K Channel Subconductance Levels Result from Heteromeric Pore Conformations

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (Kv2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the “open” state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open–close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled

Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis

Summary: Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS), it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN)-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential. : In this article, Bhinge, Stanton, and colleagues use genome editing of patient-derived iPSCs to model ALS phenotypic defects in vitro. Transcriptomic analysis of disease MNs reveals activation of MAPK, AP1, WNT, cell-cycle, and p53 signaling in ALS MNs. Pharmacological screening uncovers activated ERK and JNK signaling as therapeutic targets in ALS. Keywords: ALS, SOD1, FUS, CRISPR-Cas9, p38, ERK, JNK, WNT, TP53, JU

Molecular Features Underlying Neurodegeneration Identified through In Vitro Modeling of Genetically Diverse Parkinson’s Disease Patients

The fact that Parkinson’s disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis, we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro, we generated neurons harboring disease-causing mutations from patient-specific, induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons, reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro, including oxidative stress vulnerability and altered neuronal activity. Notably, PD neurons show that elevated RBFOX1, a gene previously linked to neurodevelopmental diseases, underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes

Stimulus information stored in lasting active and hidden network states is destroyed by network bursts.

In both humans and animals brief synchronizing bursts of epileptiform activity known as interictal epileptiform discharges (IEDs) can, even in the absence of overt seizures, cause transient cognitive impairments (TCI) that include problems with perception or short-term memory. While no evidence from single units is available, it has been assumed that IEDs destroy information represented in neuronal networks. Cultured neuronal networks are a model for generic cortical microcircuits, and their spontaneous activity is characterized by the presence of synchronized network bursts (SNBs), which share a number of properties with IEDs, including the high degree of synchronization and their spontaneous occurrence in the absence of an external stimulus. As a model approach to understanding the processes underlying IEDs, optogenetic stimulation and multielectrode array recordings of cultured neuronal networks were used to study whether stimulus information represented in these networks survives SNBs. When such networks are optically stimulated they encode and maintain stimulus information for as long as one second. Experiments involved recording the network response to a single stimulus and trials where two different stimuli were presented sequentially, akin to a paired pulse trial. We broke the sequential stimulus trials into encoding, delay and readout phases and found that regardless of which phase the SNB occurs, stimulus-specific information was impaired. SNBs were observed to increase the mean network firing rate, but this did not translate monotonically into increases in network entropy. It was found that the more excitable a network, the more stereotyped its response was during a network burst. These measurements speak to whether SNBs are capable of transmitting information in addition to blocking it. These results are consistent with previous reports and provide baseline predictions concerning the neural mechanisms by which IEDs might cause TCI

Constitutive Activation of the N-Methyl-d-aspartate Receptor via Cleft-spanning Disulfide Bonds*

Although the N-methyl-d-aspartate (NMDA) receptor plays a critical role in the central nervous system, many questions remain regarding the relationship between its structure and functional properties. In particular, the involvement of ligand-binding domain closure in determining agonist efficacy, which has been reported in other glutamate receptor subtypes, remains unresolved. To address this question, we designed dual cysteine point mutations spanning the NR1 and NR2 ligand-binding clefts, aiming to stabilize these domains in closed cleft conformations. Two mutants, E522C/I691C in NR1 (EI) and K487C/N687C in NR2 (KN) were found to exhibit significant glycine- and glutamate-independent activation, respectively, and co-expression of the two subunits produced a constitutively active channel. However, both individual mutants could be activated above constitutive levels in a concentration-dependent manner, indicating that cleft closure does not completely prevent agonist association. Interestingly, whereas the NR2 KN disulfide was found to potentiate channel gating and M3 accessibility, NR1 EI exhibited the opposite phenotype, suggesting that the EI disulfide may trap the NR1 ligand-binding domain in a lower efficacy conformation. Furthermore, both mutants affected agonist sensitivity at the opposing subunit, suggesting that closed cleft stabilization may contribute to coupling between the subunits. These results support a correlation between cleft stability and receptor activation, providing compelling evidence for the Venus flytrap mechanism of glutamate receptor domain closure