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.

Polycystin 2 (PC2 or TRPP1, formerly TRPP2) is a calcium-permeant Transient Receptor Potential (TRP) cation channel expressed primarily on the endoplasmic reticulum (ER) membrane and primary cilia of all cell and tissue types. Despite its ubiquitous expression throughout the body, studies of PC2 have focused primarily on its role in the kidney, as mutations in PC2 lead to the development of autosomal dominant polycystic kidney disease (ADPKD), a debilitating condition for which there is no cure. However, the endogenous role that PC2 plays in the regulation of general cellular homeostasis remains unclear. In this study, we measure how PC2 expression changes in different pathological states, determine that its abundance is increased under conditions of cellular stress in multiple tissues including human disease, and conclude that PC2-deficient cells have increased susceptibility to cell death induced by stress. Our results offer new insight into the normal function of PC2 as a ubiquitous stress-sensitive protein whose expression is up-regulated in response to cell stress to protect against pathological cell death in multiple diseases. Polycystin-2 (PC2 or TRPP1, formerly TRPP2) is a Transient Receptor Potential (TRP) channel most well-known for its associated pathology. When mutated, PC2 causes autosomal dominant polycystic kidney disease (ADPKD), a debilitating condition leading to bilateral renal cyst formation and eventual kidney failure 1. Located primarily on the endoplasmic reticulum (ER) and primary cilia of all cell and tissue types 2-5 , PC2 is a calcium (Ca 2+)-permeant cation channel whose expression level directly affects Ca 2+ release from the ER 5. As such, PC2 is thought to play a key role in regulating Ca 2+-regulated homeostasis and signaling pathways 6. This is supported by findings showing that polycystin-deficient cells exhibit dysregulated Ca 2+ mobilization and Ca 2+-regulated signaling pathways 5,7 , including pathologically increased cAMP levels 8,9 and changes in mitochondrial Ca 2+ uptake 10,11. The aberrant Ca 2+ signaling caused by loss of polycystins is therefore often pointed to as a central cause of enhanced apoptosis 12,13 , excess fluid secretion 14,15 , and metabolic abnormalities 10,11,16-18 seen in cystic kidney cells. Given the importance of PC2 in ADPKD development, most studies of PC2 have focused on its function in the kidney. However, the ubiquitous expression of PC2 in all cell types suggests that it is important in maintaining Ca 2+ homeostasis in tissues beyond the kidney. The tight regulation of intracellular Ca 2+ is necessary for many physiological functions, including protecting cells against outside stressors. Oxidative and ER stress responses require Ca 2+ influx from both the extracellular

Objective STriatal‐Enriched protein tyrosine Phosphatase (STEP) is a brain‐specific tyrosine phosphatase. Membrane‐bound STEP61 is the only isoform expressed in hippocampus and cortex. Genetic deletion of STEP enhances excitatory synaptic currents and long‐term potentiation in the hippocampus. However, whether STEP61 affects seizure susceptibility is unclear. Here we investigated the effects of STEP inhibitor TC‐2153 on seizure propensity in a murine model displaying kainic acid (KA)–induced status epilepticus and its effect on hippocampal excitability. Methods Adult male and female C57BL/6J mice received intraperitoneal injection of either vehicle (2.8% dimethylsulfoxide [DMSO] in saline) or TC‐2153 (10 mg/kg) and then either saline or KA (30 mg/kg) 3 h later before being monitored for behavioral seizures. A subset of female mice was ovariectomized (OVX). Acute hippocampal slices from Thy1‐GCaMP6s mice were treated with either DMSO or TC‐2153 (10 μM) for 1 h, and then incubated in artificial cerebrospinal fluid (ACSF) and potassium chloride (15 mM) for 2 min prior to live calcium imaging. Pyramidal neurons in dissociated rat hippocampal culture (DIV 8–10) were pre‐treated with DMSO or TC‐2153 (10 µM) for 1 h before whole‐cell patch‐clamp recording. Results TC‐2153 treatment significantly reduced KA‐induced seizure severity, with greater trend seen in female mice. OVX abolished this TC‐2153‐induced decrease in seizure severity in female mice. TC‐2153 application significantly decreased overall excitability of acute hippocampal slices from both sexes. Surprisingly, TC‐2153 treatment hyperpolarized resting membrane potential and decreased firing rate, sag voltage, and hyperpolarization‐induced current (Ih) of cultured hippocampal pyramidal neurons. Significance This study is the first to demonstrate that pharmacological inhibition of STEP with TC‐2153 decreases seizure severity and hippocampal activity in both sexes, and dampens hippocampal neuronal excitability and Ih. We propose that the antiseizure effects of TC‐2153 are mediated by its unexpected action on suppressing neuronal intrinsic excitability.