PROJECT SUMMARY
Neuronal Kv7/KCNQ channels are homotetramers of Kv7.2 and heterotetramers of Kv7.2 and Kv7.3 that are
highly expressed in the cortex and hippocampus, key brain regions for seizure, cognition and behavior. They
produce voltage-dependent outward K+ current (IM) which potently suppresses neuronal excitability. Dominant
mutations in Kv7.2 and Kv7.3 cause early-onset epileptic encephalopathy (EE) with severe cognitive and
behavioral deficits, stressing a critical need to understand how EE variants dysregulate Kv7 channels. Our
published studies show that Kv7 channels are preferentially enriched at the axonal plasma membrane via
calmodulin (CaM) binding to intracellular helices A and B of Kv7.2, which mediates their trafficking from the
endoplasmic reticulum to the axonal surface. Epilepsy variants in these helices reduce their axonal enrichment
and seizures in mice, underscoring the key role of axonal Kv7 channels in excitability. Importantly, membrane
lipid PIP2 is an essential cofactor for opening Kv7 channels as they are potently inhibited by its membrane
depletion. However, the PIP2 binding residues that regulate neuronal Kv7 channels in different states (open or
closed) and complex (homomers, heteromers, or CaM-bound) are unknown. Our recent work has revealed
that the PIP2-binding residues in open Kv7.2 channels are different from those in closed state and CaM-bound
open channels, and that select EE mutations of these sites induce both loss and gain of PIP2 sensitivity, and
reduce their axonal enrichment. Thus, the PIP2-binding landscape is dynamic and may regulate both function
and trafficking of Kv7 channels. The goals of this project are to identify (i) dynamic changes in PIP2 binding
residues of neuronal Kv7 channels that control their axonal enrichment and function, (ii) mechanisms by which
EE variants disrupt this modulation, and (iii) compounds that reverse this dysregulation. Our central
hypothesis is that dynamic and coordinated binding of PIP2 and CaM regulates activation and trafficking of
axonal Kv7 channels, whereas EE mutations increase neuronal excitability by impairing formation of this
complex. To test this, the present project will execute 3 specific aims using interdisciplinary approach
including molecular dynamic simulations, biochemistry, imaging, and electrophysiology. Aim 1 will identify PIP2
binding residues in CaM-bound and unbound Kv7 channels and test if their PIP2 binding and sensitivity are
regulated by EE mutations, Kv7 agonists and PIP2 mimetic compounds. Aim 2 will identify how PIP2 binding
modulates axonal surface enrichment of CaM-bound and unbound Kv7 channels by examining their exocytosis,
endocytosis, and plasma membrane retention. Aim 3 will test if loss- and gain-of PIP2 modulations of axonal
Kv7 channels lead to neuronal hyperexcitability in culture and conditional knock-in mice. In contrast to a well-
established role of PIP2 in gating modulation of Kv7 channels, this project will provide novel concepts that their
PIP2 binding sites change dynamically and modulate both function and trafficking of axonal Kv7 channels to
impact IM and neuronal excitability, and reveal novel pathogenic mechanisms of EE variants in Kv7.2 and Kv7.3.
