Research ArticleNeuroscience

Peripheral motor neuropathy is associated with defective kinase regulation of the KCC3 cotransporter

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Science Signaling  02 Aug 2016:
Vol. 9, Issue 439, pp. ra77
DOI: 10.1126/scisignal.aae0546


  • Fig. 1 Brain and muscle imaging of a patient with a KCC3 T991A mutation.

    (A) T1 sequence brain MRI, mid-sagittal view. Corpus callosum is indicated by red arrow. (B) Fluid-attenuated inversion recovery sequence brain MRI, axial view, demonstrating normal brain volume. (C) Muscle ultrasound (performed on Siemens Acuson S2000) of the tibialis anterior muscle from a healthy 10-year-old boy to represent normal echogenicity and bulk. (D) Abnormal muscle ultrasound of the tibialis anterior muscle from our 7-year-old patient. In (C) and (D), the green line indicates depth of muscle, and the red line indicates the subcutaneous fat layer.

  • Fig. 2 Identification of a de novo KCC3 T991A mutation in a patient with an early-onset, progressive, and severe axonal motor neuron neuropathy.

    (A) DNA chromatograms illustrating the detection of a heterozygous c.2971A>G mutation in exon 22 of SLC12A6, encoding a T991A substitution in KCC3. (B) Evolutionary conservation of amino acid Thr991 in KCC3 across the indicated species. Jap, Japanese. (C) Conservation of the homologous residues of amino acid Thr991 in KCC3 in other human KCC family members. (D) Cartoon of the modeled structure of the human KCC3 C-terminal domain (CTD; residues 733 to 1150), based on homology modeling by I-TASSER using the prokaryotic cation Cl cotransporter [Protein Data Bank (PDB) ID: 3G40] as the template. Residues Thr991 and Thr1048 are presented in space fill and colored.

  • Fig. 3 T991A decreases KCC3 phosphorylation by the WNK1-SPAK pathway in HEK293 cells and patient fibroblasts.

    (A) Phosphorylation of wild-type KCC3 (WT) or KCC3 T991A expressed in HEK293 cells. HEK293 cells were transfected with the indicated constructs and exposed to hypotonic low-Cl conditions for 30 min. Lysates were subjected to immunoblot (IB) with antibodies reocognizing the indicated proteins or phosphorylated proteins. ERK1 served as a loading control. (B) Phosphorylation of endogenous KCC3 and KCC3 T991A in human fibroblasts. Human fibroblast cells derived from the affected patient (heterozygous for KCC3 T991A) or his unaffected parental controls (WT) were exposed to hypotonic low-Cl conditions for 30 min. Lysates were subjected to immunoprecipitation (IP) with antibodies recognizing either KCC3 pThr991 or KCC3 pThr1048, and immunoprecipitates were immunoblotted with KCC3 total antibody. Lysates were also analyzed for the presence of the indicated proteins and phosphorylated proteins. (C) Quantification of the results of the Western blots shown in (B), statistically significant differences are indicated [repeated-measures one-way analysis of variance (ANOVA); F(3, 4) = 14.54, P = 0.0129]. Data are shown as means ± SEM. The quantification (ratio calculation) is based on phosphorylated species of KCC3/total KCC3. ns, not significant.

  • Fig. 4 T991A increases KCC3 activity and affects cell volume regulation in HEK293 cells and patient fibroblasts.

    (A) Transport activity of WT KCC3 and KCC3 T991A expressed in HEK293 cells. HEK293 cells were transfected and exposed to low-Cl in isotonic conditions (isotonic low Cl), hypotonic low-Cl conditions, high K+ in isotonic conditions (isotonic high K+), or hypotonic high-K+ conditions in the presence or absence of STOCK1S-50699 (STOCK; WNK-SPAK/OSR1 inhibitor), for an additional 30 min in the presence of 1 mM ouabain (Na+/K+ ATPase inhibitor) and 0.1 mM bumetanide (NKCC1 inhibitor), to functionally isolate KCC activity. K+ influx is presented in picomoles of K+ per milligram of protein per minute and plotted for both isotonic and hypotonic conditions. K+ uptake was significantly increased upon WNK/SPAK inhibition in untransfected and transfected cells [repeated-measures two-way ANOVA; F(15, 40) = 38.43, P < 0.0001]. KCC3-Thr991Ala–transfected cells exhibited significantly higher activity than WT KCC3 in all conditions [F(5, 40) = 813.9, P < 0.0001]. (B) Activity of endogenous WT KCC3 (WT1 and WT2) and KCC3 T991A in human fibroblasts. Cells were exposed to the indicated conditions and then treated in the same conditions with 1 mM furosemide (Furo; a KCC inhibitor) for an additional 30 min in the presence of 1 mM ouabain and 0.1 mM bumetanide. K+ influx was measured and analyzed as in (A). Data from a single representative experiment are shown as means ± SD. Statistical significance was determined by two-way ANOVA followed by Bonferroni post hoc tests (*P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant or P > 0.05). Similar results were obtained in three separate experiments. (C) Relative change in cell water volume during acute hypotonic stress in KCC3 WT parental control fibroblasts and KCC3 T991A patient fibroblasts. Both cell types were exposed to isotonic Hepes–minimum essential medium (MEM) (310 mosmol/kg H2O), followed by hypotonic Hepes-MEM (150 mosmol/kg H2O) for 20 min, and then isotonic Hepes-MEM for 5 min. (D) Summary data of cell volume increase. KCC3 T991A patient cells exhibited abnormal regulatory volume decrease compared to KCC3 WT cells. Data are means ± SEM; n = 3 coverslips or experiments. *P = 0.02 versus WT.

  • Fig. 6 Genetically modified KCC3-T991A mice exhibit hindlimb movement and nerve conduction deficits.

    (A) Response of WT, heterozygous (T991A/+), and homozygous (T991A/T991A) mice to the 6-mm-wide beam walk task. Mice were placed on the beam and allowed to cross to a safe platform. A performance score (1 to 7, see text) was given to each mouse (left axis). The time was also recorded (right axis). (B) Grip strength force was measured in all three genotypes using a bar attached to a force transducer. Data were corrected for body weight. The values (y axis) are in Newtons divided by gram of body weight. (C) Sensory (left axis) and motor (right axis) amplitudes of dorsal caudal tail nerve responses to 20 or 25 mA stimuli in WT, hetereozygous, and homozygous KCC3-T991A mice. (D) Sensory (left axis) and motor (right axis) amplitudes for sciatic nerves. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc tests; statistics are provided in Table 4. (E and F) Selected traces of motor conduction in dorsal caudal (tail) nerves from of WT and homozygous mice. The amplitude (in mV) is measured from the onset of the response peak to the top of the response peak. The latency to response is determined from the onset of the stimulus to the onset of the response peak. Tail nerves in this experiment were stimulated at 25 mA.

  • Fig. 7 Electron micrographs of sciatic nerve fibers isolated from KCC3 WT and T991A mice.

    Dissected fragments of sciatic nerves were fixed and processed for electron microscopy. (A) Typical view of a transversally cut nerve fascicle (from WT mouse) showing most of the myelinated fibers and a few unmyelinated fibers (arrowheads). (B and C) Higher magnification of WT fibers showing packed myelin sheaths. (D and E) Similar views from homozygous T991A/T991A nerves. (F and G) Double myelination pathology observed in nerves from T991A/T991A mice. (H and I) Breakage in myelin observed in nerves from T991A/T991A mice. Scale bars, 500 nm.

  • Fig. 8 Finely tuned KCC3 activity is required for structure and function of the human peripheral nervous system (PNS).

    KCC3 activity, schematically represented on a scale from none (0) to maximal (max) activity, is contingent on the amount of KCC3 and a balance between the phosphorylated (inhibited) and dephosphorylated (activated) species of KCC3 in the neuronal plasma membrane. Insufficient KCC3 (for example, as occurs in ACCPN; OMIM #218000) due to LOF KCC3 mutations (or as seen in KCC3 KO mice) or excessive, unregulated KCC3 activity (as in the patient described here with a de novo GOF KCC3 T991A mutation that abolishes a WNK1 kinase–dependent inhibitory phosphorylation event) results in severe and progressive peripheral axonal neuropathy with secondary demyelinating features, likely from impaired cell volume regulation and subsequent neurodegeneration. Normal humans and mice, as well as ACCPN carriers and KCC3 heterozygous KO mice, fall within a “functional range” that is free of significant pathology.


  • Table 1 Nerve conduction studies with patient.

    Abnormal results are highlighted in bold. Amp, amplitude; DL, distal latency; CV, conduction velocity; LLN, lower limit of normal; ULN, upper limit of normal; N/A, not applicable; EDB, extensor digitorum brevis muscle (peroneal motor innervated muscle in foot); TA, tibialis anterior muscle (peroneal motor innervated muscle in leg); AHL, abductor hallucis longus muscle (tibial motor innervated muscle in foot); ADM, abductor digitorum minimi (ulnar motor innervated muscle in hand); APB, abductor pollicis brevis (median motor innervated muscle in hand); NR, no response; NRA, no response (no conduction velocity was calculated because the proximal site recording could not be elicited and a velocity could therefore not be calculated).

    Motor nerve (and muscle)
    7 years of age
    Amp (mV); (LLN)
    9 years of age
    CV (m/s); (LLN)
    DL (ms);
    Amp (mV);
    CV (m/s);
    DL (ms);
    Peroneal (EDB)Not doneNot doneNot doneNRNRNR
    Peroneal (TA)Not doneNot doneNot doneNRNRNR
    Tibial (AHL)0.4 (>2.5)NRA (>40)6.1 (<6)Not doneNot doneNot done
    Median (APB)1.1 (>4.5)31 (>50)4.8 (<4.5)0.2 (>4.5)14 (>50)5.5 (<4.5)
    Ulnar (ADM)0.5 (>4.5)14 (>50)4.3 (<3.5)0.1 (>4.5)NR6.3 (<3.5)
    Facial (nasalis)0.3 (>1.0)N/A5.8 (<4.2)Not doneNot doneNot done
    Sensory nerveAmp (μV); (LLN)CV (m/s); (LLN)Amp (mV); (LLN)CV (m/s); (LLN)
    Sural17 (>6)42 (>40)8 (>6)27 (>40)
    Median13 (>15)44 (>50)9 (>15)44 (>50)
    Ulnar7 (>15)33 (>50)6 (>15)41 (>50)
  • Table 2 Summary of clinical studies of a patient with a KCC3 T991A mutation.
    EMG*Abnormal: Motor > sensory
    axonal neuropathy
    MRI brainNormal
    MR spectroscopy brainNormal
    Serum electrolytesNormal (except low creatinine)
    Urine electrolytes§Normal
    HearingNormal in speech and pure tones
    and normal tympanometry and
    auditory brainstem response
    Osmotic fragilityNormal erythrocyte osmotic fragility
    Peripheral blood smearNo acanthocytes and normal smear
    Creatine kinaseNormal (136)

    *See Table 1 for specific nerve conduction study data and interpretation.

    †See Fig. 1 for picture of normal MRI brain including normal brain volume and corpus callosum.

    ‡Specific values are as follows: Na, 138; K, 3.9; Cl, 100; HCO3, 23; Blood Urea Nitrogen (BUN), 9; and creatinine, 0.12 liters (normal, 0.3 to 0.7 liters).

    §Specific values are as follows: urine Na, 93; urine K, 86.5; and urine Cl, 124.

    • Table 3 Size and strength properties of the engineered mice (second cohort).

      au, arbitrary units; WT, wild type.

      MeasureNumber of mice
      per genotype tested
      or experiments
      WTT991A/+T991A/T991AWT versus
      WT versus
      T991A/+ versus
      Mass (g)6, 7, and 627.1 ± 1.931.3 ± 1.622.7 ± .480.150.140.002
      Rotarod7, 7, and 7169 ± 11191 ± 1183 ± 60.730.020.0045
      12-mm beam (time to
      cross the beam, s)
      6, 7, and 66.3 ± 0.955 ± 0.316.38 ± 1.60.730.0040.0005
      12-mm beam
      (paw slips)
      6, 7, and 60.05 ± 0.060.08 ± 0.069.9 ± 1.040.740.00010.0001
      6-mm beam
      (failure to cross)
      6, 7, and 61 of 6 mice in 1 of 3 trials02 of 6 mice in 2 of 3 trialsN/AN/AN/A
      6-mm beam
      (neurological score)
      6, 7, and 66.4 ± 0.336.6 ± 0.212.1 ± 0.260.840.00010.0001
      Grip test by wire
      hang (time)
      6, 7, and 627 ± 4.817.3 ± 3.238.8 ± 5.60.550.460.07
      Force grip
      test (N/g)
      6, 7, and 6−0.045 ± 0.002−0.041 ± 0.002−0.051 ± 0.0010.850.100.03
    • Table 4 Nerve conduction measurements in engineered mice.

      Sensory and motor signals were measured in the dorsal caudal nerve (tail) and sciatic nerve (nerve) in WT, heterozygous (T991A/+), and homozygous (T991A/T991A) mice. Significance was tested by one-way ANOVA followed by Tukey’s multiple comparison tests.

      WTT991A/+T991A/T991AMice per
      WT versus
      WT versus
      T991A/+ versus
      AmplitudeCaudal/sensory265 ± 30218 ± 61213 ± 346, 6, and 60.730.680.99
      Sciatic/sensory235 ± 78192 ± 16272 ± 775, 5, and 60.890.920.67
      Caudal/motor2657 ± 6342578 ± 770943 ± 2155, 6, and 60.990.140.14
      Sciatic/motor6521 ± 14204223 ± 11185393 ± 16616, 6, and 60.500.840.83
      LatencyCaudal/sensory1.58 ± 0.061.61 ± 0.041.70 ± 0.095, 6, and 60.950.380.51
      Sciatic/sensory1.88 ± 0.331.71 ± 0.362.11 ± 0.155, 5, and 60.910.830.58
      Caudal/motor3.20 ± 0.273.20 ± 0.534.80 ± 0.406, 6, and 60.990.030.03
      Sciatic/motor2.74 ± 0.372.34 ± 0.113.50 ± 0.486, 6, and 60.720.320.09

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