Early-onset epileptic encephalopathy caused by a reduced sensitivity of Kv7.2 potassium channels to phosphatidylinositol 4,5-bisphosphate

Abstract

Kv7.2 and Kv7.3 subunits underlie the M-current, a neuronal K+ current characterized by an absolute functional requirement for phosphatidylinositol 4,5-bisphosphate (PIP2). Kv7.2 gene mutations cause early-onset neonatal seizures with heterogeneous clinical outcomes, ranging from self-limiting benign familial neonatal seizures to severe early-onset epileptic encephalopathy (Kv7.2-EE). In this study, the biochemical and functional consequences prompted by a recurrent variant (R325G) found independently in four individuals with severe forms of neonatal-onset EE have been investigated. Upon heterologous expression, homomeric Kv7.2 R325G channels were non-functional, despite biotin-capture in Western blots revealed normal plasma membrane subunit expression. Mutant subunits exerted dominant-negative effects when incorporated into heteromeric channels with Kv7.2 and/or Kv7.3 subunits. Increasing cellular PIP2 levels by co-expression of type 1γ PI(4)P5-kinase (PIP5K) partially recovered homomeric Kv7.2 R325G channel function. Currents carried by heteromeric channels incorporating Kv7.2 R325G subunits were more readily inhibited than wild-type channels upon activation of a voltage-sensitive phosphatase (VSP), and recovered more slowly upon VSP switch-off. These results reveal for the first time that a mutation-induced decrease in current sensitivity to PIP2 is the primary molecular defect responsible for Kv7.2-EE in individuals carrying the R325G variant, further expanding the range of pathogenetic mechanisms exploitable for personalized treatment of Kv7.2-related epilepsies.

Homomeric Kv7.2 current suppression is a common feature in KCNQ2 epileptic encephalopathy

“…An assumption is that since all the mutations studied led to encephalopathy, a common functional property will be impaired, and the extent of the deficiency may correlate with the severity of the phenotype. However, the functional impact of these mutations was diverse, except for the almost complete obliteration under the homomeric Kv7.2 configuration, in line with electrophysiologic characterization of other EIEE‐causing mutations outside the voltage sensor . Several studies have highlighted the importance of the homomeric Kv7.2 subunits, suggesting that they regulate neurotransmitter release at presynaptic sites and nerve conduction in large fibers of rat sciatic nerve .…”

“…A clear difference was the dependency on PIP 2 when coexpressed with Kv7.3. Recently, the impact on PIP 2 sensitivity has been proposed to underlie the EIEE phenotype of the R325G Kv7.2 mutant . It seems reasonable to consider that the impact on PIP 2 sensitivity of W270R when combined with Kv7.3 may correlate with the phenotype severity, although such configuration is expected to represent only about 25% of the Kv7.2/Kv7.3 combinations …”

De novo gain‐of‐function variants in KCNT2 as a novel cause of developmental and epileptic encephalopathy

“…Closed (PDB 5U76) and open (PDB 5U70) configurations of chicken KCNT1 17 served as templates for homology models of human KCNT2, using previously described tools . The H185‐T191 loop in the S 4 to S 5 linker, whose cryo‐EM density was missing in the PDB 5U76 entry, was refined (together with all side chains within 7.5 Å) using the extended sampling protocol of the Prime program …”

Lack of correlation between surface expression and currents in epileptogenic AB-calmodulin binding domain Kv7.2 potassium channel mutants

“…The amino acids corresponding to helices A and B are shadowed in green [6,26]. The CaM binding domains are indicated in blue, while putative residues of Helix A [28,29] and Helix B [30] implicated in the interaction with PIP 2 are boxed in brown. The residues mutated R333 and K526 are in red.…”