Temperature and pharmacological rescue of a folding-defective, dominantl-negative KV7.2 mutation associated with neonatal seizures

Abstract

Benign familial neonatal seizures (BFNS) is a dominant epilepsy syndrome caused by mutations in the voltage-gated potassium channels K V 7.2 and K V 7.3. We examined the molecular pathomechanism of a BFNS-causing mutation (p.N258S) in the extracellular S5-H5 loop of K V 7.2. Wild type (WT) and mutant channels, expressed in both Xenopus laevis oocytes and CHO cells, were studied using electrophysiological techniques. The results revealed a pronounced loss-offunction with a dominant-negative effect of the mutant on WT K V 7.2 and K V 7.3 channels. Since single channel recordings of K V 7.3-K V 7.2 and K V 7.3-N285S concatemers showed similar properties for both constructs, we hypothesized that the observed reduction in current amplitude was due to a folding and trafficking defect, which was confirmed by biochemical and immunocytochemical experiments revealing a reduced number of mutant channels in the surface membrane. Furthermore, rescuing experiments revealed that upon specific incubation of transfected CHO cells -either at lower temperatures of <30 o C or in presence of the agonist retigabine -the N258S-derived currents increased 5-fold in contrast to the WT. The obtained results represent a first example of temperature and pharmacological rescue of a K V 7 mutation and suggest a folding and trafficking deficiency as the cause of reduced current amplitudes with a dominant-negative effect of N258S mutant proteins.

Inside job: ligand-receptor pharmacology beneath the plasma membrane

“…While it had been assumed to act only by shifting the voltage activation curve of the channel, recent evidence has also suggested the antiepileptic retigabine (RTG) may function as a specific pharmacological chaperone 69 . Incubation with RTG has been shown to correct processing of a folding-defective mutant linked to neonatal seizures, independent of effects on the open-probability of the channel, as demonstrated by increased current density even following RTG washout 69 . While molecular determinants of RTG have been linked to the channel pore 70,71,72,73,74 , direct evidence of compound-ligand interaction is unavailable.…”

KCNQ2 mutations in childhood nonlesional epilepsy: Variable phenotypes and a novel mutation in a case series

“…Despite some case‐series reports (Grinton et al, ; Kato et al, ; Weckhuysen et al, , ) and some functional studies (Maljevic et al, , ; Wuttke et al, ), however, the phenotypes and genotypes are still complex and noteworthy. We previously reported (Lee, Yang, Liang, Chang, & Li, ) in a functional study of HEK293 cells that genotype is a major determinant of phenotype.…”

“…Patients usually have intellectual developmental delays despite seizure remission. A loss (Maljevic et al, 2011;Maljevic, Wuttke, & Lerche, 2008;Wuttke et al, 2008) or gain (Miceli et al, 2015;Millichap et al, 2017) of KCNQ2 gene function is presumed to be the major mechanism for KCNQ2-associated neonatal-onset EE. Recent analyses of data from genomewide association studies (GWASs) of humans and animals indicate that KCNQ2 mutations contribute to schizophrenia susceptibility (Choi et al, 2018;Lee, Kim, & Song, 2013).…”

Potassium channel genes and benign familial neonatal epilepsy

“…The current reduction in 1:1:2 coexpression experiments for these mutants is > 50%, whereas a mere 20-30% reduction of the K V 7.2/K V 7.3 current amplitude corresponding to a haploinsufficiency mechanism is typical for the majority of BNFS-causing mutations ( Jentsch, 2000;Maljevic et al, 2008). Moreover, for the only two BFNS mutations causing a dominant-negative effect by an overall current amplitude reduction, the effect on the 1:1:2 currents was less pronounced (Maljevic et al, 2011;Singh et al, 2003), marking a close genotypephenotype correlation. The third pore mutant yields small currents on its own and an almost 50% reduction in the 1:1:2 coexpression experiments (Orhan et al, 2014).…”