Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C50
Mutations of the same conserved glutamate residue in NBD2 of the SUR1 subunit of the KATP channel can result in either hyperinsulinism or neonatal diabetes
R. Mannikko1, S. E. Flanagan2, X. Sim1, D. Segal3, K. Hussain4, S. Ellard2, A. T. Hattersley2, F. M. Ashcroft1
1. Department of Physiology Anatomy & Genetics, University of Oxford, Oxford, United Kingdom. 2. Institute of Biomedical and Clinical Research, Peninsula Medical School, Exeter, United Kingdom. 3. Centre for Diabetes and Endocrinology, Johannesburg, South Africa. 4. Institute of Child Health, UCL, London, United Kingdom.
Patients with neonatal diabetes (ND) or hyperinsulinemia (HI) often carry gain- or loss-of-function mutations in their KATP channels, respectively. KATP channels are octameric complexes composed of four SUR and four Kir6.2 subunits. Binding of ATP or ADP to Kir6.2 inhibits the channel whereas interaction of Mg-nucleotides with the nucleotide-binding domains (NBDs) of SUR stimulates channel activity. Mutation of a conserved glutamate (E1506) in the Walker B motif of NDB2 of SUR1 to lysine is known to cause HI by impairing Mg-nucleotide activation of the KATP channel. We now report two novel heterozygous SUR1 mutations (E1506D, E1506G) in separate patients with ND. To understand why mutations at the same residue can cause either ND or HI, we expressed wild-type (WT) and mutant channels in Xenopus oocytes and studied KATP currents with electrophysiological methods. Resting whole-cell KATP currents in oocytes expressing Kir6.2 and equal amounts of wild-type or ND mutant SUR1 (to simulate the heterozygous state) were larger than for WT channels. In pancreatic beta-cells, a similar increase in KATP current would impair insulin release, consistent with the diabetic phenotype. No E1506K currents were recorded at rest or following metabolic inhibition, explaining the HI phenotype. The sensitivity of ND mutant channels to MgATP inhibition was slightly reduced: IC50 were 21±2µM (S.E., n=11), 19±2µM (n=10), 13±1µM (n=23) for homomeric E1506D, E1506G and WT, respectively. All mutations decreased channel activation by MgADP as assessed using WT or an ATP-insensitive Kir6.2 subunit, but had little effect on MgATP activation. Using wild-type Kir6.2, a 30s-preconditioning exposure to 10mM MgATP caused a marked reduction in the ATP sensitivity of ND channels (IC50=78±3µM (n=8) for E1506D; 108±10µM (n=7) for E1506G), a small decrease in that of wild-type channels (IC50=30±3 µM, n=11) and no change for E1506K channels. Smaller shifts were observed with concentrations of MgATP >300µM. Thus differentially altered nucleotide regulation (MgADP for E1506K; MgATP for E1506G and E1506D) by NBD2 of SUR1 may explain the difference in resting whole-cell currents of ND and HI mutant channels and the respective clinical phenotypes.
Where applicable, experiments conform with Society ethical requirements