molecule FRET (smFRET) distributions, with outward motions seen prior to the slide helix (residue 59) and an inward motion seen at the base (residues 286 and 288) consistent with a twisting motion of the Kir domain that was previously shown to be associated with opening in KirBac1.1. In the absence of PIP2, smFRET signals are very dynamic with relatively large (>20A) motions at specific residues. PIP2 stabilizes the conformational dynamics of the Kir domain, with less transitions between structural states, and reduction in the occurrence of large conformational transitions. Potassium (K) channels play a critical role in bacterial electrolyte homeostasis and control of membrane potential, and are thus im...