Impact of disease-causing SUR1 mutations on the KATP channel subunit interface probed with a rhodamine protection assay.
J. Biol. Chem.. 2009-11-20; 285(5): 3084-3091
DOI: 10.1074/jbc.m109.043307
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1. J Biol Chem. 2010 Jan 29;285(5):3084-91. doi: 10.1074/jbc.M109.043307. Epub 2009
Nov 20.
Impact of disease-causing SUR1 mutations on the KATP channel subunit interface
probed with a rhodamine protection assay.
Hosy E(1), Dupuis JP, Vivaudou M.
Author information:
(1)Institut de Biologie Structurale (CEA, CNRS, UJF), Laboratoire des Protéines
Membranaires, 41 Rue Jules Horowitz, 38027 Grenoble, France.
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.
DOI: 10.1074/jbc.M109.043307
PMCID: PMC2823424
PMID: 19933268 [Indexed for MEDLINE]