Studies of NMDA receptor function and stoichiometry with truncated and tandem subunits

The subunits that compose eukaryotic glutamate ion channel receptors have three transmembrane domains (TMs) and terminate with intracellular tails that are important for controlling channel expression and localization. Truncation of NMDA receptor subunits before the final TM showed that this TM and intracellular tail region are necessary to form functional channels. However, it is shown here that these truncated subunits may be partially rescued by coexpressing the final TM and tail as a separate protein. The whole-cell currents so produced are somewhat lower than with full-length subunits, and they do not show the sag characteristic of currents from channels containing NR1 and NR2A subunits in the continued presence of an agonist. In addition, these truncated subunits were joined to full-length subunits to generate tandems. The functional expression of these tandems confirmed the tetrameric structure of NMDA receptors and also suggested that the subunits making up NMDA receptors are arranged as a dimer of dimers in the receptors with a 1-1-2-2 orientation of the subunits in the channel, and not in an alternating pattern of subunits around the pore. These results may redirect future studies into the mechanism of binding and gating in these receptors toward schemes including dimers, and may also be relevant to studies of glutamate receptor ion channels in general

Tackling obstacles for gene therapy targeting neurons: disrupting perineural nets with hyaluronidase improves transduction.

Gene therapy has been proposed for many diseases in the nervous system. In most cases for successful treatment, therapeutic vectors must be able to transduce mature neurons. However, both in vivo, and in vitro, where preliminary characterisation of viral particles takes place, transduction of neurons is typically inefficient. One possible explanation is that the extracellular matrix (ECM), forming dense perineural nets (PNNs) around neurons, physically blocks access to the cell surface. We asked whether co-administration of lentiviral vectors with an enzyme that disrupts the ECM could improve transduction efficiency. Using hyaluronidase, an enzyme which degrades hyaluronic acid, a high molecular weight molecule of the ECM with mainly a scaffolding function, we show that in vitro in mixed primary cortical cultures, and also in vivo in rat cortex, hyaluronidase co-administration increased the percentage of transduced mature, NeuN-positive neurons. Moreover, hyaluronidase was effective at doses that showed no toxicity in vitro based on propidium iodide staining of treated cultures. Our data suggest that limited efficacy of neuronal transduction is partly due to PNNs surrounding neurons, and further that co-applying hyaluronidase may benefit applications where efficient transduction of neurons in vitro or in vivo is required

Conservation of alternative splicing in sodium channels reveals evolutionary focus on release from inactivation and structural insights into gating

Voltage-gated sodium channels are critical for neuronal activity, and highly intolerant to variation. Even mutations that cause subtle changes in the activity these channels are sufficient to cause devastating inherited neurological diseases, such as epilepsy and pain. However, these channels do vary in healthy tissue. Alternative splicing modifies sodium channels, but the functional relevance and adaptive significance of this splicing remain poorly understood. Here we use a conserved alternate exon encoding part of the first domain of sodium channels to compare how splicing modifies different channels, and to ask whether the functional consequences of this splicing have been preserved in different genes. Although the splicing event is highly conserved, one splice variant has been selectively removed from Nav1.1 in multiple mammalian species, suggesting that the functional variation in Nav1.1 is less well-tolerated. We show for three human channels (Nav1.1, Nav1.2 and Nav1.7) splicing modifies the return from inactivated to deactivated states, and the differences between splice variants are occluded by antiepileptic drugs that bind to and stabilize inactivated states. A model based on structural data can replicate these changes, and indicates that splicing may exploit a distinct role of the first domain to change channel availability, and that the first domain of all three sodium channels plays a role in determining the rate at which the inactivation domain dissociates. Taken together, our data suggest that the stability of inactivated states is under tight evolutionary control, but that in Nav1.1 faster recovery from inactivation is associated with negative selection in mammals

The voltage-gated channelopathies as a paradigm for studying epilepsy-causing genes

‘Channelopathies’, or mutations in ion channels, are long-established causes of epilepsy. Comprehensive genetic, mechanistic and clinical data for SCN1A, a voltage-gated sodium channel, has highlighted the differing contributions of neuronal sub-types in epilepsy and confirmed that genotype-phenotype relations, even for monogenic epilepsies, are strongly influenced by modifier genes and environmental factors. An emerging population of de novo mutations in voltage-gated potassium channels has defined two novel potassium channelopathies (KCNA2 and KCNC1), which may benefit from mechanistic insights from SCN1A. Meanwhile, increasing genetic evidence has strengthened the long-standing association of voltage-gated calcium channels with epilepsy. Finally, an integrative approach for the characterisation of genetic variation in NMDA receptors has created a new standard for predicting functional effects of novel epilepsy genes

Changing channels in pain and epilepsy: Exploiting ion channel gene therapy for disorders of neuronal hyperexcitability

Chronic pain and epilepsy together affect hundreds of millions of people worldwide. While traditional pharmacotherapy provides essential relief to the majority of patients, a large proportion remains resistant, and surgical intervention is only possible for a select few. As both disorders are characterised by neuronal hyperexcitability, manipulating the expression of the most direct modulators of excitability - ion channels - represents an attractive common treatment strategy. A number of viral gene therapy approaches have been explored to achieve this. These range from the up- or down-regulation of channels that control excitability endogenously, to the delivery of exogenous channels that permit manipulation of excitability via optical or chemical means. In this review we highlight the key experimental successes of each approach and discuss the challenges facing their clinical translation

A novel synaptopathy-defective synaptic vesicle protein trafficking in the mutant CHMP2B mouse model of frontotemporal dementia

Mutations in the ESCRT-III subunit CHMP2B cause frontotemporal dementia (FTD) and lead to impaired endolysosomal trafficking and lysosomal storage pathology in neurons. We investigated the effect of mutant CHMP2B on synaptic pathology, as ESCRT function was recently implicated in the degradation of synaptic vesicle (SV) proteins. We report here that expression of C-terminally truncated mutant CHMP2B results in a novel synaptopathy. This unique synaptic pathology is characterised by selective retention of presynaptic SV trafficking proteins in aged mutant CHMP2B transgenic mice, despite significant loss of postsynaptic proteins. Furthermore, ultrastructural analysis of primary cortical cultures from transgenic CHMP2B mice revealed a significant increase in the number of presynaptic endosomes, while neurons expressing mutant CHMP2B display defective SV recycling and alterations to functional SV pools. Therefore, we reveal how mutations in CHMP2B affect specific presynaptic proteins and SV recycling, identifying CHMP2B FTD as a novel synaptopathy. This novel synaptopathic mechanism of impaired SV physiology may be a key early event in multiple forms of FTD, since proteins that mediate the most common genetic forms of FTD all localise at the presynapse

Activity clamp provides insights into paradoxical effects of the anti-seizure drug carbamazepine

A major challenge in experimental epilepsy research is to reconcile the effects of anti-epileptic drugs (AEDs) on individual neurons with their network-level actions. Highlighting this difficulty, it is unclear why carbamazepine (CBZ), a front-line AED with a known molecular mechanism, has been reported to increase epileptiform activity in several clinical and experimental studies. We confirmed in an in vitro mouse model (both sexes) that the frequency of interictal bursts increased following CBZ perfusion. To address the underlying mechanisms we developed a method, activity clamp, to distinguish the response of individual neurons from network-level actions of CBZ. We first recorded barrages of synaptic conductances from neurons during epileptiform activity, and then replayed them in pharmacologically isolated neurons under control conditions and in the presence of CBZ. CBZ consistently decreased the reliability of the second action potential in each burst of activity. Conventional current clamp recordings using excitatory ramp or square step current injections failed to reveal this effect. Network modelling showed that a CBZ-induced decrease of neuron recruitment during epileptic bursts can lead to an increase in burst frequency at the network level, by reducing the refractoriness of excitatory transmission. By combining activity clamp with computer simulations, the present study provides a potential explanation for the paradoxical effects of CBZ on epileptiform activity.SIGNIFICANCE STATEMENTThe effects of anti-epileptic drugs on individual neurons are difficult to separate from their network-level actions. Although carbamazepine has a known anti-epileptic mechanism, it has also been reported to paradoxically increase epileptiform activity in clinical and experimental studies. To investigate this paradox during realistic neuronal epileptiform activity we developed a method, activity clamp, to distinguish effects of carbamazepine on individual neurons from network-level actions. We demonstrate that carbamazepine consistently decreases the reliability of the second action potential in each burst of epileptiform activity. Network modelling shows that this effect on individual neuronal responses could explain the paradoxical effect of carbamazepine at the network level

Nongenomic actions of progesterone and 17β-estradiol on the chloride conductance of skeletal muscle.

Myotonia congenita, caused by mutations in ClC-1, tends to be more severe in men and is often exacerbated by pregnancy. We performed whole-cell patch clamp of mouse muscle chloride currents in the absence/presence of 100 μM progesterone or 17β-estradiol. 100 μM progesterone rapidly and reversibly shifted the ClC-1 activation curve of mouse skeletal muscle (V50 changed from -52.6 ± 9.3 to +35.5 ± 6.7; P < 0.01) and markedly reduced chloride currents at depolarized potentials. 17β-estradiol at the same concentration had a similar but smaller effect (V50 change from -57.2 ± 7.6 to -40.5 ± 9.8; P < 0.05). 1 μM progesterone produced no significant effect. Although the data support the existence of a nongenomic mechanism in mammalian skeletal muscle through which sex hormones at high concentration can rapidly modulate ClC-1, the influence of hormones on muscle excitability in vivo remains an open question. Copyright © 2013 Wiley Periodicals, Inc