HMSN/ACC truncation mutations disrupt brain-type creatine kinase-dependant activation of K+/Cl- co-transporter 3.
The potassium-chloride co-transporter 3 (KCC3) is mutated in hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC); however, the molecular mechanisms of HMSN/ACC pathogenesis and the exact role of KCC3 in the development of the nervous system remain poorly understood. The functional regulation of this transporter by protein partners is also largely unknown. Using a yeast two-hybrid approach, we discovered that the C-terminal domain (CTD) of KCC3, which is lost in most HMSN/ACC-causing mutations, directly interacts with brain-specific creatine kinase (CK-B), an ATP-generating enzyme that is also a partner of KCC2. The interaction of KCC3 with CK-B was further confirmed by in vitro glutathione S-transferase pull-down assay, followed by sequencing of the pulled-down complexes. In transfected cultured cells, immunofluorescence labeling showed that CK-B co-localizes with wild-type KCC3, whereas the kinase fails to interact with the inactive truncated KCC3. Finally, CK-B's inhibition by DNFB results in reduction of activity of KCC3 in functional assays using Xenopus laevis oocytes. This physical and functional association between the co-transporter and CK-B is, therefore, the first protein-protein interaction identified to be potentially involved in the pathophysiology of HMSN/ACC.
Department of Medicine, Centre of Excellence in Neuromics, CHUM Research Centre, University of Montreal, Montreal, QC, Canada.
Adèle
A
Salin-Cantegrel
Masoud
M
Shekarabi
Sébastien
S
Holbert
Patrick
P
Dion
Daniel
D
Rochefort
Janet
J
Laganière
Sandra
S
Dacal
Pascale
P
Hince
Liliane
L
Karemera
Claudia
C
Gaspar
Jean-Yves
JY
Lapointe
Guy A
GA
Rouleau
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D052276:Creatine Kinase, BB Form / Q000378:metabolism*; D005260:Female; D006367:HeLa Cells; D015417:Hereditary Sensory and Motor Neuropathy / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D027981:Symporters / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014982:Xenopus laevis; D000096922:K Cl- Cotransporters
D052276:Creatine Kinase, BB Form; D027981:Symporters; C119650:SLC12A6 protein, human; D000096922:K Cl- Cotransporters; C492280:Slc12a6 protein, mouse