Distinct cellular expression and subcellular localization of Kv2 voltage-gated K+ channel subtypes in dorsal root ganglion neurons conserved between mice and humans.

The distinct organization of Kv2 voltage-gated potassium channels on and near the cell body of brain neurons enables their regulation of action potentials and specialized membrane contact sites. Somatosensory neurons have a pseudounipolar morphology and transmit action potentials from peripheral nerve endings through axons that bifurcate to the spinal cord and the cell body within ganglia including the dorsal root ganglia (DRG). Kv2 channels regulate action potentials in somatosensory neurons, yet little is known about where Kv2 channels are located. Here, we define the cellular and subcellular localization of the Kv2 paralogs, Kv2.1 and Kv2.2, in DRG somatosensory neurons with a panel of antibodies, cell markers, and genetically modified mice. We find that relative to spinal cord neurons, DRG neurons have similar levels of detectable Kv2.1 and higher levels of Kv2.2. In older mice, detectable Kv2.2 remains similar, while detectable Kv2.1 decreases. Both Kv2 subtypes adopt clustered subcellular patterns that are distinct from central neurons. Most DRG neurons co-express Kv2.1 and Kv2.2, although neuron subpopulations show preferential expression of Kv2.1 or Kv2.2. We find that Kv2 protein expression and subcellular localization are similar between mouse and human DRG neurons. We conclude that the organization of both Kv2 channels is consistent with physiological roles in the somata and stem axons of DRG neurons. The general prevalence of Kv2.2 in DRG as compared to central neurons and the enrichment of Kv2.2 relative to detectable Kv2.1 in older mice, proprioceptors, and axons suggest more widespread roles for Kv2.2 in DRG neurons.

A Kv2 inhibitor combination reveals native neuronal conductances consistent with Kv2/KvS heteromers.

KvS proteins are voltage-gated potassium channel subunits that form functional channels when assembled into heterotetramers with Kv2.1 ( KCNB1 ) or Kv2.2 ( KCNB2 ). Mammals have 10 KvS subunits: Kv5.1 ( KCNF1 ), Kv6.1 ( KCNG1 ), Kv6.2 ( KCNG2 ), Kv6.3 ( KCNG3 ), Kv6.4 ( KCNG4 ), Kv8.1 ( KCNV1 ), Kv8.2 ( KCNV2 ), Kv9.1 ( KCNS1 ), Kv9.2 ( KCNS2 ), and Kv9.3 ( KCNS3 ). Electrically excitable cells broadly express channels containing Kv2 subunits and most neurons have substantial Kv2 conductance. However, whether KvS subunits contribute to these conductances has not been clear, leaving the physiological roles of KvS subunits poorly understood. Here, we identify that two potent Kv2 inhibitors, used in combination, can distinguish conductances of Kv2/KvS channels and Kv2-only channels. We find that Kv5, Kv6, Kv8, or Kv9-containing channels are resistant to the Kv2-selective pore-blocker RY785 yet remain sensitive to the Kv2-selective voltage sensor modulator guangxitoxin-1E (GxTX). Using these inhibitors in mouse superior cervical ganglion neurons, we find that little of the Kv2 conductance is carried by KvS-containing channels. In contrast, conductances consistent with KvS-containing channels predominate over Kv2-only channels in mouse and human dorsal root ganglion neurons. These results establish an approach to pharmacologically distinguish conductances of Kv2/KvS heteromers from Kv2-only channels, enabling investigation of the physiological roles of endogenous KvS subunits. These findings suggest that drugs targeting KvS subunits could modulate electrical activity of subsets of Kv2-expressing cell types.

Electrically silent KvS subunits associate with native Kv2 channels in brain and impact diverse properties of channel function.

Voltage-gated K+ channels of the Kv2 family are highly expressed in brain and play dual roles in regulating neuronal excitability and in organizing endoplasmic reticulum - plasma membrane (ER-PM) junctions. Studies in heterologous cells suggest that the two pore-forming alpha subunits Kv2.1 and Kv2.2 assemble with "electrically silent" KvS subunits to form heterotetrameric channels with distinct biophysical properties. Here, using mass spectrometry-based proteomics, we identified five KvS subunits as components of native Kv2.1 channels immunopurified from mouse brain, the most abundant being Kv5.1. We found that Kv5.1 co-immunoprecipitates with Kv2.1 and to a lesser extent with Kv2.2 from brain lysates, and that Kv5.1 protein levels are decreased by 70% in Kv2.1 knockout mice and 95% in Kv2.1/2.2 double knockout mice. Multiplex immunofluorescent labelling of rodent brain sections revealed that in neocortex Kv5.1 immunolabeling is apparent in a large percentage of Kv2.1 and Kv2.2-positive layer 2/3 neurons, and in a smaller percentage of layer 5 and 6 neurons. At the subcellular level, Kv5.1 is co-clustered with Kv2.1 and Kv2.2 at ER-PM junctions in cortical neurons, although clustering of Kv5.1-containing channels is reduced relative to homomeric Kv2 channels. We also found that in heterologous cells coexpression with Kv5.1 reduces the clustering and alters the pharmacological properties of Kv2.1 channels. Together, these findings demonstrate that the Kv5.1 electrically silent subunit is a component of a substantial fraction of native brain Kv2 channels, and that its incorporation into heteromeric channels can impact diverse aspects of Kv2 channel function.

Structural modeling of hERG channel-drug interactions using Rosetta.

The human ether-a-go-go-related gene (hERG) not only encodes a potassium-selective voltage-gated ion channel essential for normal electrical activity in the heart but is also a major drug anti-target. Genetic hERG mutations and blockage of the channel pore by drugs can cause long QT syndrome, which predisposes individuals to potentially deadly arrhythmias. However, not all hERG-blocking drugs are proarrhythmic, and their differential affinities to discrete channel conformational states have been suggested to contribute to arrhythmogenicity. We used Rosetta electron density refinement and homology modeling to build structural models of open-state hERG channel wild-type and mutant variants (Y652A, F656A, and Y652A/F656 A) and a closed-state wild-type channel based on cryo-electron microscopy structures of hERG and EAG1 channels. These models were used as protein targets for molecular docking of charged and neutral forms of amiodarone, nifekalant, dofetilide, d/l-sotalol, flecainide, and moxifloxacin. We selected these drugs based on their different arrhythmogenic potentials and abilities to facilitate hERG current. Our docking studies and clustering provided atomistic structural insights into state-dependent drug-channel interactions that play a key role in differentiating safe and harmful hERG blockers and can explain hERG channel facilitation through drug interactions with its open-state hydrophobic pockets.

Molecular determinants of µ-conotoxin KIIIA interaction with the human voltage-gated sodium channel NaV1.7.

The voltage-gated sodium (NaV) channel subtype NaV1.7 plays a critical role in pain signaling, making it an important drug target. Here we studied the molecular interactions between µ-Conotoxin KIIIA (KIIIA) and the human NaV1.7 channel (hNaV1.7). We developed a structural model of hNaV1.7 using Rosetta computational modeling and performed in silico docking of KIIIA using RosettaDock to predict residues forming specific pairwise contacts between KIIIA and hNaV1.7. We experimentally validated these contacts using mutant cycle analysis. Comparison between our KIIIA-hNaV1.7 model and the cryo-EM structure of KIIIA-hNaV1.2 revealed key similarities and differences between NaV channel subtypes with potential implications for the molecular mechanism of toxin block. The accuracy of our integrative approach, combining structural data with computational modeling, experimental validation, and molecular dynamics simulations, suggests that Rosetta structural predictions will be useful for rational design of novel biologics targeting specific NaV channels.

Distinct cellular expression and subcellular localization of Kv2 voltage-gated K+ channel subtypes in dorsal root ganglion neurons conserved between mice and humans.

The distinct organization of Kv2 voltage-gated potassium channels on and near the cell body of brain neurons enables their regulation of action potentials and specialized membrane contact sites. Somatosensory neurons have a pseudounipolar morphology and transmit action potentials from peripheral nerve endings through axons that bifurcate to the spinal cord and the cell body within ganglia including the dorsal root ganglia (DRG). Kv2 channels regulate action potentials in somatosensory neurons, yet little is known about where Kv2 channels are located. Here we define the cellular and subcellular localization of the Kv2 paralogs, Kv2.1 and Kv2.2, in DRG somatosensory neurons with a panel of antibodies, cell markers, and genetically modified mice. We find that relative to spinal cord neurons, DRG neurons have similar levels of detectable Kv2.1, and higher levels of Kv2.2. In older mice, detectable Kv2.2 remains similar while detectable Kv2.1 decreases. Both Kv2 subtypes adopt clustered subcellular patterns that are distinct from central neurons. Most DRG neurons co-express Kv2.1 and Kv2.2, although neuron subpopulations show preferential expression of Kv2.1 or Kv2.2. We find that Kv2 protein expression and subcellular localization is similar between mouse and human DRG neurons. We conclude that the organization of both Kv2 channels is consistent with physiological roles in the somata and stem axons of DRG neurons. The general prevalence of Kv2.2 in DRG as compared to central neurons and the enrichment of Kv2.2 relative to detectable Kv2.1, in older mice, proprioceptors, and axons suggest more widespread roles for Kv2.2 in DRG neurons.

Computational design of peptides to target NaV1.7 channel with high potency and selectivity for the treatment of pain.

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

Structural modeling of the hERG potassium channel and associated drug interactions.

The voltage-gated potassium channel, KV11.1, encoded by the human Ether-à-go-go-Related Gene (hERG), is expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. Gating of the hERG channel is characterized by rapid, voltage-dependent, C-type inactivation, which blocks ion conduction and is suggested to involve constriction of the selectivity filter. Mutations S620T and S641A/T within the selectivity filter region of hERG have been shown to alter the voltage dependence of channel inactivation. Because hERG channel blockade is implicated in drug-induced arrhythmias associated with both the open and inactivated states, we used Rosetta to simulate the effects of hERG S620T and S641A/T mutations to elucidate conformational changes associated with hERG channel inactivation and differences in drug binding between the two states. Rosetta modeling of the S641A fast-inactivating mutation revealed a lateral shift of the F627 side chain in the selectivity filter into the central channel axis along the ion conduction pathway and the formation of four lateral fenestrations in the pore. Rosetta modeling of the non-inactivating mutations S620T and S641T suggested a potential molecular mechanism preventing F627 side chain from shifting into the ion conduction pathway during the proposed inactivation process. Furthermore, we used Rosetta docking to explore the binding mechanism of highly selective and potent hERG blockers - dofetilide, terfenadine, and E4031. Our structural modeling correlates well with much, but not all, existing experimental evidence involving interactions of hERG blockers with key residues in hERG pore and reveals potential molecular mechanisms of ligand interactions with hERG in an inactivated state.

Pore opening, not voltage sensor movement, underpins the voltage-dependence of facilitation by a hERG blocker.

A drug that blocks the cardiac myocyte voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene (hERG) carries a potential risk of long QT syndrome and life-threatening cardiac arrhythmia, including Torsade de Points Interestingly, certain hERG blockers can also facilitate hERG activation to increase hERG currents, which may reduce proarrhythmic potential. However, the molecular mechanism involved in the facilitation effect of hERG blockers remains unclear. The hallmark feature of the facilitation effect by hERG blockers is that a depolarizing preconditioning pulse shifts voltage-dependence of hERG activation to more negative voltages. Here we utilize a D540K hERG mutant to study the mechanism of the facilitation effect. D540K hERG is activated by not only depolarization but also hyperpolarization. This unusual gating property enables tests of the mechanism by which voltage induces facilitation of hERG by blockers. With D540K hERG, we find that nifekalant, a hERG blocker and Class III antiarrhythmic agent, blocks and facilitates not only current activation by depolarization but also current activation by hyperpolarization, suggesting a shared gating process upon depolarization and hyperpolarization. Moreover, in response to hyperpolarizing conditioning pulses, nifekalant facilitates D540K hERG currents but not wild-type currents. Our results indicate that induction of facilitation is coupled to pore opening, not voltage per se We propose that gated access to the hERG central cavity underlies the voltage-dependence of induction of facilitation. This study identifies hERG channel pore gate opening as the conformational change facilitated by nifekalant, a clinically important antiarrhythmic agent. Significance Statement Nifekalant is a clinically important antiarrhythmic agent and a hERG blocker which can also facilitate voltage-dependent activation of hERG channels after a preconditioning pulse. Here we show that the mechanism of action of the preconditioning pulse is to open a conductance gate to enable drug access to a facilitation site. Moreover, we find that facilitation increases hERG currents by altering pore dynamics, rather than acting through voltage sensors.

Mechanism of use-dependent Kv2 channel inhibition by RY785.

Understanding the mechanism by which ion channel modulators act is critical for interpretation of their physiological effects and can provide insight into mechanisms of ion channel gating. The small molecule RY785 is a potent and selective inhibitor of Kv2 voltage-gated K+ channels that has a use-dependent onset of inhibition. Here, we investigate the mechanism of RY785 inhibition of rat Kv2.1 (Kcnb1) channels heterologously expressed in CHO-K1 cells. We find that 1 µM RY785 block eliminates Kv2.1 current at all physiologically relevant voltages, inhibiting ≥98% of the Kv2.1 conductance. Both onset of and recovery from RY785 inhibition require voltage sensor activation. Intracellular tetraethylammonium, a classic open-channel blocker, competes with RY785 inhibition. However, channel opening itself does not appear to alter RY785 access. Gating current measurements reveal that RY785 inhibits a component of voltage sensor activation and accelerates voltage sensor deactivation. We propose that voltage sensor activation opens a path into the central cavity of Kv2.1 where RY785 binds and promotes voltage sensor deactivation, trapping itself inside. This gated-access mechanism in conjunction with slow kinetics of unblock supports simple interpretation of RY785 effects: channel activation is required for block by RY785 to equilibrate, after which trapped RY785 will simply decrease the Kv2 conductance density.

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors.

Kv2 voltage-gated potassium channels are modulated by amphoterin-induced gene and open reading frame (AMIGO) neuronal adhesion proteins. Here, we identify steps in the conductance activation pathway of Kv2.1 channels that are modulated by AMIGO1 using voltage-clamp recordings and spectroscopy of heterologously expressed Kv2.1 and AMIGO1 in mammalian cell lines. AMIGO1 speeds early voltage-sensor movements and shifts the gating charge-voltage relationship to more negative voltages. The gating charge-voltage relationship indicates that AMIGO1 exerts a larger energetic effect on voltage-sensor movement than is apparent from the midpoint of the conductance-voltage relationship. When voltage sensors are detained at rest by voltage-sensor toxins, AMIGO1 has a greater impact on the conductance-voltage relationship. Fluorescence measurements from voltage-sensor toxins bound to Kv2.1 indicate that with AMIGO1, the voltage sensors enter their earliest resting conformation, yet this conformation is less stable upon voltage stimulation. We conclude that AMIGO1 modulates the Kv2.1 conductance activation pathway by destabilizing the earliest resting state of the voltage sensors.

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues.

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.

Fluorescent toxins as ion channel activity sensors.

Voltage gated ion channels (VGICs) shape the electrical character of cells by undergoing structural changes in response to membrane depolarization. High-resolution techniques have provided a wealth of data on individual VGIC structures, but the conformational changes of endogenous channels in live cell membranes have remained unexplored. Here, we describe methods for imaging structural changes of voltage-gated K+ channels in living cells, using peptidyl toxins labeled with fluorophores that report specific protein conformations. These Endogenous Voltage-sensor Activity Probes (EVAPs) enable study of both VGIC allostery and function in the context of endogenous live-cell membranes under different physiological states. In this chapter, we describe methods for the synthesis, imaging, and analysis of dynamic EVAPs, which can report K+ channel activity in complex tissue preparations via 2-photon excitation microscopy, and environment-sensitive EVAPs, which report voltage-dependent conformational changes at the VGIC-toxin interface. The methods here present the utility of current EVAPs and lay the groundwork for the development of other probes that act by similar mechanisms. EVAPs can be correlated with electrophysiology, offering insight into the molecular details of endogenous channel function and allostery in live cells. This enables investigation of conformational changes of channels in their native, functional states, putting structures and models into a context of live-cell membranes. The expansive array of state-dependent ligands and optical probes should enable probes more generally for investigating the molecular motions of endogenous proteins.

Molecular determinants of pro-arrhythmia proclivity of d- and l-sotalol via a multi-scale modeling pipeline.

Drug isomers may differ in their proarrhythmia risk. An interesting example is the drug sotalol, an antiarrhythmic drug comprising d- and l- enantiomers that both block the hERG cardiac potassium channel and confer differing degrees of proarrhythmic risk. We developed a multi-scale in silico pipeline focusing on hERG channel - drug interactions and used it to probe and predict the mechanisms of pro-arrhythmia risks of the two enantiomers of sotalol. Molecular dynamics (MD) simulations predicted comparable hERG channel binding affinities for d- and l-sotalol, which were validated with electrophysiology experiments. MD derived thermodynamic and kinetic parameters were used to build multi-scale functional computational models of cardiac electrophysiology at the cell and tissue scales. Functional models were used to predict inactivated state binding affinities to recapitulate electrocardiogram (ECG) QT interval prolongation observed in clinical data. Our study demonstrates how modeling and simulation can be applied to predict drug effects from the atom to the rhythm for dl-sotalol and also increased proarrhythmia proclivity of d- vs. l-sotalol when accounting for stereospecific beta-adrenergic receptor blocking.

Distinguishing Potassium Channel Resting State Conformations in Live Cells with Environment-Sensitive Fluorescence.

Ion channels are polymorphic membrane proteins whose high-resolution structures offer images of individual conformations, giving us starting points for identifying the complex and transient allosteric changes that give rise to channel physiology. Here, we report live-cell imaging of voltage-dependent structural changes of voltage-gated Kv2.1 channels using peptidyl tarantula toxins labeled with an environment-sensitive fluorophore, whose spectral shifts enable identification of voltage-dependent conformation changes in the resting voltage sensing domain (VSD) of the channel. We synthesize a new environment-sensitive, far-red fluorophore, julolidine phenoxazone (JP) azide, and conjugate it to tarantula toxin GxTX to characterize Kv2.1 VSD allostery during membrane depolarization. JP has an inherent response to the polarity of its immediate surroundings, offering site-specific structural insight into each channel conformation. Using voltage-clamp spectroscopy to collect emission spectra as a function of membrane potential, we find that they vary with toxin labeling site, the presence of Kv2 channels, and changes in membrane potential. With a high-affinity conjugate in which the fluorophore itself interacts closely with the channel, the emission shift midpoint is 50 mV more negative than the Kv2.1 gating current midpoint. This suggests that substantial conformational changes at the toxin-channel interface are associated with early gating charge transitions and these are not concerted with VSD motions at more depolarized potentials. These fluorescent probes enable study of conformational changes that can be correlated with electrophysiology, putting channel structures and models into a context of live-cell membranes and physiological states.

Facilitation of I Kr current by some hERG channel blockers suppresses early afterdepolarizations.

Drug-induced block of the cardiac rapid delayed rectifying potassium current (I Kr), carried by the human ether-a-go-go-related gene (hERG) channel, is the most common cause of acquired long QT syndrome. Indeed, some, but not all, drugs that block hERG channels cause fatal cardiac arrhythmias. However, there is no clear method to distinguish between drugs that cause deadly arrhythmias and those that are clinically safe. Here we propose a mechanism that could explain why certain clinically used hERG blockers are less proarrhythmic than others. We demonstrate that several drugs that block hERG channels, but have favorable cardiac safety profiles, also evoke another effect; they facilitate the hERG current amplitude in response to low-voltage depolarization. To investigate how hERG facilitation impacts cardiac safety, we develop computational models of I Kr block with and without this facilitation. We constrain the models using data from voltage clamp recordings of hERG block and facilitation by nifekalant, a safe class III antiarrhythmic agent. Human ventricular action potential simulations demonstrate the ability of nifekalant to suppress ectopic excitations, with or without facilitation. Without facilitation, excessive I Kr block evokes early afterdepolarizations, which cause lethal arrhythmias. When facilitation is introduced, early afterdepolarizations are prevented at the same degree of block. Facilitation appears to prevent early afterdepolarizations by increasing I Kr during the repolarization phase of action potentials. We empirically test this prediction in isolated rabbit ventricular myocytes and find that action potential prolongation with nifekalant is less likely to induce early afterdepolarization than action potential prolongation with dofetilide, a hERG channel blocker that does not induce facilitation. Our data suggest that hERG channel blockers that induce facilitation increase the repolarization reserve of cardiac myocytes, rendering them less likely to trigger lethal ventricular arrhythmias.

The tarantula toxin GxTx detains K+ channel gating charges in their resting conformation.

Allosteric ligands modulate protein activity by altering the energy landscape of conformational space in ligand-protein complexes. Here we investigate how ligand binding to a K+ channel's voltage sensor allosterically modulates opening of its K+-conductive pore. The tarantula venom peptide guangxitoxin-1E (GxTx) binds to the voltage sensors of the rat voltage-gated K+ (Kv) channel Kv2.1 and acts as a partial inverse agonist. When bound to GxTx, Kv2.1 activates more slowly, deactivates more rapidly, and requires more positive voltage to reach the same K+-conductance as the unbound channel. Further, activation kinetics are more sigmoidal, indicating that multiple conformational changes coupled to opening are modulated. Single-channel current amplitudes reveal that each channel opens to full conductance when GxTx is bound. Inhibition of Kv2.1 channels by GxTx results from decreased open probability due to increased occurrence of long-lived closed states; the time constant of the final pore opening step itself is not impacted by GxTx. When intracellular potential is less than 0 mV, GxTx traps the gating charges on Kv2.1's voltage sensors in their most intracellular position. Gating charges translocate at positive voltages, however, indicating that GxTx stabilizes the most intracellular conformation of the voltage sensors (their resting conformation). Kinetic modeling suggests a modulatory mechanism: GxTx reduces the probability of voltage sensors activating, giving the pore opening step less frequent opportunities to occur. This mechanism results in K+-conductance activation kinetics that are voltage-dependent, even if pore opening (the rate-limiting step) has no inherent voltage dependence. We conclude that GxTx stabilizes voltage sensors in a resting conformation, and inhibits K+ currents by limiting opportunities for the channel pore to open, but has little, if any, direct effect on the microscopic kinetics of pore opening. The impact of GxTx on channel gating suggests that Kv2.1's pore opening step does not involve movement of its voltage sensors.

Remodeling neuronal ER-PM junctions is a conserved nonconducting function of Kv2 plasma membrane ion channels.

The endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is localized at ER-PM junctions in brain neurons and is unique among PM proteins in its ability to remodel these specialized membrane contact sites. Here, we show that this function is conserved between Kv2.1 and Kv2.2, which differ in their biophysical properties, modulation, and cellular expression. Kv2.2 ER-PM junctions are present at sites deficient in the actin cytoskeleton, and disruption of the actin cytoskeleton affects their spatial organization. Kv2.2-containing ER-PM junctions overlap with those formed by canonical ER-PM tethers. The ability of Kv2 channels to remodel ER-PM junctions is unchanged by point mutations that eliminate their ion conduction but eliminated by point mutations within the Kv2-specific proximal restriction and clustering (PRC) domain that do not impact their ion channel function. The highly conserved PRC domain is sufficient to transfer the ER-PM junction-remodeling function to another PM protein. Last, brain neurons in Kv2 double-knockout mice have altered ER-PM junctions. Together, these findings demonstrate a conserved in vivo function for Kv2 family members in remodeling neuronal ER-PM junctions that is distinct from their canonical role as ion-conducting channels shaping neuronal excitability.