Abstract
Heterozygous missense variants in KCNQ2, which encodes the potassium channel subunit Kv7.2, are among the most common genetic causes of severe neonatal-onset epileptic encephalopathy. Because about 20% of known severe Kv7.2 missense changes lie within the intracellular C-terminal region, improving understanding of the underlying pathogenic mechanisms is important. We analyzed the basis for the severe phenotypes of Kv7.2 A337T and A337G, variants in the C-terminal’s calmodulin (CaM)-binding Helix A. When expressed heterologously in mammalian cells, alone or in combination with wild type Kv7.2 or with wild type Kv7.2 and Kv7.3, both variants strongly suppressed channel currents. A337T channels expressed alone exhibited significantly reduced protein half-life and surface trafficking and co-immunoprecipitated less CaM. For both variants, increasing cellular phosphatidylinositol 4,5-bisphosphate (PIP2) by overexpression of PI(4)P5-kinase restored current densities. For both variants, the fraction of current suppressed by activation of M1 muscarinic receptors with 10 μM oxotremorine methiodide, which depletes PIP2, was less than for controls. During voltage-sensitive phosphatase-induced transient PIP2 depletion and resynthesize, potassium current inhibition and recovery kinetics were both markedly slowed. These results suggest that these variants may reduce currents by a mechanism not previously described: slowing of PIP2 migration between the bulk membrane and binding sites mediating channel electromechanical coupling. A novel Kv7.2/3-selective opener, SF0034, rescued current amplitudes. Our findings show that these two Helix A variants suppress channel current density strongly, consistent with their severe heterozygous phenotypes, implicate impairment of CaM and PIP2 regulation in KCNQ2 encephalopathy pathogenesis, and highlight the potential usefulness of selective Kv7 openers for this distinctive pathogenic mechanism and patient subgroup.
Highlights
Variants in KCNQ2 can underlie a severe syndrome consisting of intractable neonatal onset seizures and profound global developmental delay called KCNQ2 encephalopathy (Weckhuysen et al, 2012; Millichap et al, 2016)
At +100 mV, we found that inclusion of A337T or A337G significantly delayed C. intestinalis voltage sensitive phosphatase (ciVSP)-induced current declines compared to controls (Figure 6C)
To begin to assess whether such “second generation” openers merit exploration as candidate therapeutic agents for KCNQ2 encephalopathy, we studied the effects of SF0034 on channels including the A337T pathogenic variant
Summary
Variants in KCNQ2 can underlie a severe syndrome consisting of intractable neonatal onset seizures and profound global developmental delay called KCNQ2 encephalopathy (Weckhuysen et al, 2012; Millichap et al, 2016). Recent cohort studies have shown that 10–30% of previously undiagnosed patients with early infantile epileptic encephalopathy (EIEE) have de novo heterozygous missense or single codon deletion variants in KCNQ2 (Saitsu et al, 2012; Weckhuysen et al, 2012; Kato et al, 2013; Milh et al, 2013; Olson et al, 2017). KCNQ2 variants causing BFNE are usually inherited as an autosomal dominant trait with high penetrance but sometimes appears de novo. It is important to understand KCNQ2 genotypephenotype relationships so that individuals with BFNE or with rare non-pathogenic variants can be rapidly distinguished from those with EIEE-causing variants. BFNE infants have a good prognosis, and ill infants with non-pathogenic KCNQ2 variants need further diagnostic efforts. Individuals with pathogenic KCNQ2 encephalopathy variants have ended their diagnostic odyssey, have a poor long-term prognosis, and are appropriate candidates for novel therapeutic trials
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