Muscle Dysfunction Caused by a KATP Channel Mutation in Neonatal Diabetes Is Neuronal in Origin

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Science  23 Jul 2010:
Vol. 329, Issue 5990, pp. 458-461
DOI: 10.1126/science.1186146


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 β-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.

Heterozygous gain-of-function mutations in the gene (KCNJ11) encoding the Kir6.2 subunit of the adenosine triphosphate (ATP)–sensitive potassium (KATP) channel can give rise to intermediate DEND (iDEND) syndrome, a rare genetic disorder characterized by neonatal diabetes accompanied by muscle hypotonia, delayed speech and motor milestones, and balance problems (1). Hypotonia (lack of muscle tone), which is usually most severe in the lower limbs, may account in part for the gait impairments. KATP channels are widely expressed (2, 3), and it is not clear whether the hypotonia in iDEND results from overactive KATP channels in muscle or in nerve. In the former case, muscle excitability would be directly reduced by excess hyperpolarizing KATP conductance, whereas in the latter neuronal regulation of muscle contraction would be compromised. To distinguish between these possibilities, we selectively expressed in mice a human gain-of-function Kir6.2 mutation in either muscle or nerve and evaluated its effect on muscle strength and motor coordination. We selected the Kir6.2-Val59→Met59 (V59M) mutation, which is the most common cause of iDEND (>50% of cases). Mice expressing this mutation selectively in pancreatic β-cells exhibit severe diabetes but no neurological problems (4).

We used a Cre-lox approach in combination with the muscle creatine kinase promoter (Mck-Cre mice) or the nestin promoter (Nes-Cre mice) to selectively target expression of Kir6.2-V59M to either muscle (m-V59M mice) or nerve (n-V59M mice), respectively (5). Nestin is expressed in neuronal precursor cells, and nestin-Cre is thus expected to target all neurons. However, because KATP channels comprise both Kir6.2 (pore-forming) and SUR (regulatory) subunits (3) and SUR is essential for both proper channel function and plasma membrane targeting (6), only those neurons that endogenously express SUR will express functional transgenic KATP channels. SUR1 mRNA expression was unaffected in mutant mice, suggesting that channel density will also be unaltered (fig. S1).

The analysis in Fig. 1A confirms that Kir6.2-V59M mRNA is expressed in the muscle but not the brain of m-V59M mice, in the brain but not the muscle of n-V59M mice, and not at all in control mice (5). Furthermore, endogenous wild-type and introduced mutant Kir6.2 mRNAs are expressed at comparable levels (fig. S1). This establishes our hemizygous mouse as a plausible model for human patients with the V59M mutation, all of whom are heterozygotes (1).

Fig. 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 indicate the presence of the wild-type gene only, and three bands indicate both wild-type and mutant genes. sk1, quadriceps muscle; sk2, triceps muscle; sk3, diaphragm. Data are representative of experiments on four ROSA and four m-V59M mice done in parallel and three ROSA and three n-V59M mice performed in parallel [supporting online material (SOM) text and additional control data in fig. S1A]. (B) Weight lifting, inverted screen, and horizontal bar tests on 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 (ANOVA) on ranks]. Additional control data in fig. S2.

On three tests of muscle strength (5), mice selectively expressing the Kir6.2-V59M mutation in muscle performed equally well to control mice (Fig. 1B and fig. S2). Likewise, their motor control and balance were unaffected (5) (Fig. 2A and fig. S3). In contrast, mice in which Kir6.2-V59M was targeted to neurons performed less well in all tests of muscle function: they were unable to lift weights as effectively or to hang as long from an inverted screen or a horizontal bar (Fig. 1B and fig. S2). n-V59M mice also showed impaired balance control (Fig. 2A and fig. S3). For example, they took three times longer than controls to turn around on a thin rod suspended above the ground, and many fell off while attempting to do so (movies S1 and S2). The n-V59M mice also fell off a rotating rod earlier than control mice.

Fig. 2

Motor coordination and locomotor activity are impaired by expression of Kir6.2-V59M in neurons but not in muscle. (A) (Left) Orientation time and (middle) percentage of falls on the static rod test and (right) time before falling from a rotating rod for m-V59M, n-V59M, and control (Mck-Cre, Nes-Cre) mice. Same mice as in Fig. 1. (B and C) Duration of time spent in spontaneous physical activity (B) or using a free-running wheel (C) over a 23-hour period for 12-week-old n-V59M (n = 13), Nes-Cre (n = 14), ROSA (n = 14), and wild-type (WT) (n = 4) littermates. Mean ± SEM. **P < 0.01. ***P < 0.001 (one-way ANOVA). Additional control data are in fig. S3.

These results demonstrate that n-V59M mice are impaired in muscle strength, balance, and motor coordination. Their motor problems could be due either to reduced excitability of central nervous system (CNS) neurons or to decreased transmitter release at the neuromuscular junction. Studies of isolated nerve-muscle preparations (5) ruled out the latter possibility, because no differences were observed in the amplitude and frequency of miniature endplate potentials or evoked endplate potentials between control and n-V59M mice (fig. S4). There was also no difference in the muscle resting membrane potential. Similar results were found for m-V59M mice (fig. S5). This suggests that the motor difficulties experienced by the mice originate in the CNS.

The n-V59M mice also displayed hyperactivity, spontaneously moving around more frequently (Fig. 2B) and staying significantly longer than controls in free-running wheels (5) (Fig. 2C and fig. S6). Although hyperactivity is not included as a characteristic feature of iDEND syndrome (1), several reports have indicated that once iDEND children have learned to walk they exhibit pronounced hyperactivity (79). Our results with mice raise the possibility that this is caused by KATP channel overactivity and might therefore be included as a feature of iDEND.

To confirm that neurons are indeed affected by the Kir6.2-V59M mutation, we recorded the electrical activity of cerebellar Purkinje cells in acute brain slices (5). KATP channels are highly expressed in multiple brain regions, including those involved in movement (e.g., cortex and cerebellum) (10, 11), but we selected Purkinje cells because of their importance for motor control. In both cell-attached (Fig. 3, A and B, and fig. S7) and whole-cell (fig. S7C) recordings, action potential frequency was substantially lower in Purkinje cells of n-V59M mice than of controls. Tolbutamide, a specific inhibitor of KATP channels, increased the firing rate of n-V59M cells to that of control neurons but had no effect on control cells (Fig. 3A). The resting membrane potential of n-V59M Purkinje cells was also more hyperpolarized than in controls and was restored by tolbutamide (Fig. 3B). This suggests that, in n-V59M mice, an increased KATP current in Purkinje cells suppresses firing and may thereby impair motor function. Neurons other than Purkinje cells are also likely to contribute to the motor phenotype.

Fig. 3

KATP channel activity in Purkinje cells of n-V59M mice and skeletal muscle of m-V59M mice. (A) Mean (± SEM) action potential frequency of control (gray) and n-V59M (black) neurons in the absence (for control, n = 9 mice, 34 neurons; for n-V59M, n = 3 mice, 26 neurons) and presence (for control, n = 7 mice, 16 neurons; for n-V59M, n = 3 mice, 15 neurons) of 500 μM tolbutamide. Cell-attached recordings. **P = 0.002 (t test). (B) Mean (± SEM) resting membrane potential of control (gray, n = 17 neurons, six mice) and n-V59M neurons (black, n = 13 neurons, five mice) in the absence and then the presence of 200 μM tolbutamide. Whole-cell recordings. ***P < 0.001 (t test). (C) KATP channel currents recorded at –60 mV in inside-out patches from control (above) or m-V59M (below) flexor digitalis brevis (FDB) muscle. (D) Mean (± SEM) MgATP sensitivity of KATP channels in inside-out patches from control (●, n = 4; IC50 = 15 μM) or m-V59M (◯, n = 4; IC50 = 66 μM) FDB muscle.

Patch-clamp recordings of isolated muscle fibers from m-V59M mice revealed that the ATP sensitivity of the native muscle KATP channel is reduced compared with that of control mice (Fig. 3, C and D, and table S1). Why should the Kir6.2-V59M mutation fail to affect the muscle membrane potential, yet hyperpolarize neurons? We reasoned this might reflect differences in tissue metabolism (and thus intracellular ATP concentrations) or, alternatively, that it may be due to the different SUR isoforms that compose muscle (SUR2A) and nerve (SUR1) KATP channels (3). To distinguish between these possibilities, we compared the responses of muscle and neuronal types of KATP channel heterologously expressed in the same cell type (Xenopus oocytes) (5). Channels composed of wild-type Kir6.2 and either SUR1 or SUR2A were closed under resting conditions because of the high oocyte intracellular ATP concentration. Lowering intracellular ATP concentration by metabolic inhibition activated large whole-cell Kir6.2/SUR1 currents but had no effect on Kir6.2/SUR2A channels (Fig. 4A). The Kir6.2-V59M mutation enhanced the resting Kir6.2/SUR1 current but had no effect on Kir6.2/SUR2A currents, which remained closed even when metabolism was inhibited. Thus, differences in SUR isoforms explain why muscle is unaffected by the Kir6.2-V59M mutation, whereas neuronal electrical activity is reduced.

Fig. 4

Kir6.2/SUR1 and Kir6.2/SUR2A show different sensitivities to metabolic inhibition. (A) Mean (± SEM) steady-state whole-cell KATP currents evoked by a voltage step from –10 to –30 mV before (black) and after (pale gray) application of 3 mM azide for oocytes injected with Kir6.2 (WT) or a 1:1 mixture of Kir6.2 and Kir6.2-V59M (hetV59M) mRNA, plus either SUR1 or SUR2A mRNAs. White, 3 mM azide plus 300 μM diazoxide (SUR1) or 100 μM pinacidil (SUR2A). Dark-gray, 3 mM azide, 300 μM diazoxide, and 0.5 mM tolbutamide (SUR1) or 3 mM azide, 100 μM pinacidil, and 10 μM glibenclamide (SUR2A). The number of oocytes is indicated. **P < 0.001 with respect to WT (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. KATP conductance (G) is expressed relative to that in the absence of nucleotides (GC). The curves are the best fit of the Hill equation to the mean data (SOM text and table S1). (C) MgADP activation of channels composed of WT Kir6.2 (left) or Kir6.2-V59M (right) plus either SUR1 (black) or SUR2A (white). Increase in the KATP conductance in the presence of 100 μM MgADP. Conductance (G) is expressed as a fraction of the maximal conductance in the absence of MgADP (GC). Mean ± SEM. (D and 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) or presence (□; SUR1, n = 14; SUR2A, n = 7) of 100 μM MgADP (SOM text and table S2).

Adenine nucleotides inhibit KATP channels by binding to Kir6.2 and (when Mg2+ is present) activate them via interaction with SUR (12, 13). MgATP block (Fig. 4B) and magnesium adenosine diphosphate (MgADP) activation in the absence of MgATP (Fig. 4C) were similar for Kir6.2/SUR1 and Kir6.2/SUR2A channels whether wild-type or mutant. By contrast, whereas MgADP activated SUR1-containing channels when MgATP was present, markedly shifting the ATP dose-response curve (Fig. 4D), this was not the case for SUR2A-containing channels (Fig. 4E). This difference may underlie the different sensitivities of mutant Kir6.2/SUR1 and Kir6.2/SUR2A channels to metabolic inhibition.

Our results demonstrate that hemizygous mice carrying the Kir6.2-V59M mutation provide a faithful model for the muscle weakness, balance problems, and hyperactivity seen in iDEND children. They also confirm that analogous problems in human patients are not a secondary consequence of diabetes (or of insulin-induced hypoglycemia), because the mice have normal blood glucose levels (fig. S8). Importantly, the data indicate that the motor deficits result from impairment of neuronal, not muscle, function.

Over the past 4 years, sulphonylurea therapy has become the treatment of choice for neonatal diabetes and iDEND (14). These drugs stimulate insulin secretion by blocking open KATP channels (15). They also improve the neurological problems in many (but not all) iDEND patients. Some children begin to walk or talk for the first time shortly after starting therapy (16); in most cases, hypotonia and fine motor control are improved (17) and hyperactivity is reduced (9, 10). Our data suggest that these improvements reflect sulphonylurea block of inappropriately active KATP channels in brain neurons.

Most iDEND patients are currently treated with glibenclamide, which interacts with both SUR1 and SUR2A. Our hypothesis that their muscle problems are likely neuronal in origin means that it may also be possible to use SUR1-specific sulphonylureas such as tolbutamide, gliclazide, or nateglinide (15). Indeed, there is one report that gliclazide can enhance movement control in a patient (17).

The choice of sulphonylurea used for type 2 diabetes therapy has long been debated (1820). In particular, there has been concern that these drugs may interact adversely with cardiac (SUR2A-containing) KATP channels. KATP channel activation plays an important role in cardiac ischemic preconditioning in the heart (3), and this effect is prevented by glibenclamide (21). Neonatal diabetes patients require higher sulphonylurea doses than type 2 diabetes patients, and they may be expected to take the drugs over a much longer time period. Our finding that the motor problems in iDEND are neuronal in origin suggests that it is unnecessary to use blockers of muscle (SUR2A-containing) KATP channels to ameliorate the motor problems. Thus, it may be worth considering whether iDEND patients would be better treated with SUR1-specific drugs to avoid potential cardiac cross-reactivity.

Lastly, when examined in the light of our mouse data, the success of sulphonylureas in treating the muscle impairments of iDEND children suggests that these drugs gain access to the brain. This means that efforts to improve sulphonylurea therapy for motor problems in iDEND patients should focus on identifying those that have high blood-brain barrier permeability.

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S10

Tables S1 to S4

Movies S1 and S2

References and Notes

  1. Materials and methods are available as supporting material on Science Online.
  2. We thank C. Girard for generating the ROSA mouse and help with early experiments; J. Brüning for the Mck-Cre mice; R. Deacon for help with the mouse behavioral tests and for the use of the OXION mouse behavior facility; and the Wellcome Trust, the Medical Research Council, the Royal Society, the European Union (LHSB-CT-2004-005137 and HEALTH-F4-2007-201924), the Muscular Dystrophy Campaign, and Myasthenia Gravis Association for support. R.H.C. holds an OXION Wellcome Trust prize studentship and J.S.M. holds a Medical Research Council studentship. F.M.A. is a Royal Society University Research Professor.
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