Reduced axonal surface expression and phosphoinositide sensitivity in K v 7 channels disrupts their function to inhibit neuronal excitability in Kcnq2 epileptic encephalopathy

Abstract

Neuronal K7/KCNQ channels are voltage-gated potassium channels composed of K7.2/KCNQ2 and K7.3/KCNQ3 subunits. Enriched at the axonal membrane, they potently suppress neuronal excitability. De novo and inherited dominant mutations in K7.2 cause early onset epileptic encephalopathy characterized by drug resistant seizures and profound psychomotor delay. However, their precise pathogenic mechanisms remain elusive. Here, we investigated selected epileptic encephalopathy causing mutations in calmodulin (CaM)-binding helices A and B of K7.2. We discovered that R333W, K526N, and R532W mutations located peripheral to CaM contact sites decreased axonal surface expression of heteromeric channels although only R333W mutation reduced CaM binding to K7.2. These mutations also altered gating modulation by phosphatidylinositol 4,5-bisphosphate (PIP), revealing novel PIP binding residues. While these mutations disrupted K7 function to suppress excitability, hyperexcitability was observed in neurons expressing K7.2-R532W that displayed severe impairment in voltage-dependent activation. The M518 V mutation at the CaM contact site in helix B caused most defects in K7 channels by severely reducing their CaM binding, K currents, and axonal surface expression. Interestingly, the M518 V mutation induced ubiquitination and accelerated proteasome-dependent degradation of K7.2, whereas the presence of K7.3 blocked this degradation. Furthermore, expression of K7.2-M518V increased neuronal death. Together, our results demonstrate that epileptic encephalopathy mutations in helices A and B of K7.2 cause abnormal K7 expression and function by disrupting K7.2 binding to CaM and/or modulation by PIP. We propose that such multiple K7 channel defects could exert more severe impacts on neuronal excitability and health, and thus serve as pathogenic mechanisms underlying Kcnq2 epileptic encephalopathy.

Identifying mutation hotspots reveals pathogenetic mechanisms of KCNQ2 epileptic encephalopathy

“…Therefore, we next tested if selected EE mutations alter gating modulation of K v 7 channels by PIP 2 . Previous studies have shown that the activation of K v 7 channels is far from saturated by the endogenous membrane level of PIP 2 39 and that supplying exogeneous PIP 2 can enhance single-channel open probability and whole-cell current densities of homomeric K v 7.2 channels 12,14,37,40 .…”

“…To determine their effects on voltage-dependent activation of homomeric K v 7.2 channels, we performed whole-cell patch clamp recording in Chinese hamster ovary (CHOhm1) cells, which display very low expression of endogenous K + channels and depolarized resting membrane potential of −10 mV 12,33 . Application of depolarizing voltage steps from −100 to +20 mV in GFP-transfected CHOhm1 cells produces very little voltage-dependent currents that reverse around −26 mV 12,34 . In contrast, the same voltage steps in cells transfected with GFP and K v 7.2 wild-type (WT) generated slowly activating voltage-dependent outward K + currents that reached peak current densities of 17.3 ± 1.1 pA/pF at +20 mV ( Fig.…”

“…3g,h, Supplementary Tables S4-5). pip 2 -induced potentiation of K v 7.2 current is blocked by selected EE variants. PIP 2 is a critical cofactor required for the opening of K v 7 channels 14,16,17,36 and is proposed to bind to the intracellular side of S4, the S2-S3 and S4-S5 linkers, and intracellular region from pre-helix A to the helix B-C linker [11][12][13][14]28,[36][37][38] . Therefore, we next tested if selected EE mutations alter gating modulation of K v 7 channels by PIP 2 .…”

“…At right, the model is viewed from the extracellular space, with two opposing subunits shown as ribbons and their neighbors shown as transparent surfaces. Sites of pathogenic mutations are www.nature.com/scientificreports www.nature.com/scientificreports/ are still under investigation [11][12][13][14][15] . They are also called 'M-channels' because their currents are inhibited by PIP 2 depletion upon activation of Gq/11-coupled receptors such as the M1 muscarinic acetylcholine receptor 14,16,17 .…”

Sexually Divergent Mortality and Partial Phenotypic Rescue After Gene Therapy in a Mouse Model of Dravet Syndrome

“…NaVb1 is a promiscuous and multifunctional protein that (1) potentiates channel activity of the pore-forming voltagegated sodium channel a subunits, [43][44][45][46] (2) functions as a chaperone of sodium channel a subunits from the intracellular pool to the neuronal membrane, 47 (3) interacts with and modulates the activity of voltage-gated potassium channels, [48][49][50] and (4) acts as a cell adhesion molecule to enhance neurite outgrowth. 51 A population of NaVb1 subunits are localized at the axon initial segment, 52 as is NaV1.1.…”