Abstract
The function of the ATP-sensitive potassium (K(ATP)) channel relies on the proper coupling between its two subunits: the pore-forming Kir6.2 and the regulator SUR. The conformation of the interface between these two subunits can be monitored using a rhodamine 123 (Rho) protection assay because Rho blocks Kir6.2 with an efficiency that depends on the relative position of transmembrane domain (TMD) 0 of the associated SUR (Hosy, E., Dérand, R., Revilloud, J., and Vivaudou, M. (2007) J. Physiol. 582, 27-39). Here we find that the natural and synthetic K(ATP) channel activators MgADP, zinc, and SR47063 induced a Rho-insensitive conformation. The activating mutation F132L in SUR1, which causes neonatal diabetes, also rendered the channel resistant to Rho block, suggesting that it stabilized an activated conformation by uncoupling TMD0 from the rest of SUR1. At a nearby residue, the SUR1 mutation E128K impairs trafficking, thereby reducing surface expression and causing hyperinsulinism. To augment channel density at the plasma membrane to investigate the effect of mutating this residue on channel function, we introduced the milder mutation E126A at the matching residue of SUR2A. Mutation E126A imposed a hypersensitive Rho phenotype indicative of a functional uncoupling between TMD0 and Kir6.2. These results suggest that the TMD0-Kir6.2 interface is mobile and that the gating modes of Kir6.2 correlate with distinct positions of TMD0. They further demonstrate that the second intracellular loop of SUR, which contains the two residues studied here, is a key structural element of the TMD0-Kir6.2 interface.
Highlights
Because of this pivotal role, the KATP channel is a prime target for pharmacological intervention to correct insulin secretion dysfunction, and there exist a number of molecules, several in clinical use, that are able to block or activate the channel by binding to SUR
MgADP Binding to SUR Protects Kir6.2 from Rhodamine Block—Xenopus oocytes injected with cRNA of Kir6.2⌬C36 or with a mixture of cRNAs coding SUR2A or SUR1 and Kir6.2expressed exogenous potassium currents inhibited by ATP
The full KATP channel constituted of SUR1 or SUR2A and Kir6.2 were strongly blocked by rhodamine 123 (Rho) (ϳ80%) in control but only weakly in the presence of MgADP (13 Ϯ 4 and 22 Ϯ 5% inhibition for SUR2A and SUR1, respectively; Fig. 1, B, C, and F)
Summary
Because of this pivotal role, the KATP channel is a prime target for pharmacological intervention to correct insulin secretion dysfunction, and there exist a number of molecules, several in clinical use, that are able to block or activate the channel by binding to SUR. In excised inside-out patches, MgADP (300 M), the main physiological KATP channel opener, inhibited SUR-less Kir6.2⌬C36 currents by ϳ40%, but it did not change SURϩKir6.2 currents. The full KATP channel constituted of SUR1 or SUR2A and Kir6.2 were strongly blocked by Rho (ϳ80%) in control but only weakly in the presence of MgADP (13 Ϯ 4 and 22 Ϯ 5% inhibition for SUR2A and SUR1, respectively; Fig. 1, B, C, and F).
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
