Predicted Loss of Cellular Function in KCNC2 Developmental and Epileptic Encephalopathy
Abstract number :
1.012
Submission category :
1. Basic Mechanisms / 1B. Epileptogenesis of genetic epilepsies
Year :
2022
Submission ID :
2204219
Source :
www.aesnet.org
Presentation date :
12/3/2022 12:00:00 PM
Published date :
Nov 22, 2022, 05:23 AM
Authors :
Ethan Goldberg, MD, PhD – The Children's Hospital of Philadelphia; Christopher Currin, PhD – IST Austria; Shavonne Massey, MD – Pediatrics – The Children's Hospital of Philadelphia; Ingo Helbig, MD – Pediatrics – The Children's Hospital of Philadelphia; Tim Vogels, PhD – IST Austria; Jerome Clatot, PhD – Pediatrics – The Children's Hospital of Philadelphia
Rationale: De novo heterozygous variants in KCNC2 encoding the voltage-gated potassium (K+) channel subunit Kv3.2 are an emerging cause of developmental and epileptic encephalopathy (DEE). However, the mechanism whereby variants in KCNC2 lead to epilepsy are unclear. We recently identified a novel variant in KCNC2-p.Cys125Tyr via exome sequencing in a patient with DEE. Here, we attempted to further investigate the mechanisms of KCNC2 encephalopathy.
Methods: The human wild-type (WT) KCNC2 cDNA and KCNC2-p.Cys125Tyr variant were synthesized and subcloned into a pCMV-IRES-EGFP plasmid. HEK-293T cells were then transfected with plasmid containing the WT or variant KCNC2 cDNA. Outward potassium current was recorded from GFP positive cells using whole-cell voltage clamp. A computational model of a Kv3.2-expressing parvalbumin (PV) positive fast-spiking GABAergic interneuron (PV-IN) was then used to simulate the effects of the identified variant-associated abnormalities in K+ current on PV-IN excitability.
Results: We demonstrate a profound increase in current density, a leftward/hyperpolarized shift in the voltage dependence of activation, accelerated channel activation and delayed deactivation of K+ channels formed by KCNC2-p.Cys125Tyr subunits relative to WT, consistent with gain of function (GoF). The nonspecific K+ modulator 4-aminopyridine (4-AP) and verapamil (a non-dihydropyridine calcium channel blocker both blocked Kv1.2-WT and Kv1.2-Cys125Tyr channels. Results of computational modeling suggest that such GoF at the level of the ion channel will exert a loss of function effect on Kv3.2-expressing fast-spiking PV-INs.
Conclusions: KCNC2 represents a rare cause of DEE (KCNC2 encephalopathy). The biophysical abnormalities of identified variants support a GoF mechanism at the level of the ion channel, with a predicted disease pathomechanism of impaired high-frequency firing of Kv3.2-expressing neurons. Thus, selective blockade or down-regulation of variant KCNC2 may represent a viable approach to therapy for KCNC2 DEE that requires further development.
Funding: This work was supported by The Hartwell Foundation Individual Biomedical Research Award and NIH NINDS K02 NS112600 to I.H., an ERC Consolidator Grant (SYNAPSEEK) to T.P.V., the NOMIS Foundation through the NOMIS Fellowships program at IST Austria to C.B.C., NIH NINDS U54 NS108874 (PI, A.L.G.), and NIH NINDS K08 NS097633 and R01 NS122887 and the Burroughs Wellcome Fund Career Award for Medical Scientists to E.M.G.
Basic Mechanisms