Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC217
A Conserved Tryptophan at the Membrane-Water Interface Acts as a Gatekeeper for Kir6.2/SUR1 Channels and Causes Neonatal Diabetes when Mutated
R. Mannikko1, P. J. Stansfeld2, A. S. Ashcroft1, A. T. Hattersley3, M. Sansom2, S. Ellard3, F. M. Ashcroft1
1. Department of Physiology Anatomy & Genetics, University of Oxford, Oxford, United Kingdom. 2. Department of Biochemistry, University of Oxford, Oxford, United Kingdom. 3. Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, United Kingdom.
We identified a novel heterozygous mutation, W68R, in the Kir6.2 subunit of the ATP-sensitive potassium (KATP) channel in a patient with remitting relapsing neonatal diabetes. This tryptophan is absolutely conserved in mammalian Kir channels. The functional effects of mutations at residue 68 of Kir6.2 were studied by heterologous expression in Xenopus oocytes and by homology modelling. We found the Kir6.2-W68R mutation increased the whole-cell KATP current, and reduced MgATP inhibition in both the homomeric (hom) and heterozygous (het) state: IC50 were 142±12µM (S.E., n=13) for homW68R, 41±7µM for hetW68R and 16±1µM (n=26) for wild-type (WT) channels. Substitution of a glutamate residue (homW68E) produced an identical effect: IC50=153±18 µM (n=7). Replacement with tyrosine (Y) rendered the KATP channel almost completely insensitive to ATP block. The effect of mutations at W68 was not charge or size dependent, the order of potency for ATP inhibition being W<M~L<R~E~K~A<C~F<Y. Mutation of W68 dramatically increased the unliganded channel open probability (Po(0)) which was ~0.8 for W68A, W68F and W68Y channels versus ~0.4 for WT channels. The PIP2 affinity, as judged by the sensitivity of channel to inhibition by neomycin, was also affected, but did not explain the reduced ATP sensitivity: IC50 were WT, 129±15μM (n=8); W68R, 11.3±2.1μM (n=4); W68E, 8.2±0.9μM (n=4); W68F,148±32μM (n=5); W68Y: <25% block at 10 mM neomycin. In different Kir crystal structures the residue corresponding to W68 adopts two distinct positions. In most Kir, the tryptophan packs against the second transmembrane domain (TM2), so impeding its movement and opening of the gate (flipped-in). In Kir2.2, this residue is ‘flipped-out’, allowing movement of TM2 and opening of the channel gate. This suggests W68 may act as a molecular 'gatekeeper' for Kir channels.
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