ML277 specifically enhances the fully activated open state of KCNQ1 by modulating VSD-pore coupling

Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form

Electro-mechanical coupling of KCNQ channels is a target of epilepsy-associated mutations and retigabine

KCNQ2 and KCNQ3 form the M-channels that are important in regulating neuronal excitability. Inherited mutations that alter voltage-dependent gating of M-channels are associated with neonatal epilepsy. In the homolog KCNQ1 channel, two steps of voltage sensor activation lead to two functionally distinct open states, the intermediate-open (IO) and activated-open (AO), which define the gating, physiological, and pharmacological properties of KCNQ1. However, whether the M-channel shares the same mechanism is unclear. Here, we show that KCNQ2 and KCNQ3 feature only a single conductive AO state but with a conserved mechanism for the electro-mechanical (E-M) coupling between voltage sensor activation and pore opening. We identified some epilepsy-linked mutations in KCNQ2 and KCNQ3 that disrupt E-M coupling. The antiepileptic drug retigabine rescued KCNQ3 currents that were abolished by a mutation disrupting E-M coupling, suggesting that modulating the E-M coupling in KCNQ channels presents a potential strategy for antiepileptic therapy

Evaluation of gliovascular functions of AQP4 readthrough isoforms

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels