• AMIGO
• ER–PM junction
• Kv2.1
• Kv2.2
• Shab family
• human neurons
• potassium channel
• presynaptic terminals

• Humans
• Shab Potassium Channels/metabolism
• Shab Potassium Channels/genetics
• Neurons/metabolism
• Neurons/cytology
• Protein Processing, Post-Translational
• Presynaptic Terminals/metabolism
• Neurogenesis
• Glycosylation
• Endoplasmic Reticulum/metabolism
• Phosphorylation

• Genetic Code
• Humans
• Ion Channels/chemistry
• Ion Channels/metabolism
• Ion Channels/genetics
• Amino Acids/chemistry
• Amino Acids/genetics
• Animals

• F30 HL165692 NHLBI NIH HHS
• F32 HL149184 NHLBI NIH HHS
• R24 NS104617 NINDS NIH HHS
• R35 GM148239 NIGMS NIH HHS
• National Heart, Lung, and Blood Institute
• National Institute of Neurological Disorders and Stroke
• National Institute of General Medical Sciences

• DEE
• KCNB
• KV2.1
• neurodevelopmental delay
• seizures

• R01 NS107671 NINDS NIH HHS

• Behavioral neuroscience
• Neuroscience
• Sensory neuroscience

• Wellcome Trust
• 110193/Z/15/Z Wellcome Trust

• cryoelectron microscopy
• ion channels in the nervous system

• Animals
• Humans
• Cryoelectron Microscopy
• Hydrophobic and Hydrophilic Interactions
• Ion Channel Gating/genetics
• Mutation
• Shab Potassium Channels/genetics
• Shab Potassium Channels/metabolism
• Shab Potassium Channels/ultrastructure
• Spasms, Infantile/genetics

• Z01 NS002945 Intramural NIH HHS

• Integrin
• K(+) channel
• Microglia
• Migration
• PI3K/AKT
• Proliferation
• Ras-MAPK

• Humans
• Potassium Channels/metabolism
• Neurodegenerative Diseases
• Neoplasms/drug therapy
• Signal Transduction
• Cell Proliferation/physiology

• R01 AG060919 NIA NIH HHS

• Animals
• CHO Cells
• Cricetinae
• Cricetulus
• Ion Channel Gating/physiology
• Kinetics
• Rats
• Shab Potassium Channels/metabolism

• R01 HL128537 NHLBI NIH HHS
• R01 NS096317 NINDS NIH HHS
• National Institutes of Health

• sodium channels
• patch clamp

• Amino Acid Sequence
• Animals
• Brain/physiology
• HEK293 Cells
• Humans
• Ion Channel Gating/radiation effects
• Light
• Mice, Inbred C57BL
• Neurons/physiology
• Neurons/radiation effects
• Peptides/chemical synthesis
• Peptides/chemistry
• Peptides/toxicity
• Protein Engineering
• Time Factors
• Toxins, Biological/toxicity
• Ultraviolet Rays
• Voltage-Gated Sodium Channels/metabolism
• Zebrafish
• Mice

• R01 NS091352 NINDS NIH HHS
• Agence Nationale de la Recherche (French National Research Agency)
• Fondation Leducq
• AFM-Téléthon (French Muscular Dystrophy Association)

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

• Excitability
• Kv2.2
• PKC
• Phosphorylation
• Pyramidal neurons

• aerobic glycolysis
• bioengineering
• disease modeling
• iPSC
• inherited retinal degenerations
• photoreceptors
• retinal organoids
• subretinal space
• transplantation

• Animals
• Bioengineering/methods
• Cell Differentiation
• Humans
• Induced Pluripotent Stem Cells/cytology
• Organoids/cytology
• Retinal Degeneration/pathology
• Retinal Degeneration/therapy
• Retinal Pigment Epithelium/cytology

• Analgesic
• Electrophysiology
• Peptide pharmacophore
• Spider-venom peptide
• Tethered toxin
• Voltage-gated sodium channel

The voltage-gated K⁺ channel Kv2.1 serves a major structural role in the soma and proximal dendrites of mammalian brain neurons, tethering the plasma membrane (PM) to endoplasmic reticulum (ER). Although Kv2.1 clustering at neuronal ER-PM junctions (EPJs) is tightly regulated and highly conserved, its function remains unclear. By identifying and evaluating proteins in close spatial proximity to Kv2.1-containing EPJs, we discovered that a significant role of Kv2.1 at EPJs is to promote the clustering and functional coupling of PM L-type Ca²⁺ channels (LTCCs) to ryanodine receptor (RyR) ER Ca²⁺ release channels. Kv2.1 clustering also unexpectedly enhanced LTCC opening at polarized membrane potentials. This enabled Kv2.1-LTCC-RyR triads to generate localized Ca²⁺ release events (i.e., Ca²⁺ sparks) independently of action potentials. Together, these findings uncover a novel mode of LTCC regulation and establish a unique mechanism whereby Kv2.1-associated EPJs provide a molecular platform for localized somatodendritic Ca²⁺ signals in mammalian brain neurons.

• calcium signaling
• ion channel
• membrane contact sites
• molecular biophysics
• mouse
• neuroscience
• rat
• structural biology

The potassium channels Kv2.1 and Kv2.2 are widely expressed throughout the mammalian brain. Kv2.1 provides the majority of delayed rectifying current in rat hippocampus while both channels are differentially expressed in cortex. Particularly unusual is their neuronal surface localization pattern: while half the channel population is freely-diffusive on the plasma membrane as expected from the generalized Singer & Nicolson fluid mosaic model, the other half localizes into micron-sized clusters on the soma, dendrites, and axon initial segment. These clusters contain hundreds of channels, which for Kv2.1, are largely non-conducting. Competing theories of the mechanism underlying Kv2.1 clustering have included static tethering to being corralled by an actin fence. Now, recent work has demonstrated channel clustering is due to formation of endoplasmic reticulum/plasma membrane (ER/PM) junctions through interaction with ER-resident VAMP-associated proteins (VAPs). Interaction between surface Kv2 channels and ER VAPs groups channels together in clusters. ER/PM junctions play important roles in inter-organelle communication: they regulate ion flux, are involved in lipid transfer, and are sites of endo- and exocytosis. Kv2-induced ER/PM junctions are regulated through phosphorylation of the channel C-terminus which in turn regulates VAP binding, providing a rapid means to create or dismantle these microdomains. In addition, insults such as hypoxia or ischemia disrupt this interaction resulting in ER/PM junction disassembly. Kv2 channels are the only known plasma membrane protein to form regulated, injury sensitive junctions in this manner. Furthermore, it is likely that concentrated VAPs at these microdomains sequester additional interactors whose functions are not yet fully understood.

• ER/PM junction
• Kv2.1
• Kv2.2
• VAP
• membrane contact site

• ion channel
• membrane contact sites
• neuron
• subcellular

Voltage-gated K⁺ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

• auxiliary subunit
• brain
• cell adhesion molecule
• immunohistochemistry
• ion channel

• excitability
• phosphorylation
• potassium channel
• sorting
• targeting

Previous studies established different responses between proximal and distal portions of Schaffer collateral axons during high‐frequency and burst stimulation, with distal axons demonstrating biphasic changes in excitability (hyperexcitability followed by depression), but proximal axons showing only monophasic depression. Voltage‐dependent potassium (K_V) channels are important determinants of axonal excitability, and block of K_V channels can promote axon hyperexcitability. We therefore hypothesized that block of K_V channels should lead to biphasic response changes in proximal Schaffer collaterals, like those seen in distal Schaffer collaterals. To test this hypothesis, we made extracellular recordings of distal Schaffer collateral responses in stratum radiatum of hippocampal area CA1 and proximal Schaffer collateral responses in stratum pyramidale of area CA3 during high‐frequency stimulation (HFS) at 100 Hz and burst stimulation at 200 msec intervals (5 Hz or theta frequency). We then applied a nonselective K_V channel blocker, tetraethlylammonium (TEA, 10 mmol/L) or 4‐aminopyridine (4‐AP, 100 μmol/L), and assessed effects on Schaffer collateral responses. Surprisingly, block of K_V channels had little or no effect on proximal Schaffer collateral responses during high‐frequency or burst stimulation. In contrast, K_V channel blockade caused more rapid depression of distal Schaffer collateral responses during both high‐frequency and burst stimulation. These findings indicate that K_V channels are important for maintaining distal, but not proximal, Schaffer collateral excitability during period of sustained high activity. Differential sensitivity of distal versus proximal Schaffer collaterals to K_V channel block may reflect differences in channel density, diversity, or subcellular localization.

• Axon excitability
• burst stimulation
• high‐frequency stimulation
• voltage‐gated potassium channel

• cyclization
• inhibitory cystine knot
• sortase A
• κ-PVIIA

• AMME contiguous gene syndrome
• Atrial fibrillation
• Brugada syndrome
• Cardiac arrhythmia
• KCNQ1
• Long QT syndrome
• Potassium channel

• Kv2.1
• Tityus serrulatus
• autoimmune diseases
• memory T cell
• scorpion

Uterine smooth muscle cells remain quiescent throughout most of gestation, only generating spontaneous action potentials immediately prior to, and during, labor. This study presents a method that combines transcriptomics with biophysical recordings to characterise the conductance repertoire of these cells, the ‘conductance repertoire’ being the total complement of ion channels and transporters expressed by an electrically active cell. Transcriptomic analysis provides a set of potential electrogenic entities, of which the conductance repertoire is a subset. Each entity within the conductance repertoire was modeled independently and its gating parameter values were fixed using the available biophysical data. The only remaining free parameters were the surface densities for each entity. We characterise the space of combinations of surface densities (density vectors) consistent with experimentally observed membrane potential and calcium waveforms. This yields insights on the functional redundancy of the system as well as its behavioral versatility. Our approach couples high-throughput transcriptomic data with physiological behaviors in health and disease, and provides a formal method to link genotype to phenotype in excitable systems. We accurately predict current densities and chart functional redundancy. For example, we find that to evoke the observed voltage waveform, the BK channel is functionally redundant whereas hERG is essential. Furthermore, our analysis suggests that activation of calcium-activated chloride conductances by intracellular calcium release is the key factor underlying spontaneous depolarisations.

A well-known problem in electrophysiologal modeling is that the parameters of the gating kinetics of the ion channels cannot be uniquely determined from observed behavior at the cellular level. One solution is to employ simplified “macroscopic” currents that mimic the behavior of aggregates of distinct entities at the protein level. The gating parameters of each channel or pump can be determined by studying it in isolation, leaving the general problem of finding the densities at which the channels occur in the plasma membrane. We propose an approach, which we apply to uterine smooth muscle cells, whereby we constrain the list of possible entities by means of transcriptomics and chart the indeterminacy of the problem in terms of the kernel of the corresponding linear transformation. A graphical representation of this kernel visualises the functional redundancy of the system. We show that the role of certain conductances can be fulfilled, or compensated for, by suitable combinations of other conductances; this is not always the case, and such “non-substitutable” conductances can be regarded as functionally non-redundant. Electrogenic entities belonging to the latter category are suitable putative clinical targets.

• Action Potentials
• Biophysical Phenomena
• Calcium/metabolism
• Cell Membrane/metabolism
• Computational Biology
• Computer Simulation
• Female
• Gene Expression Profiling
• Humans
• Ion Channel Gating
• Ion Channels/genetics
• Ion Channels/metabolism
• Ion Pumps/genetics
• Ion Pumps/metabolism
• Ion Transport
• Kinetics
• Membrane Potentials
• Models, Biological
• Myocytes, Smooth Muscle/metabolism
• Myometrium/cytology
• Myometrium/metabolism
• Patch-Clamp Techniques
• RNA, Messenger/genetics
• RNA, Messenger/metabolism

• G0800138-2 Medical Research Council
• G0901801 Medical Research Council

Few gating-modifier toxins have been reported to target low-voltage-activated (LVA) calcium channels, and the structural basis of toxin sensitivity remains incompletely understood. Studies of voltage-gated potassium (Kv) channels have identified the S3b–S4 “paddle motif,” which moves at the protein-lipid interface to drive channel opening, as the target for these amphipathic neurotoxins. Voltage-gated calcium (Cav) channels contain four homologous voltage sensor domains, suggesting multiple toxin binding sites. We show here that the S3–S4 segments within Cav3.1 can be transplanted into Kv2.1 to examine their individual contributions to voltage sensing and pharmacology. With these results, we now have a more complete picture of the conserved nature of the paddle motif in all three major voltage-gated ion channel types (Kv, Nav, and Cav). When screened with tarantula toxins, the four paddle sequences display distinct toxin binding properties, demonstrating that gating-modifier toxins can bind to Cav channels in a domain specific fashion. Domain III was the most commonly and strongly targeted, and mutagenesis revealed an acidic residue that is important for toxin binding. We also measured the lipid partitioning strength of all toxins tested and observed a positive correlation with their inhibition of Cav3.1, suggesting a key role for membrane partitioning.

• Dorsal root ganglion neurons
• Kv2
• Kv5‐Kv9
• postnatal development

• electrophysiology
• immunohistochemistry
• localization
• modulation
• neocortex
• network

• endoplasmic reticulum (ER)
• membrane structure
• mitosis
• plasma membrane
• potassium channel
• protein phosphorylation
• subcellular organelle

• Aging
• Alzheimer’s disease
• Apoptosis
• Kv2.1
• Reactive oxygen species

• Actin
• Golgi
• Intracellular Trafficking
• Ion Channels
• Myosin
• Protein Sorting

Peripheral nerve injuries caused by trauma are associated with increased sensory neuron excitability and debilitating chronic pain symptoms. Axotomy-induced alterations in the function of ion channels are thought to largely underlie the pathophysiology of these phenotypes. Here, we characterise the mRNA distribution of Kv2 family members in rat dorsal root ganglia (DRG) and describe a link between Kv2 function and modulation of sensory neuron excitability. Kv2.1 and Kv2.2 were amply expressed in cells of all sizes, being particularly abundant in medium-large neurons also immunoreactive for neurofilament-200. Peripheral axotomy led to a rapid, robust and long-lasting transcriptional Kv2 downregulation in the DRG, correlated with the onset of mechanical and thermal hypersensitivity. The consequences of Kv2 loss-of-function were subsequently investigated in myelinated neurons using intracellular recordings on ex vivo DRG preparations. In naïve neurons, pharmacological Kv2.1/Kv2.2 inhibition by stromatoxin-1 (ScTx) resulted in shortening of action potential (AP) after-hyperpolarization (AHP). In contrast, ScTx application on axotomized neurons did not alter AHP duration, consistent with the injury-induced Kv2 downregulation. In accordance with a shortened AHP, ScTx treatment also reduced the refractory period and improved AP conduction to the cell soma during high frequency stimulation. These results suggest that Kv2 downregulation following traumatic nerve lesion facilitates greater fidelity of repetitive firing during prolonged input and thus normal Kv2 function is postulated to limit neuronal excitability. In summary, we have profiled Kv2 expression in sensory neurons and provide evidence for the contribution of Kv2 dysfunction in the generation of hyperexcitable phenotypes encountered in chronic pain states.

•

Kv2.1 and Kv2.2 are expressed in rat dorsal root ganglion neurons.

• AHPD50
• AP
• AP half width
• APD50
• ATF3
• CGRP
• CNS
• DRG
• Dorsal root ganglia
• GAPDH
• IB4
• IHC
• IR
• ISH
• Kv channel
• NF200
• Neuropathic pain
• Potassium channel
• RP
• SNT
• ScTx
• action potential
• activating transcription factor 3
• after-hyperpolarization half width
• calcitonin gene-related peptide
• central nervous system
• dorsal root ganglion
• glyceraldehyde 3-phosphate dehydrogenase
• immunohistochemistry
• in situ hybridization
• input resistance
• isolectin B4
• neurofilament 200
• refractory period
• spinal nerve transection
• stromatoxin-1
• voltage-gated potassium channel

Voltage-activated ion channels open and close in response to changes in membrane voltage, a property that is fundamental to the roles of these channels in electrical signaling. Protein toxins from venomous organisms commonly target the S1–S4 voltage-sensing domains in these channels and modify their gating properties. Studies on the interaction of hanatoxin with the Kv2.1 channel show that this tarantula toxin interacts with the S1–S4 domain and inhibits opening by stabilizing a closed state. Here we investigated the interaction of hanatoxin with the Shaker Kv channel, a voltage-activated channel that has been extensively studied with biophysical approaches. In contrast to what is observed in the Kv2.1 channel, we find that hanatoxin shifts the conductance–voltage relation to negative voltages, making it easier to open the channel with membrane depolarization. Although these actions of the toxin are subtle in the wild-type channel, strengthening the toxin–channel interaction with mutations in the S3b helix of the S1-S4 domain enhances toxin affinity and causes large shifts in the conductance–voltage relationship. Using a range of previously characterized mutants of the Shaker Kv channel, we find that hanatoxin stabilizes an activated conformation of the voltage sensors, in addition to promoting opening through an effect on the final opening transition. Chimeras in which S3b–S4 paddle motifs are transferred between Kv2.1 and Shaker Kv channels, as well as experiments with the related tarantula toxin GxTx-1E, lead us to conclude that the actions of tarantula toxins are not simply a product of where they bind to the channel, but that fine structural details of the toxin–channel interface determine whether a toxin is an inhibitor or opener.

The voltage-activated sodium (Nav) channel Nav1.9 is expressed in dorsal root ganglion (DRG) neurons where it is believed to play an important role in nociception. Progress in revealing the functional properties and pharmacological sensitivities of this non-canonical Nav channel has been slow because attempts to express this channel in a heterologous expression system have been unsuccessful. Here, we use a protein engineering approach to dissect the contributions of the four Nav1.9 voltage sensors to channel function and pharmacology. We define individual S3b–S4 paddle motifs within each voltage sensor, and show that they can sense changes in membrane voltage and drive voltage sensor activation when transplanted into voltage-activated potassium channels. We also find that the paddle motifs in Nav1.9 are targeted by animal toxins, and that these toxins alter Nav1.9-mediated currents in DRG neurons. Our results demonstrate that slowly activating and inactivating Nav1.9 channels have functional and pharmacological properties in common with canonical Nav channels, but also show distinctive pharmacological sensitivities that can potentially be exploited for developing novel treatments for pain.