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Epilepsy-causing mutations in Kv7.2 C-terminus affect binding and functional modulation by calmodulin
Paolo Ambrosino1, Alessandro Alaimo2, Silvia Bartollino1
1Dept. of Medicine and Health Sciences, University of Molise, Campobasso, Italy.
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.
Insights
Mutations in the KCNQ2 gene cause epilepsy by disrupting calmodulin binding to Kv7.2 channels. Modulating calmodulin shows potential for treating KCNQ2-related epilepsies.
Area of Science:
- Neuroscience
- Molecular Biology
- Genetics
Background:
- Mutations in the KCNQ2 gene lead to early-onset epilepsies, including Benign Familial Neonatal Seizures (BFNS).
- The KCNQ2 gene encodes Kv7.2 potassium channel subunits, crucial for neuronal excitability.
- Calmodulin (CaM) is a key regulator of Kv7.2 channel function.
Purpose of the Study:
- To investigate how specific BFNS-causing mutations in KCNQ2 affect calmodulin binding and CaM-mediated channel regulation.
- To elucidate the pathogenetic mechanisms underlying KCNQ2-related epilepsies at the molecular and functional levels.
- To explore the therapeutic potential of modulating CaM-dependent Kv7.2 channel activity.
Main Methods:
- Biochemical assays including Far-Western blotting, Surface Plasmon Resonance, and dansylated CaM fluorescence were used to assess CaM binding to wild-type and mutant Kv7.2 C-terminal fragments.
- Electrophysiological recordings (patch-clamp) in CHO cells were performed to evaluate CaM-dependent regulation of Kv7.2 and Kv7.2/Kv7.3 currents in the presence of CaM or a CaM mutant.
Main Results:
- Each KCNQ2 mutation exhibited distinct effects on CaM interaction and channel regulation.
- Mutations ranged from causing minor CaM affinity changes with no functional impact (L351V) to significantly reduced CaM affinity and modulation (L351F, Y362C, R553Q).
- One mutation (W344R) led to complete functional loss without altering CaM affinity, while CaM overexpression partially or fully restored function for some mutants, suggesting a therapeutic avenue.
Conclusions:
- BFNS-causing KCNQ2 mutations result in specific molecular and functional deficits related to CaM interaction.
- The varying impacts of mutations on CaM binding and regulation provide insights into the diverse phenotypes of KCNQ2-related epilepsies.
- Enhancing CaM-dependent Kv7.2 channel modulation represents a promising therapeutic strategy for KCNQ2-related epilepsies.
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