Authors :
Presenting Author: Jerome Clatot, PhD – CHOP
Ethan Goldberg, MD. PhD. – Professor, Neurology, The Children's Hospital of Philadelphia
Rationale:
Variants in KCNC1 encoding the voltage-gated potassium (K+) channel subunit Kv3.1 are an emerging cause of a spectrum of neurological disease including intellectual disability, isolated myoclonus, progressive myoclonus epilepsy, and developmental and epileptic encephalopathy. Here we characterize the recurrent de novo missense variant KCNC1 c.1196C >T (p.Thr399Met) associated with mild global developmental delay and treatment-resistant myoclonic epilepsy with onset at age one year. The variant is localized to a highly conserved threonine residue in the extracellular S5-S6 linker at the C-terminal end of the pore helix on the intracellular side of the selectivity filter. This threonine may play an important role in solvating/desolvating potassium as it enters/leaves the selectivity filter.
Methods:
We use whole-cell voltage clamp electrophysiological techniques to record homomeric wild-type (WT) vs. variant Kv3.1, or heteromeric WT:variant in a 1:1 ratio expressed in HEK-293T cells.
Results:
We demonstrate a complete loss of function (LoF), as shown previously. However, when expressed 1:1 with Kv3.1-WT – to mimic the heterozygous state of KCNC1 in the patient – current density was recovered (14.7 ± 1 pA/pF for homomeric Kv3.1-Thr399Met; 1185.7 ± 124.2 pA/pF for Kv3.1-WT:Kv3.1-Thr399Met; p < 0.001 via one way ANOVA), yet exhibited a prominent leftward (hyperpolarized) shift of 23.4 mV of the voltage dependence of activation along with slowed deactivation kinetics. Thus, when expressed in the heterozygous state, the loss-of-function effects of homomeric Kv3.1-p.Thr399Met lead to a marked gain of function (GoF) (i.e., a “dominant-positive” effect that rescues conduction). To better understand the mechanism whereby this dominant-positive effect might occur, we co-express Kv3.1-p.Thr399Met with the recurrent variant Kv3.1-p.Ala421Val known to be a profound LoF previously described as dominant-negative. Co-expression of the two variants -- each of which is non-functional alone – yield an increase in K+ current, albeit with a left-shifted (hyperpolarized) voltage-dependence of activation.