Abstract
Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Mutations in Kv7.2 and Kv7.3 subunits cause epileptic encephalopathy (EE), yet the underlying pathogenetic mechanism is unclear. Here, we used novel statistical algorithms and structural modeling to identify EE mutation hotspots in key functional domains of Kv7.2 including voltage sensing S4, the pore loop and S6 in the pore domain, and intracellular calmodulin-binding helix B and helix B-C linker. Characterization of selected EE mutations from these hotspots revealed that L203P at S4 induces a large depolarizing shift in voltage dependence of Kv7.2 channels and L268F at the pore decreases their current densities. While L268F severely reduces expression of heteromeric channels in hippocampal neurons without affecting internalization, K552T and R553L mutations at distal helix B decrease calmodulin-binding and axonal enrichment. Importantly, L268F, K552T, and R553L mutations disrupt current potentiation by increasing phosphatidylinositol 4,5-bisphosphate (PIP2), and our molecular dynamics simulation suggests PIP2 interaction with these residues. Together, these findings demonstrate that each EE variant causes a unique combination of defects in Kv7 channel function and neuronal expression, and suggest a critical need for both prediction algorithms and experimental interrogations to understand pathophysiology of Kv7-associated EE.
Highlights
Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability
They generate slowly activating and non-inactivating voltage-dependent K+ currents that contribute to resting membrane potential, prevent repetitive and burst firing of action potentials (APs), and modulate AP threshold[3,5,6,7].They are enriched at the plasma membrane of axonal initial segments (AIS) and distal axons[8,9], where APs initiate and propagate[10]
PIP2 molecules interacted with K552-R553-K554 within 100 ns and remained stably bound throughout the simulations (Fig. 4e, Supplementary Video 1, Supplementary Fig. S4), consistent with previous in vitro biochemical studies and molecular docking simulations that demonstrated PIP2 binding to the corresponding residues in the C-terminal helix A-B fragments of Kv7.137. These findings suggest that K552T and R553L mutations are located in helix B of Kv7.2 that interacts with the phosphate head group of PIP2
Summary
Kv7 channels are enriched at the axonal plasma membrane where their voltage-dependent potassium currents suppress neuronal excitability. Recent discoveries of epilepsy-related genes in multiple laboratories and through large consortia have revealed a diverse array of proteins that may contribute to epileptogenesis[1,2] Among these proteins, neuronal KCNQ/Kv7 potassium (K+) channels have been implicated in epilepsy since mutations in the principle subunits, KCNQ2/Kv7.2 and KCNQ3/Kv7.3, cause Benign Familial Neonatal Epilepsy (BFNE [MIM: 121200]) and Epileptic Encephalopathy (EE [MIM: 613720]) (RIKEE database www.rikee.org). Neuronal Kv7 channels are mainly composed of heterotetramers of Kv7.2 and Kv7.33, which show overlapping distribution in the hippocampus and cortex[4] They generate slowly activating and non-inactivating voltage-dependent K+ currents that contribute to resting membrane potential, prevent repetitive and burst firing of action potentials (APs), and modulate AP threshold[3,5,6,7].They are enriched at the plasma membrane of axonal initial segments (AIS) and distal axons[8,9], where APs initiate and propagate[10]. They are called ‘M-channels’ because their currents are inhibited by PIP2 depletion upon activation of Gq/11-coupled receptors such as the M1 muscarinic acetylcholine receptor[14,16,17]
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