Dataset Information

Two KCNQ2 Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP2 Interaction.

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 ?M 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.

SUBMITTER: Tran B

PROVIDER: S-EPMC7518097 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Two KCNQ2 Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP2 Interaction.

Tran Baouyen B Ji Zhi-Gang ZG Xu Mingxuan M Tsuchida Tammy N TN Cooper Edward C EC

Frontiers in physiology 20200911

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)-bindin ...[more]

PMID: 33041849

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Project description:KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.

Dataset Information

Two KCNQ2 Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP2 Interaction.

Publications

Two <i>KCNQ2</i> Encephalopathy Variants in the Calmodulin-Binding Helix A Exhibit Dominant-Negative Effects and Altered PIP<sub>2</sub> Interaction.

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