The downregulation of Kv1 channels in Lgi1-/-mice is accompanied by a profound modification of its interactome and a parallel decrease in Kv2 channels.

In animal models of LGI1-dependent autosomal dominant lateral temporal lobe epilepsy, Kv1 channels are downregulated, suggesting their crucial involvement in epileptogenesis. The molecular basis of Kv1 channel-downregulation in LGI1 knock-out mice has not been elucidated and how the absence of this extracellular protein induces an important modification in the expression of Kv1 remains unknown. In this study we analyse by immunofluorescence the modifications in neuronal Kv1.1 and Kv1.2 distribution throughout the hippocampal formation of LGI1 knock-out mice. We show that Kv1 downregulation is not restricted to the axonal compartment, but also takes place in the somatodendritic region and is accompanied by a drastic decrease in Kv2 expression levels. Moreover, we find that the downregulation of these Kv channels is associated with a marked increase in bursting patterns. Finally, mass spectrometry uncovered key modifications in the Kv1 interactome that highlight the epileptogenic implication of Kv1 downregulation in LGI1 knock-out animals.

Homeostatic regulation of axonal Kv1.1 channels accounts for both synaptic and intrinsic modifications in the hippocampal CA3 circuit.

Homeostatic plasticity of intrinsic excitability goes hand in hand with homeostatic plasticity of synaptic transmission. However, the mechanisms linking the two forms of homeostatic regulation have not been identified so far. Using electrophysiological, imaging, and immunohistochemical techniques, we show here that blockade of excitatory synaptic receptors for 2 to 3 d induces an up-regulation of both synaptic transmission at CA3-CA3 connections and intrinsic excitability of CA3 pyramidal neurons. Intrinsic plasticity was found to be mediated by a reduction of Kv1.1 channel density at the axon initial segment. In activity-deprived circuits, CA3-CA3 synapses were found to express a high release probability, an insensitivity to dendrotoxin, and a lack of depolarization-induced presynaptic facilitation, indicating a reduction in presynaptic Kv1.1 function. Further support for the down-regulation of axonal Kv1.1 channels in activity-deprived neurons was the broadening of action potentials measured in the axon. We conclude that regulation of the axonal Kv1.1 channel constitutes a major mechanism linking intrinsic excitability and synaptic strength that accounts for the functional synergy existing between homeostatic regulation of intrinsic excitability and synaptic transmission.

Patient-derived antibodies reveal the subcellular distribution and heterogeneous interactome of LGI1.

Autoantibodies against leucine-rich glioma-inactivated 1 (LGI1) occur in patients with encephalitis who present with frequent focal seizures and a pattern of amnesia consistent with focal hippocampal damage. To investigate whether the cellular and subcellular distribution of LGI1 may explain the localization of these features, and hence gain broader insights into LGI1's neurobiology, we analysed the detailed localization of LGI1 and the diversity of its protein interactome, in mouse brains using patient-derived recombinant monoclonal LGI1 antibodies. Combined immunofluorescence and mass spectrometry analyses showed that LGI1 is enriched in excitatory and inhibitory synaptic contact sites, most densely within CA3 regions of the hippocampus. LGI1 is secreted in both neuronal somatodendritic and axonal compartments, and occurs in oligodendrocytic, neuro-oligodendrocytic and astro-microglial protein complexes. Proteomic data support the presence of LGI1-Kv1-MAGUK complexes, but did not reveal LGI1 complexes with postsynaptic glutamate receptors. Our results extend our understanding of regional, cellular and subcellular LGI1 expression profiles and reveal novel LGI1-associated complexes, thus providing insights into the complex biology of LGI1 and its relationship to seizures and memory loss.

Development of a selective inhibitor for Kv1.1 channels prevalent in demyelinated nerves.

Members of the voltage-gated K+ channel subfamily (Kv1), involved in regulating transmission between neurons or to muscles, are associated with human diseases and, thus, putative targets for neurotherapeutics. This applies especially to those containing Kv1.1 α subunits which become prevalent in murine demyelinated axons and appear abnormally at inter-nodes, underlying the perturbed propagation of nerve signals. To overcome this dysfunction, akin to the consequential debilitation in multiple sclerosis (MS), small inhibitors were sought that are selective for the culpable hyper-polarising K+ currents. Herein, we report a new semi-podand - compound 3 - that was designed based on the modelling of its interactions with the extracellular pore region in a deduced Kv1.1 channel structure. After synthesis, purification, and structural characterisation, compound 3 was found to potently (IC50 = 8 µM) and selectively block Kv1.1 and 1.6 channels. The tested compound showed no apparent effect on native Nav and Cav channels expressed in F-11 cells. Compound 3 also extensively and selectively inhibited MS-related Kv1.1 homomer but not the brain native Kv1.1- or 1.6-containing channels. These collective findings highlight the therapeutic potential of compound 3 to block currents mediated by Kv1.1 channels enriched in demyelinated central neurons.

LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels.

Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current. LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.

Role of the Excitability Brake Potassium Current IKD in Cold Allodynia Induced by Chronic Peripheral Nerve Injury.

Cold allodynia is a common symptom of neuropathic and inflammatory pain following peripheral nerve injury. The mechanisms underlying this disabling sensory alteration are not entirely understood. In primary somatosensory neurons, cold sensitivity is mainly determined by a functional counterbalance between cold-activated TRPM8 channels and Shaker-like Kv1.1-1.2 channels underlying the excitability brake current IKD Here we studied the role of IKD in damage-triggered painful hypersensitivity to innocuous cold. We found that cold allodynia induced by chronic constriction injury (CCI) of the sciatic nerve in mice, was related to both an increase in the proportion of cold-sensitive neurons (CSNs) in DRGs contributing to the sciatic nerve, and a decrease in their cold temperature threshold. IKD density was reduced in high-threshold CSNs from CCI mice compared with sham animals, with no differences in cold-induced TRPM8-dependent current density. The electrophysiological properties and neurochemical profile of CSNs revealed an increase of nociceptive-like phenotype among neurons from CCI animals compared with sham mice. These results were validated using a mathematical model of CSNs, including IKD and TRPM8, showing that a reduction in IKD current density shifts the thermal threshold to higher temperatures and that the reduction of this current induces cold sensitivity in former cold-insensitive neurons expressing low levels of TRPM8-like current. Together, our results suggest that cold allodynia is largely due to a functional downregulation of IKD in both high-threshold CSNs and in a subpopulation of polymodal nociceptors expressing TRPM8, providing a general molecular and neural mechanism for this sensory alteration.SIGNIFICANCE STATEMENT This paper unveils the critical role of the brake potassium current IKD in damage-triggered cold allodynia. Using a well-known form of nerve injury and combining behavioral analysis, calcium imaging, patch clamping, and pharmacological tools, validated by mathematical modeling, we determined that the functional expression of IKD is reduced in sensory neurons in response to peripheral nerve damage. This downregulation not only enhances cold sensitivity of high-threshold cold thermoreceptors signaling cold discomfort, but it also transforms a subpopulation of polymodal nociceptors signaling pain into neurons activated by mild temperature drops. Our results suggest that cold allodynia is linked to a reduction of IKD in both high-threshold cold thermoreceptors and nociceptors expressing TRPM8, providing a general model for this form of cold-induced pain.

IKD Current in Cold Transduction and Damage-Triggered Cold Hypersensitivity.

In primary sensory neurons of the spinal and trigeminal somatosensory system, cold-sensitivity is strongly dependent on the functional balance between TRPM8 channels, the main molecular entity responsible for the cold-activated excitatory current, and Shaker-like Kv1.1-1.2 potassium channels, the molecular counterpart underlying the excitability brake current IKD. This slow-inactivating outward K+ current reduces the excitability of cold thermoreceptor neurons increasing their thermal threshold, and prevents unspecific activation by cold of neurons of other somatosensory modalities. Here we examine the main biophysical properties of this current in primary sensory neurons, its central role in cold thermotransduction, and its contribution to alterations in cold sensitivity triggered by peripheral nerve damage.

Postnatal development of temporal integration, spike timing and spike threshold regulation by a dendrotoxin-sensitive K⁺ current in rat CA1 hippocampal cells.

Spike timing and network synchronization are important for plasticity, development and maturation of brain circuits. Spike delays and timing can be strongly modulated by a low-threshold, slowly inactivating, voltage-gated potassium current called D-current (ID ). ID can delay the onset of spiking, cause temporal integration of multiple inputs, and regulate spike threshold and network synchrony. Recent data indicate that ID can also undergo activity-dependent, homeostatic regulation. Therefore, we have studied the postnatal development of ID -dependent mechanisms in CA1 pyramidal cells in hippocampal slices from young rats (P7-27), using somatic whole-cell recordings. At P21-27, these neurons showed long spike delays and pronounced temporal integration in response to a series of brief depolarizing current pulses or a single long pulse, whereas younger cells (P7-20) showed shorter discharge delays and weak temporal integration, although the spike threshold became increasingly negative with maturation. Application of α-dendrotoxin (α-DTX), which blocks ID , reduced the spiking latency and temporal integration most strongly in mature cells, while shifting the spike threshold most strongly in a depolarizing direction in these cells. Voltage-clamp analysis revealed an α-DTX-sensitive outward current (ID ) that increased in amplitude during development. In contrast to P21-23, ID in the youngest group (P7-9) showed smaller peri-threshold amplitude. This may explain why long discharge delays and robust temporal integration only appear later, 3 weeks postnatally. We conclude that ID properties and ID -dependent functions develop postnatally in rat CA1 pyramidal cells, and ID may modulate network activity and plasticity through its effects on synaptic integration, spike threshold, timing and synchrony.

Association of the Kv1 family of K+ channels and their functional blueprint in the properties of auditory neurons as revealed by genetic and functional analyses.

Developmental plasticity in spiral ganglion neurons (SGNs) ensues from profound alterations in the functional properties of the developing hair cell (HC). For example, prehearing HCs are spontaneously active. However, at the posthearing stage, HC membrane properties transition to graded receptor potentials. The dendrotoxin (DTX)-sensitive Kv1 channel subunits (Kv1.1, 1.2, and 1.6) shape the firing properties and membrane potential of SGNs, and the expression of the channel undergoes developmental changes. Because of the stochastic nature of Kv subunit heteromultimerization, it has been difficult to determine physiologically relevant subunit-specific interactions and their functions in the underlying mechanisms of Kv1 channel plasticity in SGNs. Using Kcna2 null mutant mice, we demonstrate a surprising paradox in changes in the membrane properties of SGNs. The resting membrane potential of Kcna2(-/-) SGNs was significantly hyperpolarized compared with that of age-matched wild-type (WT) SGNs. Analyses of outward currents in the mutant SGNs suggest an apparent approximately twofold increase in outward K(+) currents. We show that in vivo and in vitro heteromultimerization of Kv1.2 and Kv1.4 α-subunits underlies the striking and unexpected alterations in the properties of SGNs. The results suggest that heteromeric interactions of Kv1.2 and Kv1.4 dominate the defining features of Kv1 channels in SGNs.

AgTx2-GFP, Fluorescent Blocker Targeting Pharmacologically Important Kv1.x (x = 1, 3, 6) Channels.

The growing interest in potassium channels as pharmacological targets has stimulated the development of their fluorescent ligands (including genetically encoded peptide toxins fused with fluorescent proteins) for analytical and imaging applications. We report on the properties of agitoxin 2 C-terminally fused with enhanced GFP (AgTx2-GFP) as one of the most active genetically encoded fluorescent ligands of potassium voltage-gated Kv1.x (x = 1, 3, 6) channels. AgTx2-GFP possesses subnanomolar affinities for hybrid KcsA-Kv1.x (x = 3, 6) channels and a low nanomolar affinity to KcsA-Kv1.1 with moderate dependence on pH in the 7.0-8.0 range. Electrophysiological studies on oocytes showed a pore-blocking activity of AgTx2-GFP at low nanomolar concentrations for Kv1.x (x = 1, 3, 6) channels and at micromolar concentrations for Kv1.2. AgTx2-GFP bound to Kv1.3 at the membranes of mammalian cells with a dissociation constant of 3.4 ± 0.8 nM, providing fluorescent imaging of the channel membranous distribution, and this binding depended weakly on the channel state (open or closed). AgTx2-GFP can be used in combination with hybrid KcsA-Kv1.x (x = 1, 3, 6) channels on the membranes of E. coli spheroplasts or with Kv1.3 channels on the membranes of mammalian cells for the search and study of nonlabeled peptide pore blockers, including measurement of their affinity.

Modeling the kinetics of heteromeric potassium channels.

Mechanistic mathematical modeling has long been used as a tool for answering questions in cellular physiology. To mathematically describe cellular processes such as cell excitability, volume regulation, neurotransmitter release, and hormone secretion requires accurate descriptions of ion channel kinetics. One class of ion channels currently lacking a physiological model framework is the class of channels built with multiple different potassium protein subunits called heteromeric voltage gated potassium channels. Here we present a novel mathematical model for heteromeric potassium channels that captures both the number and type of protein subunits present in each channel. Key model assumptions are validated by showing our model is the reduction of a Markov model and through observations about voltage clamp data. We then show our model's success in replicating kinetic properties of concatemeric channels with different numbers of K v 1.1 and K v 1.2 subunits. Finally, through comparisons with multiple expression experiments across multiple voltage gated potassium families, we use the model to make predictions about the importance and effect of genetic mutations in heteromeric channel formation.

Identification of a critical binding site for local anaesthetics in the side pockets of Kv 1 channels.

BACKGROUND AND PURPOSE: Local anaesthetics block sodium and a variety of potassium channels. Although previous studies identified a residue in the pore signature sequence together with three residues in the S6 segment as a putative binding site, the precise molecular basis of inhibition of Kv channels by local anaesthetics remained unknown. Crystal structures of Kv channels predict that some of these residues point away from the central cavity and face into a drug binding site called side pockets. Thus, the question arises whether the binding site of local anaesthetics is exclusively located in the central cavity or also involves the side pockets. EXPERIMENTAL APPROACH: A systematic functional alanine mutagenesis approach, scanning 58 mutants, together with in silico docking experiments and molecular dynamics simulations was utilized to elucidate the binding site of bupivacaine and ropivacaine. KEY RESULTS: Inhibition of Kv 1.5 channels by local anaesthetics requires binding to the central cavity and the side pockets, and the latter requires interactions with residues of the S5 and the back of the S6 segments. Mutations in the side pockets remove stereoselectivity of inhibition of Kv 1.5 channels by bupivacaine. Although binding to the side pockets is conserved for different local anaesthetics, the binding mode in the central cavity and the side pockets shows considerable variations. CONCLUSION AND IMPLICATIONS: Local anaesthetics bind to the central cavity and the side pockets, which provide a crucial key to the molecular understanding of their Kv channel affinity and stereoselectivity, as well as their spectrum of side effects.

The palmitoyl acyltransferase ZDHHC14 controls Kv1-family potassium channel clustering at the axon initial segment.

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type-I PDZ domain-containing proteins. However, ZDHHC14's neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the axon initial segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14's importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability.

Assembly and Function of the Juxtaparanodal Kv1 Complex in Health and Disease.

The precise axonal distribution of specific potassium channels is known to secure the shape and frequency of action potentials in myelinated fibers. The low-threshold voltage-gated Kv1 channels located at the axon initial segment have a significant influence on spike initiation and waveform. Their role remains partially understood at the juxtaparanodes where they are trapped under the compact myelin bordering the nodes of Ranvier in physiological conditions. However, the exposure of Kv1 channels in de- or dys-myelinating neuropathy results in alteration of saltatory conduction. Moreover, cell adhesion molecules associated with the Kv1 complex, including Caspr2, Contactin2, and LGI1, are target antigens in autoimmune diseases associated with hyperexcitability such as encephalitis, neuromyotonia, or neuropathic pain. The clustering of Kv1.1/Kv1.2 channels at the axon initial segment and juxtaparanodes is based on interactions with cell adhesion molecules and cytoskeletal linkers. This review will focus on the trafficking and assembly of the axonal Kv1 complex in the peripheral and central nervous system (PNS and CNS), during development, and in health and disease.

IA channels: diverse regulatory mechanisms.

In many peripheral and central neurons, A-type K(+) currents, IA, have been identified and shown to be key determinants in shaping action potential waveforms and repetitive firing properties, as well as in the regulation of synaptic transmission and synaptic plasticity. The functional properties and physiological roles of native neuronal IA, however, have been shown to be quite diverse in different types of neurons. Accumulating evidence suggests that this functional diversity is generated by multiple mechanisms, including the expression and subcellular distributions of IA channels encoded by different voltage-gated K(+) (Kv) channel pore-forming (α) subunits, interactions of Kv α subunits with cytosolic and/or transmembrane accessory subunits and regulatory proteins and post-translational modifications of channel subunits. Several recent reports further suggest that local protein translation in the dendrites of neurons and interactions between IA channels with other types of voltage-gated ion channels further expands the functional diversity of native neuronal IA channels. Here, we review the diverse molecular mechanisms that have been shown or proposed to underlie the functional diversity of native neuronal IA channels.