ORIGINAL ARTICLE doi: 10.1111/j.1463-1326.2007.00777.x Functional analysis of two Kir6.2 (KCNJ11) mutations, K170T and E322K, causing neonatal diabetes A. I. Tarasov, 1 C. A. Girard, 1 B. Larkin, 1 P. Tammaro, 1 S. E. Flanagan, 2 S. Ellard 2 and F. M. Ashcroft 1 1 University Laboratory of Physiology, Oxford University, Oxford, UK 2 Institute of Biomedical and Clinical Science, Peninsula Medical School, Exeter, UK Heterozygous activating mutations in Kir6.2 (KCNJ11), the pore-forming subunit of the adenosine triphosphate (ATP)-sensitive potassium (K ATP ) channel, are a common cause of neonatal diabetes (ND). We assessed the functional effects of two Kir6.2 mutations associated with ND: K170T and E322K. K ATP channels were expressed in Xenopus oocytes, and the heterozygous state was simulated by coexpression of wild-type and mutant Kir6.2 with SUR1 (the b cell type of sulphonylurea receptor (SUR)). Both mutations reduced the sensitivity of the K ATP channel to inhibition by MgATP and enhanced whole-cell K ATP currents. In pancreatic b cells, such an increase in the K ATP current is expected to reduce insulin secretion and thereby cause diabetes. The E322K mutation was without effect when Kir6.2 was expressed in the absence of SUR1, suggesting that this residue impairs coupling to SUR1. This is consistent with its predicted location on the outer surface of the tetrameric Kir6.2 pore. The kinetics of K170T channel opening and closing were altered by the mutation, which may contribute to the lower ATP sensitivity. Neither mutation affected the sensitivity of the channel to inhibition by the sulphonylurea tolbutamide, suggesting that patients carrying these mutations may respond to these drugs. Keywords: ATP-sensitive potassium channel, KCNJ11, Kir6.2, neonatal diabetes Received 2 March 2007; accepted 3 April 2007 Introduction ATP-dependent K þ (K ATP ) channels couple cell metabo- lism to membrane excitability in many tissues including the heart, brain, skeletal muscle and many endocrine cells [1]. In glucose-sensing tissues, they couple changes in blood glucose to cell activation: for example, they stimulate insulin secretion from pancreatic b cells [1–4]. In other tissues, they open primarily in response to ischaemic stress. K ATP channels are octameric complexes of Kir6.2 and SUR1 subunits [5,6]. Kir6.2 is a two-transmembrane- helix protein that assembles as a tetramer to form the channel pore [7]. The Kir6.2 tetramer is surrounded by four sulfonylurea receptor (SUR) subunits, with the SUR1 isoform being found in pancreatic b cells [8]. Metabolic regulation of K ATP channel activity is medi- ated by interaction of adenine nucleotides with both Kir6.2 and SUR1 subunits. K ATP channel closure is fav- oured by ATP binding to Kir6.2 [9], whereas binding and hydrolysis of MgATP at the nucleotide-binding domains of SUR1 results in opening of the K ATP chan- nel [9,10]. Consequently, when metabolism is low, K ATP channels are open, hyperpolarizing the b cell and stabilizing the quiescent state in which insulin is not secreted [1,2]. Conversely when metabolism increases, Correspondence: Prof. Frances M. Ashcroft, University Laboratory of Physiology, Oxford University, Parks Road, Oxford OX1 3PT, UK. E-mail: frances.ashcroft@physiol.ox.ac.uk Conflicts of Interest: All authors declare no conflicts of interest. 46 j Diabetes, Obesity and Metabolism, 9 (Suppl. 2), 2007, 46–55 # 2007 The Authors Journal Compilation # 2007 Blackwell Publishing Ltd