Human Molecular Genetics Advance Access originally published online on June 19, 2008
Human Molecular Genetics 2008 17(17):2703-2711; doi:10.1093/hmg/ddn172
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HMSN/ACC truncation mutations disrupt brain-type creatine kinase-dependant activation of K+/Cl– co-transporter 3
1 Centre of Excellence in Neuromics, CHUM Research Centre and the Department of Medicine, University of Montreal, Montreal, Qc, Canada, H2L 4M1 2 Department of Physics, University of Montreal, Montreal, Qc, Canada, H3C 3J7
* To whom correspondence should be addressed at: Centre of Excellence in Neuromics, CHUM Research Centre, Notre-Dame Hospital, 2099 Alexandre-de-Seve, Montreal, Qc, H2L 4M1, Canada. Tel: +1 5148908000; Fax: +1 5144127602; Email: guy.rouleau{at}umontreal.ca
Received March 3, 2008; Revised May 7, 2008; Accepted June 10, 2008
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.