Muscle dysfunction caused by a KATP channel mutation in neonatal diabetes is neuronal in origin

Abstract

Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel, cause neonatal diabetes. Many patients also suffer from hypotonia (weak and flaccid muscles) and balance problems. The diabetes arises from suppressed insulin secretion by overactive KATP channels in pancreatic beta-cells, but the source of the motor phenotype is unknown. By using mice carrying a human Kir6.2 mutation (Val59-->Met59) targeted to either muscle or nerve, we show that analogous motor impairments originate in the central nervous system rather than in muscle or peripheral nerves. We also identify locomotor hyperactivity as a feature of KATP channel overactivity. These findings suggest that drugs targeted against neuronal, rather than muscle, KATP channels are needed to treat the motor deficits and that such drugs require high blood-brain barrier permeability.

Figure 1. Muscle function is impaired by expression of Kir6.2-V59M in neurons but not in muscle.

(A) Kir6.2 expression in tissue isolated from control, m-V59M or n-V59M mice. Wild-type, but not mutant, cDNA is cut by the restriction enzyme BtsCI: two bands thus indicates the presence of the wild-type gene only, and three bands indicates both wild-type and mutant genes. sk1, quadriceps muscle; sk2, triceps muscle; sk3, diaphragm. Data are representative of experiments on 4 ROSA and 4 m-V59M mice done in parallel, and 3 ROSA and 3 n-V59M mice performed in parallel. (Supporting on-line text and additional control data in Fig.S1A). (B) Weight lifting, inverted screen and horizontal bar tests on 11-12-week old m-V59M (n=42), n-V59M (n=31) and control mice (Mck-Cre, n=35; Nes-Cre, n=53). Mean±SEM. ***, p<0.001 (Kruskal-Wallis One Way Analysis of Variance on Ranks). Additional control data in Fig.S2.

Figure 2. Motor coordination and locomotor activity are impaired by expression of Kir6.2-V59M in neurons but not in muscle.

(A) Orientation time and percentage of falls on the static rod test, and time before falling from a rotating rod, for m-V59M, n-V59M and control mice. Same mice as in Fig.1. (B,C) Duration of time spent in spontaneous physical activity (B) or using a free-running wheel (C) over a 23-hr period for 12-week old n-V59M (n=13), Nes-Cre (n=14), ROSA (n=14) and WT (n=4) littermates. Mean±SEM. **, p<0.01. ***, p<0.001 (One-way ANOVA). Additional control data in Fig.S3.

Figure 3. K_ATP channel activity in Purkinje cells of n-V59M mice and skeletal muscle of m-V59M mice

(A) Mean (±SEM) action potential frequency of control (grey bars) and n-V59M (black bars) neurons in the absence (control: n=9 mice, 34 neurons. n-V59M: n=3 mice, 26 neurons) and presence (control: n=7 mice, 16 neurons. n-V59M, n=3 mice, 15 neurons) of 500µM tolbutamide. Cell-attached recordings. **, p=0.002 (t-test). (B) Mean (±SEM) resting membrane potential of control (grey bars, n=17 neurons, 6 mice) and n-V59M neurons (black bars, n=13 neurons, 5 mice) in the absence and then the presence of 200µM tolbutamide. Whole-cell recordings. ***, p<0.001 (t-test). (C) K_ATP channel currents recorded at -60mV in inside-out patches from control (above) or m-V59M (below) FDB muscle. (D) Mean (±SEM) MgATP sensitivity of K_ATP channels in inside-out patches from control (●, n=4; IC₅₀=15µM) or m-V59M (○, n=4; IC₅₀=66µM) FDB muscle.

Figure 4. Kir6.2/SUR1 is more sensitive to metabolism than Kir6.2/SUR2A due to differential nucleotide sensitivity

(A) Mean (±SEM) steady-state whole-cell K_ATP currents evoked by a voltage step from -10 to -30 mV before (black bars) and after (pale-gray bars) application of 3mM azide for oocytes injected with Kir6.2 (WT), or 1:1 mixture of Kir6.2 and Kir6.2-V59M (hetV59M mRNA, plus either SUR1 or SUR2A mRNAs. White bars, 3mM azide plus 300µM diazoxide (SUR1) or 100µM pinacidil (SUR2A). Dark-grey bars, 3mM azide, 300µM diazoxide and 0.5mM tolbutamide (SUR1) or 3mM azide, 100µM pinacidil and 10µM glibenclamide (SUR2A). The number of oocytes is indicated. **, p<0.001 with respect to wild-type (t-test). (B) Mean (±SEM) MgATP sensitivity of Kir6.2/SUR1 (●, n=15), Kir6.2/SUR2A (○, n=15), hetKir6.2-V59M/SUR1 (■, n=12) and hetKir6.2-V59M/SUR2A (□, n=16) channels. K_ATP conductance (G) is expressed relative to that in the absence of nucleotides (G_C). The curves are the best fit of the Hill equation to the mean data (Supporting on-line text, Table S1). (C) MgADP activation of channels composed of wild-type Kir6.2 (left) or Kir6.2-V59M (right) plus either SUR1 (black bars) or SUR2A (white bars). K_ATP conductance (G) in the presence of 100µM MgADP is expressed relative to the maximal conductance in the absence of MgADP (G_C). Mean±SEM. (D,E) Mean (±SEM) MgATP sensitivity of hetKir6.2-V59M/SUR1 (D) and hetKir6.2-V59M/SUR2A (E) channels in the absence (■; SUR1, n=12; SUR2A, n=16) and presence (□; SUR1, n=14; SUR2A, n=7) of 100µM MgADP. (Supporting on-line text, TableS2).