Kir6.2 mutations causing neonatal diabetes provide new insights into Kir6.2-SUR1 interactions

Abstract

ATP-sensitive K(+) (K(ATP)) channels, comprised of pore-forming Kir6.2 and regulatory SUR1 subunits, play a critical role in regulating insulin secretion. Binding of ATP to Kir6.2 inhibits, whereas interaction of MgATP with SUR1 activates, K(ATP) channels. We tested the functional effects of two Kir6.2 mutations (Y330C, F333I) that cause permanent neonatal diabetes mellitus, by heterologous expression in Xenopus oocytes. Both mutations reduced ATP inhibition and increased whole-cell currents, which in pancreatic beta-cells is expected to reduce insulin secretion and precipitate diabetes. The Y330C mutation reduced ATP inhibition both directly, by impairing ATP binding (and/or transduction), and indirectly, by stabilizing the intrinsic open state of the channel. The F333I mutation altered ATP binding/transduction directly. Both mutations also altered Kir6.2/SUR1 interactions, enhancing the stimulatory effect of MgATP (which is mediated via SUR1). This effect was particularly dramatic for the Kir6.2-F333I mutation, and was abolished by SUR1 mutations that prevent MgATP binding/hydrolysis. Further analysis of F333I heterozygous channels indicated that at least three SUR1 must bind/hydrolyse MgATP to open the mutant K(ATP) channel.

Figure 1

(A) Whole-cell currents recorded from Xenopus oocytes coexpressing SUR1 and either Kir6.2, Kir6.2-F333I or Kir6.2-Y330C, as indicated, in response to voltage steps of ±20 mV from a holding potential of −10 mV. The bars indicate azide and tolbutamide (tolb) application. (B) Mean steady-state whole-cell currents evoked by a voltage step from −10 to −30 mV before (control, white bars) and after application of 3 mM azide (pale grey bars) and in the presence of 3 mM azide plus 0.5 mM tolbutamide (dark grey bars). The number of oocytes is indicated below the bars.

Figure 2

(A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing SUR1 and either wild type Kir6.2, Kir6.2-F333I or Kir6.2-Y330C, as indicated, in response to 3 s voltage ramps from −110 to +100 mV. ATP (100 μM) was applied as indicated by the horizontal bars. (B) Mean relationship between [ATP] and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c) for wild-type and mutant K_ATP channels. Experiments were carried out in the absence of Mg²⁺. The smooth curves are the best fit to the Hill equation (equation (4)), using the values given in Table IA. (a) Kir6.2/SUR1 (open symbols, n=10), heterozygous (semifilled symbols, n=6) and homomeric (filled symbols, n=11) Kir6.2-Y330C/SUR1 channels. (b) Kir6.2/SUR1 (open symbols, n=10), heterozygous (semifilled symbols, n=5), and homomeric (filled symbols, n=6) Kir6.2-F333I/SUR1 channels.

Figure 3

Mean relationship between [ATP] and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c) for Kir6.2ΔC (circles, n=7), Kir6.2ΔC-F333I (squares, n=5) and Kir6.2ΔC-Y330C (triangles, n=6) channels. SUR1 was not coinjected in these experiments. The smooth curves are the best fit of equation (4), using the values given in Table IB.

Figure 4

Single-channel currents recorded at −60 mV from inside-out membrane patches excised from oocytes expressing Kir6.2/SUR1, Kir6.2-F333I/SUR1, Kir6.2-Y330C/SUR1, Kir6.2ΔC and Kir6.2ΔC-Y330C. The dashed line indicates the zero current level.

Figure 5

Simulated ATP dose-inhibition curves (dashed lines) for heteromeric Kir6.2-Y330C/SUR1 (left) and Kir6.2-F333I/SUR1 (right) channels. The predicted IC₅₀ values are 43 μM (Kir6.2-Y330C/SUR1) and 25 μM (Kir6.2-F333I/SUR1). The data are the same as those in Figure 2.

Figure 6

(A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing SUR1 and either wild type, Kir6.2-F333I or Kir6.2-Y330C as indicated, in response to voltage ramps from −110 to +100 mV. MgATP (1 mM) was applied as indicated by the horizontal bars. (B) Mean relationship between [MgATP] and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c) for wild-type and mutant K_ATP channels. The smooth curves are the best fit to equation (4), using the values given in Table IA. (a) Kir6.2/SUR1 (open symbols, n=6), heterozygous (semifilled symbols, n=6) and homomeric (filled symbols, n=7) Kir6.2-Y330C/SUR1 channels. (b) Kir6.2/SUR1 (open symbols, n=6) and heterozygous Kir6.2-F333I/SUR1 (semifilled symbols, n=6) channels. The shaded bar indicates the predicted range of physiological ATP concentrations found in β-cells.

Figure 7

(A) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing Kir6.2-F333I and either SUR1 (a) or SUR1-KA/KM (b) in response to voltage ramps from −110 to +100 mV. MgATP (1 mM) was applied as indicated by the bars. (B) Mean slope conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c), plotted against time, for Kir6.2-F333I/SUR1 (open circles, n=5) and Kir6.2-F333I/SUR1-KA/KM (filled circles, n=7). MgATP (1 mM) was added as indicated by the bar. (C) Mean slope conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c), plotted against time for Kir6.2-F333I/SUR1-KA/KM. The bar indicates application of 1 mM MgATP (open circles, n=10) or of 1 mM MgATP plus 10 μM LY294002 (filled squares, n=5). (D) Currents recorded in inside-out patches excised from Xenopus oocytes coexpressing Kir6.2-F333I and SUR1 in response to voltage ramps from −110 to +100 mV. MgATP and MgADP (1 mM) were applied as indicated by the bars.

Figure 8

(A) Schematic of the different channel types expected when wild-type and mutant Kir6.2 are coexpressed with SUR1 (as in the heterozygous state). The box indicates channel types expected to have altered MgATP activation if four activated SUR1 are needed to open the channel. (B) Schematic of the different channel types expected when wild-type and mutant SUR1 are coexpressed with Kir6.2-F333I (as in the heterozygous state). The box indicates channel types expected to have altered MgATP activation if four activated SUR1 are needed to open the channel. (C) Mean slope conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c), plotted against time, for the heterozygous mixture of channel types that occur when Kir6.2-F333I is coexpressed with both SUR1 and SUR1-KA/KM subunits (semifilled circles, n=9). The blue line indicates the Kir6.2-F333I/SUR1 data and the pink line the Kir6.2-F333I/SUR1-KAKM data (same data as in Figure 7B). MgATP (1 mM) was added as indicated by the bar.

Figure 9

(A) Homology model of Kir6.2 (Antcliff et al, 2005). For clarity, only two subunits are shown, and the intracellular and transmembrane domains are from separate subunits (each subunit is shown in a different colour). ATP (yellow) is shown in its binding site. The red circle illustrates the ATP-binding site. (B) Close-up of the ATP-binding sites shown in A. ATP is shown in yellow and residues lining the ATP-binding site are labelled. Residues Y330 and F333 are shown in red.