Functional analysis of two Kir6.2 (KCNJ11) mutations, K170T and E322K, causing neonatal diabetes

Abstract

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.

Dual role of K ATP channel C-terminal motif in membrane targeting and metabolic regulation

“…Inside-out macropatch recordings of COSm6 cells expressing Kir6.2 E322K revealed a marked difference in ATP sensitivity compared with WT channels (Fig. 5 A-C), consistent with previous findings in nonmammalian cells (17). The E322K channel is significantly less sensitive to inhibition by ATP (K 1/2 for ATP inhibition is shifted from 15 M in WT to 62 M in the mutant, Fig.…”

“…Importantly, the Kir6.2 E322K mutant results in striking changes in ATP sensitivity ( Fig. 5C; K 1/2 for ATP inhibition is shifted from 15 M in WT to 62 M in the mutant), consistent with a strong gain-of-function cellular phenotype (17). Thus, as illustrated by our functional data (Figs.…”

β IV -Spectrin and CaMKII facilitate Kir6.2 regulation in pancreatic beta cells

“…For example, we previously demonstrated that a human Kir6.2 E322K permanent neonatal diabetes mutation that blocks AnkB association is associated with both defects in membrane trafficking and Kir6.2 membrane biophysical activity (12). However, Tarasov et al found that this site may also be involved in coupling Kir6.2 regulation with SUR1 (15), and individuals with the KCNJ11 E322K mutation respond to sulfonylurea treatment. Whereas additional data will be necessary to define the mechanisms underlying the regulation of these proteins at baseline and in disease, these findings ultimately support the clear in vivo roles of the cytoskeleton and cytoskeletal-associated proteins in the regulation of excitable cell function.…”

“…Ankyrin-B (AnkB) is present within the islet and necessary for the targeting and expression of the ATP-sensitive potassium channel (K ATP ) (12) and inositol 1,4,5 trisphosphate (IP3) receptor (13). Human mutations in Kir6.2 (K ATP channel α-subunit) that block binding with AnkB are associated with neonatal diabetes mellitus (12,14), although these variants may also affect K ATP Kir6.2/SUR1 subunit coupling (15). Consistent with β IV -spectrin localization, AnkB and Kir6.2 are highly Whereas CaMKIIδ associates only with β IV -spectrin fusion constructs containing the distal C-terminal CaMKII binding motif in islet lysates, β IV -spectrin-dependent association with AnkB, Kir6.2, and SUR1 requires residues 1620-2218 (includes 15th spectrin repeat).…”

Electrophysiology of Islet Cells

“…The underlying mechanisms include a reduced sensitivity of the channel to ATP, an increased ATPase activity of SUR1, or an abnormal channel activation in response to nucleotide diphosphates or long-chain acyl CoAs (Riedel et al 2003;Gloyn et al 2005;Proks et al 2004;de Wet et al 2007b;Ellard et al 2007;Schwanstecher et al 2002). It is shown that in some K IR 6.2 mutations, impaired coupling to SUR1 determines the loss of nucleotide inhibition (Tarasov et al 2007).…”