A novel high-affinity inhibitor against the human ATP-sensitive Kir6.2 channel.

The adenosine triphosphate (ATP)-sensitive (KATP) channels in pancreatic ß cells couple the blood glucose level to insulin secretion. KATP channels in pancreatic ß cells comprise the pore-forming Kir6.2 and the modulatory sulfonylurea receptor 1 (SUR1) subunits. Currently, there is no high-affinity and relatively specific inhibitor for the Kir6.2 pore. The importance of developing such inhibitors is twofold. First, in many cases, the lack of such an inhibitor precludes an unambiguous determination of the Kir6.2's role in certain physiological and pathological processes. This problem is exacerbated because Kir6.2 knockout mice do not yield the expected phenotypes of hyperinsulinemia and hypoglycemia, which in part, may reflect developmental adaptation. Second, mutations in Kir6.2 or SUR1 that increase the KATP current cause permanent neonatal diabetes mellitus (PNDM). Many patients who have PNDM have been successfully treated with sulphonylureas, a common class of antidiabetic drugs that bind to SUR1 and indirectly inhibit Kir6.2, thereby promoting insulin secretion. However, some PNDM-causing mutations render KATP channels insensitive to sulphonylureas. Conceptually, because these mutations are located intracellularly, an inhibitor blocking the Kir6.2 pore from the extracellular side might provide another approach to this problem. Here, by screening the venoms from >200 animals against human Kir6.2 coexpressed with SUR1, we discovered a small protein of 54 residues (SpTx-1) that inhibits the KATP channel from the extracellular side. It inhibits the channel with a dissociation constant value of 15 nM in a relatively specific manner and with an apparent one-to-one stoichiometry. SpTx-1 evidently inhibits the channel by primarily targeting Kir6.2 rather than SUR1; it inhibits not only wild-type Kir6.2 coexpressed with SUR1 but also a Kir6.2 mutant expressed without SUR1. Importantly, SpTx-1 suppresses both sulfonylurea-sensitive and -insensitive, PNDM-causing Kir6.2 mutants. Thus, it will be a valuable tool to investigate the channel's physiological and biophysical properties and to test a new strategy for treating sulfonylurea-resistant PNDM.

Chloroquine blocks the Kir4.1 channels by an open-pore blocking mechanism.

Kir4.1 channels have been implicated in various physiological processes, mainly in the K+ homeostasis of the central nervous system and in the control of glial function and neuronal excitability. Even though, pharmacological research of these channels is very limited. Chloroquine (CQ) is an amino quinolone derivative known to inhibit Kir2.1 and Kir6.2 channels with different action mechanism and binding site. Here, we employed patch-clamp methods, mutagenesis analysis, and molecular modeling to characterize the molecular pharmacology of Kir4.1 inhibition by CQ. We found that this drug inhibits Kir4.1 channels heterologously expressed in HEK-293 cells. CQ produced a fast-onset voltage-dependent pore-blocking effect on these channels. In inside-out patches, CQ showed notable higher potency (IC50 ≈0.5µM at +50mV) and faster onset of block when compared to whole-cell configuration (IC50 ≈7µM at +60mV). Also, CQ showed a voltage-dependent unblock with repolarization. These results suggest that the drug directly blocks Kir4.1 channels by a pore-plugging mechanism. Moreover, we found that two residues (Thr128 and Glu158), facing the central cavity and located within the transmembrane pore, are particularly important structural determinants of CQ block. This evidence was similar to what was previously reported with Kir6.2, but distinct from the interaction site (cytoplasmic pore) CQ-Kir2.1. Thus, our findings highlight the diversity of interaction sites and mechanisms that underlie amino quinolone inhibition of Kir channels.