Increased ATPase activity produced by mutations at arginine-1380 in nucleotide-binding domain 2 of ABCC8 causes neonatal diabetes

Abstract

Gain-of-function mutations in the genes encoding the ATP-sensitive potassium (K(ATP)) channel subunits Kir6.2 (KCNJ11) and SUR1 (ABCC8) are a common cause of neonatal diabetes mellitus. Here we investigate the molecular mechanism by which two heterozygous mutations in the second nucleotide-binding domain (NBD2) of SUR1 (R1380L and R1380C) separately cause neonatal diabetes. SUR1 is a channel regulator that modulates the gating of the pore formed by Kir6.2. K(ATP) channel activity is inhibited by ATP binding to Kir6.2 but is stimulated by MgADP binding, or by MgATP binding and hydrolysis, at the NBDs of SUR1. Functional analysis of purified NBD2 showed that each mutation enhances MgATP hydrolysis by purified isolated fusion proteins of maltose-binding protein and NBD2. Inhibition of ATP hydrolysis by MgADP was unaffected by mutation of R1380, but inhibition by beryllium fluoride (which traps the ATPase cycle in the prehydrolytic state) was reduced. MgADP-dependent activation of K(ATP) channel activity was unaffected. These data suggest that the R1380L and R1380C mutations enhance the off-rate of P(i), thereby enhancing the hydrolytic rate. Molecular modeling studies supported this idea. Because mutant channels were inhibited less strongly by MgATP, this would increase K(ATP) currents in pancreatic beta cells, thus reducing insulin secretion and producing diabetes.

Fig. 1.

Function and mechanism of the NBDs of SUR1. (A) Role of the K_ATP channel in glucose-stimulated insulin secretion. (B) Comparison of the amino acid sequences of the Walker A motifs (consensus: GXXXXGKT/S) in the NBDs of various ABCC-family proteins. Residues equivalent to SUR1-R1380 are in boldface. MRP, multidrug resistance-related protein; CFTR, cystic fibrosis transmembrane conductance regulator. (C) The SUR1 ATPase catalytic cycle. BeF, beryllium fluoride (BeF₃⁻ and BeF₄²⁻).

Fig. 2.

ATPase activity of SUR1-NBD2 under various conditions. ○, wild-type; ●, R1380C; and ▴, R1380L. (A) ATP activity as a function of ATP concentration. (B) Concentration dependence of MgADP inhibition of ATPase activity (at 1 mM MgATP). (C) Concentration dependence of BeF inhibition of ATPase activity (at 1 mM MgATP).

Fig. 3.

Mean steady-state whole-cell K_ATP currents evoked by a voltage step from −10 to −30 mV before (control, gray bars) and after (white bars) application of 3 mM sodium azide, and in the presence of 3 mM azide plus 0.5 mM tolbutamide (hatched bars) for wild-type channels (n = 12) and R1380L channels (n = 13). *, P = 0.05; **, P = 0.01 compared with control (t test).

Fig. 4.

Mean relationship between ATP concentration and K_ATP conductance (G), expressed relative to that in the absence of nucleotide (G_c) for wild-type (•) and R1380L (▴) channels. (A) In the presence of Mg²⁺: wild-type channels (n = 10) and R1380L channels (n = 5). (B) In the absence of Mg²⁺: wild-type channels (n = 7) and R1380L channels (n = 7).

Fig. 5.

Mean K_ATP currents recorded in the presence of 0.1 mM ADP, 0.1 mM ATP, or 0.1 mM ATP plus 0.1 mM ADP. Currents (G) are expressed relative to the mean of the currents recorded in control solution (G_c) before and after application of nucleotide. **, P < 0.01.

Fig. 6.

Wild-type and mutant ATP-binding site 2. (A) Homology model of ATP-binding site 2 of wild-type SUR1, indicating the main interactions of the posthydrolytic inorganic phosphate (CPK colors). MgADP is green, the NBD1 backbone is red, the signature sequence of NBD1 is yellow, the NBD2 backbone is gray, and the Walker A domain of NBD2 is orange. (B) Mutant model, with the posthydrolytic phosphate unbound. Unlike the wild-type simulation, K1385 hydrogen bonds to D860.