Super-resolution microscopy recently revealed that, unlike the soma and dendrites, the axon membrane skeleton is structured as a series of actin rings connected by spectrin filaments that are held under tension. Currently, the structure-function relationship of the axonal structure is unclear. Here, we used atomic force microscopy (AFM) to show that the stiffness of the axon plasma membrane is significantly higher than the stiffnesses of dendrites and somata. To examine whether the structure of the axon plasma membrane determines its overall stiffness, we introduced a coarse-grain molecular dynamics model of the axon membrane skeleton that reproduces the structure identified by super-resolution microscopy. Our proposed computational model accurately simulates the median value of the Young’s modulus of the axon plasma membrane determined by atomic force microscopy. It also predicts that because the spectrin filaments are under entropic tension, the thermal random motion of the voltage-gated sodium channels (Nav), which are bound to ankyrin particles, a critical axonal protein, is reduced compared to the thermal motion when spectrin filaments are held at equilibrium. Lastly, our model predicts that because spectrin filaments are under tension, any axonal injuries that lacerate spectrin filaments will likely lead to a permanent disruption of the membrane skeleton due to the inability of spectrin filaments to spontaneously form their initial under-tension configuration.

Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N-and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.

First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel antiseizure strategies. Impaired GABAergic inhibition is key to the development of such benzodiazepine-resistant seizures, as well as the pathophysiology of status epilepticus (SE). It is emerging that reduced or impaired neuronal K+/Cl– cotransporter 2 (KCC2) activity contributes to deficits in γ-aminobutyric acid (GABA)-mediated inhibition and increased seizure vulnerability. The with-no-lysine kinase (WNK)-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signaling pathway inhibits neuronal KCC2 via KCC2-T1007 phosphorylation. A selective WNK kinase inhibitor, WNK463, was recently synthesized by Novartis. Exploiting WNK463, we test the hypothesis that pharmacological WNK inhibition will enhance KCC2 activity, increase the efficacy of GABAergic inhibition, and thereby limit seizure activity in animal models. Immunoprecipitation and Western blot analysis were used to examine WNK463’s effects on KCC2-T1007 phosphorylation, in vitro and in vivo. A thallium (Tl+) uptake assay was used in human embryonic kidney (HEK-293) cells expressing KCC2 to test WNK463’s effects on KCC2-mediated Tl+ transport. Gramicidin-perforated- and whole-cell patch-clamp recordings in cortical rat neurons were used to examine WNK463’s effects on KCC2-mediated Cl– transport. In mouse brain slices (entorhinal cortex), field recordings were utilized to examine WNK463’s effects on 4-aminopyridine-induced seizure activity. Last, WNK463 was directly deliver to the mouse hippocampus in vivo and tested in a kainic acid model of diazepam-resistant SE. WNK463 significantly reduces KCC2-T1007 phosphorylation in vitro and in vivo (mice). In human embryonic kidney 293 (HEK-293) cells expressing KCC2, WNK463 greatly enhanced the rates Tl+ transport. However, the drug did not enhance Tl+ transport in cells expressing a KCC2-phospho null T1007 mutant. In cultured rat neurons, WNK463 rapidly reduced intracellular Cl– and consequently hyperpolarized the Cl– reversal potential (EGABA). In mature neurons that were artificially loaded with 30 mM Cl–, WNK463 significantly enhanced KCC2-mediated Cl– export and hyperpolarized EGABA. In a 4-aminopyridine model of acute seizures, WNK463 reduced the frequency and number of seizure-like events (SLEs). Finally, in an in vivo kainic acid (KA) model of diazepam-resistant SE, WNK463 slowed the onset and reduced the severity of KA-induced status epilepticus. Last, WNK463 prevented the development of pharmaco-resistance to diazepam in drug-treated mice. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. Our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant SE.

Human normal and sickle red blood cells (RBCs) adhere with high affinity to the alpha5 chain of laminin (LAMA5) via the basal cell adhesion molecule/Lutheran (BCAM/Lu) receptor, which is implicated in vasoocclusive episodes in sickle cell disease and activated through the cyclic adenosine monophosphate (cAMP) signaling pathway. However, the effect of the cAMP pathway on the expression of active BCAM/Lu receptors at the single-molecule level is unknown. We established an in vitro technique, based on atomic force microscopy, which enables detection of single BCAM/Lu proteins on the RBC surface and measures the unbinding force between BCAM/Lu and LAMA5. We showed that the expression of active BCAM/Lu receptors is higher in homozygous sickle RBCs (SS-RBCs) than normal RBCs and that it is critically dependent on the cAMP signaling pathway on both normal and SS-RBCs. Of importance, we illustrated that A-kinase anchoring proteins are crucial for BCAM/Lu receptor activation. Furthermore, we found that SS-RBCs from hydroxyurea-treated patients show a lower expression of active BCAM/Lu receptors, a lower unbinding force to LAMA5, and insignificant stimulation by epinephrine as compared to SS-RBCs from untreated patients. To our knowledge, these findings may lead to novel antiadhesive targets for vasoocclusive episodes in sickle cell disease.