Journal
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE
Volume 1852, Issue 9, Pages 1856-1866Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.bbadis.2015.06.012
Keywords
Calmodulin; Kv7.2; Epilepsy; Surface Plasmon Resonance; Far-Western blotting; Fluorescence; Electrophysiology
Funding
- Fundacion Biofisica Bizkaia
- Universidad del Pais Vasco (UPV/EHU) postdoctoral fellowship
- JAE-predoctoral CSIC fellowship
- European Social Funds [JAEPre_2010_00711]
- Italian Society for Pharmacology
- Spanish Ministry of Economy and Competitiveness [BFU2012-39883]
- Spanish Ion Channel Initiative Consolider project [CSD2008-00005]
- Basque Government [SAIOTEK SA-2006/00023, 304211ENA9]
- Telethon [GGP07125]
- Fondazione San Paolo - IMI (Project Neuroscience)
- Regione Molise (Convenzione AIFA/Regione Molise)
- Science and Technology Council of the Province of Avellino
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Mutations in the KCNQ2 gene, encoding for voltage-gated Kv7.2K(+) channel subunits, are responsible for early-onset epileptic diseases with widely-diverging phenotypic presentation, ranging from Benign Familial Neonatal Seizures (BFNS) to epileptic encephalopathy. In the present study, Kv7.2 BFNS-causing mutations (W344R, L351F, L351V, Y362C, and R553Q) have been investigated for their ability to interfere with calmodulin (CaM) binding and CaM-induced channel regulation. To this aim, semi-quantitative (Far-Western blotting) and quantitative (Surface Plasmon Resonance and dansylated CaM fluorescence) biochemical assays have been performed to investigate the interaction of CaM with wild-type or mutant Kv7.2 C-terminal fragments encompassing the CaM-binding domain; in parallel, mutation-induced changes in CaM-dependent Kv7.2 or Kv7.2/Kv7.3 current regulation were investigated by patch-clamp recordings in Chinese Hamster Ovary (CHO) cells co-expressing Kv7.2 or Kv7.2/Kv7.3 channels and CaM or CaM1234 (a CaM isoform unable to bind Ca2+). The results obtained suggest that each BFNS-causing mutation prompts specific biochemical and/or functional consequences; these range from slight alterations in CaM affinity which did not translate into functional changes (L351V), to a significant reduction in the affinity and functional modulation by CaM (L351F, Y362C or R5530J, to a complete functional loss without significant alteration in CaM affinity (W344R). CaM overexpression increased Kv7.2 and Kv7.2/Kv7.3 current levels, and partially (R553Q) or fully (L351F) restored normal channel function, providing a rationale pathogenetic mechanism for mutation-induced channel dysfunction in BFNS, and highlighting the potentiation of CaM-dependent Kv7.2 modulation as a potential therapeutic approach for Kv7.2-related epilepsies. (C) 2015 Elsevier B.V. All rights reserved.
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