title: Epilepsy-causing mutations in Kv7.2 C-terminus affect binding and functional modulation by calmodulin.
abstract: 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 Ca(2+)). 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 R553Q), 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.
authors.affiliation: Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Ambrosino
authors.affiliation: Biophysics Unit, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
authors.forename: Alessandro
authors.initials: A
authors.lastname: Alaimo
authors.affiliation: Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy.
authors.forename: Silvia
authors.initials: S
authors.lastname: Bartollino
authors.affiliation: Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy.
authors.forename: Laura
authors.initials: L
authors.lastname: Manocchio
authors.affiliation: Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: De Maria
authors.affiliation: Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Mosca
authors.affiliation: Biophysics Unit, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
authors.forename: Carolina
authors.initials: C
authors.lastname: Gomis-Perez
authors.affiliation: Biophysics Unit, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
authors.forename: Araitz
authors.initials: A
authors.lastname: Alberdi
authors.affiliation: Dept. of Obstetrics and Gynecology, Catholic University of Sacred Heart, Rome, Italy.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Scambia
authors.affiliation: Dept. of Medical Genetics, Hospices Civils de Lyon, Lyon, France.
authors.forename: Gaetan
authors.initials: G
authors.lastname: Lesca
authors.affiliation: Biophysics Unit, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain.
authors.forename: Alvaro
authors.initials: A
authors.lastname: Villarroel
authors.affiliation: Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy; Biophysics Unit, CSIC, UPV/EHU, Universidad del País Vasco, Leioa, Spain; Dept. of Neuroscience, University of Naples "Federico II", Naples, Italy. Electronic address: m.taglialatela@unimol.it.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Taglialatela
authors.affiliation: Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy.
authors.forename: Maria Virginia
authors.initials: MV
authors.lastname: Soldovieri
keywords: Calmodulin; Electrophysiology; Epilepsy; Far-Western blotting; Fluorescence; Kv7.2; Surface Plasmon Resonance