Biology subjects

Sack, J. T.

Publications and source records attributed to Sack, J. T..

14 recordsLinked to original sources

bioRxiv2026

Upregulation of Calbindin in Adult Inhibitory Neurons Reactivates Critical Period Plasticity in Mouse Visual Cortex

Critical periods are windows of peak learning performance where heightened synaptic plasticity enables fast, robust reorganization in juvenile brain circuits. Unlike adult plasticity which requires ongoing, persistent change in sensory experience, cortical representations can be rapidly changed during critical periods and have enduring effects. Transplantation of GABAergic inhibitory neurons has been shown to restore critical period plasticity to recipient circuits by triggering signalling changes within host inhibitory neurons. Here we transcriptionally profiled host inhibitory neurons in mouse primary visual cortex (V1) to detect gene expression changes during transplant-induced plasticity. Gene ontology enrichment analysis of differentially-expressed (DE) transcripts in host inhibitory neurons revealed synaptic plasticity- and inhibitory neuron development-related profiles. We assessed the protein expression of a top DE candidate, the calcium-binding protein Calbindin (Calb1), across transplant conditions and developmental stages. We found high Calb1 expression during the V1 critical period and during transplant-induced plasticity. To assess the functional significance of transplant-reactivated DE gene activity on visual cortical plasticity, we developed a set of AAVs to manipulate Calb1 expression specifically within inhibitory neurons. Using intrinsic signal imaging to measure ocular dominance plasticity, we found that Calb1 levels in V1 inhibitory neurons determine the extent of visual cortical plasticity. Our study provides evidence that direct induction of critical period-stage gene expression patterns in inhibitory neurons restores juvenile plasticity in targeted adult cortical circuits.

neuroscience↗

bioRxiv2026

A hERG Blocker Facilitates K+ Channel Current by Agonizing Pore Opening while Blocking

Many drugs that block voltage-gated K+ channels encoded by the human Ether-a-go-go-Related Gene (hERG) can cause long QT syndrome and life-threatening cardiac arrhythmias, yet the molecular mechanisms that determine this risk remain unclear. A process that may counteract arrhythmogenic hERG block, termed facilitation, is common to many clinically-approved hERG blockers including nifekalant, amiodarone, promethazine, imipramine, nortriptyline, haloperidol, verapamil, carvedilol, metoprolol, propranolol, quinidine, fluoxetine, and chlorpheniramine. Facilitation is an increase in hERG current, under certain conditions, due to these blockers. Here, we propose that an agonism-while-blocking mechanism undergirds facilitation. We focus on nifekalant, a Class III antiarrhythmic drug and exemplar hERG blocker that induces facilitation. We tested the hypothesis that nifekalant opens hERG channel gates while blocking, and that unblock of these open-yet-blocked channels results in supranormal hERG current. We develop rate-theory kinetic models to identify features of agonism-while-blocking that produce facilitation. We generate atomistic models that predict that nifekalant blocks the hERG conduction path while modulating the intracellular conduction gate. Voltage-clamp measurements reveal that agonism-while-blocking undergirds nifekalant block and facilitation. We speculate that this agonism-while-blocking mechanism contributes to the relative safety of hERG blockers that induce facilitation.

pharmacology and toxicology↗

bioRxiv2025

Kv2/Kv6.4 heteromeric potassium channels are expressed in spinal motor neurons and localized at C-bouton synapses

Voltage-gated K+ channels of the Kv2 family co-assemble with electrically silent KvS subunits in specific subpopulations of brain neurons, forming heteromeric Kv2/KvS channels with distinct functional properties. Little is known about the composition and function of Kv2 channels in spinal cord neurons, however. Here, we show that while Kv2.1 is broadly expressed in multiple classes of spinal cord neurons, the Kv6.4 "electrically-silent" subunit is specifically expressed in motor neurons. In motor neurons, we find that Kv6.4 protein is co-clustered with Kv2.1 and Kv2.2 subunits at endoplasmic reticulum-plasma membrane (ER-PM) junctions beneath C-bouton synapses. In Kv2.1 S590A mutant mice, in which Kv2.1 is unable to bind ER VAP proteins, Kv2.1 and Kv6.4 clustering at ER-PM junctions is severely reduced suggesting Kv2 channels are localized at ER-PM junctions by the same molecular mechanism in motor neurons and brain neurons. Moreover, clustering of Kv6.4, as well as the AMIGO-1 auxiliary subunit, are severely reduced in Kv2.1 knockout mice and moderately reduced in Kv2.2 knockout mice. Thus, expression and localization of Kv6.4 subunits is dependent on Kv2 subunits, likely through their co-assembly into heteromeric channels. Finally, we find that presynaptic C-boutons and postsynaptic clusters of the ER-resident sigma1-receptor are preserved in motor neurons of Kv2 knockout mice. Together, these findings identify a specific Kv2/KvS channel subtype expressed in motor neurons that localizes to C-bouton junctions where it could regulate neuronal excitability and signaling at ER-PM junctions.

neuroscience↗

bioRxiv2024

KvS regulatory subunits confer drug resistance to Kv2 channels

KvS proteins are voltage-gated potassium channel subunits that form functional channels when assembled into heteromers 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 heteromers 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 predominantly RY785-sensitive conductances consistent with channels composed entirely of Kv2 subunits. In contrast, RY785-resistant but GxTX-sensitive conductances consistent with Kv2/KvS heteromeric channels predominate 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 which distinguish KvS subunits could modulate electrical activity of subsets of Kv2-expressing cell types.

neuroscience↗

bioRxiv2024

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.

neuroscience↗

bioRxiv2023

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. Significance statementThe subcellular distribution of Kv2 voltage-gated potassium channels enable compartment-specific modulation of membrane excitability and organization of membrane contact sites. Here we identify subcellular distributions of the Kv2 paralogs, Kv2.1 and Kv2.2, in somatosensory neurons that bear similarities to and distinctions from central neurons. The distribution of Kv2 channels is similar in mouse and human somatosensory neurons. These results identify unique locations of Kv2 channels in somatosensory neurons that could enable roles in sensory information processing.

neuroscience↗

bioRxiv2022

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 1,000-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.

biophysics↗

bioRxiv2022

Facilitation by a hERG blocker is induced by pore opening and operates by pore reopening

A drug that blocks the cardiac myocyte voltage-gated K+ channels encoded by the human Ether-a-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 of the D540K hERG channel enables testing of hypotheses about 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, facilitates not only current activation by depolarization but also current activation by hyperpolarization. Our results indicate that induction of facilitation is coupled to pore opening, not voltage per se. We propose that a gated-access mechanism is involved in the voltage-dependence of induction of facilitation. This study provides a molecular mechanism for modulation of hERG channels by nifekalant, a clinically important antiarrhythmic agent. Significance statementNifekalant 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.

pharmacology and toxicology↗

bioRxiv2021

Structural modeling of the hERG potassium channel and associated drug interactions

The voltage-gated potassium channel, KV11.1, encoded by the human Ether-a-go-go- Related Gene (hERG) is expressed in cardiac myocytes, where it is crucial for the membrane repolarization of the action potential. Gating of 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 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 F627 side chain in the selectivity filter into the central channel axis along the ion conduction pathway and 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 correlate well with existing experimental evidence involving interactions of these drugs with key hERG residues Y652 and F656 inside the pore and reveal potential ligand binding interactions within fenestration region in an inactivated state. Significance StatementComputational models of hERG potassium channel provide structural insights into an inactivated state and associated drug interactions. Our computational approach will be useful to study ion channel modulation by small molecules.

biophysics↗

bioRxiv2021

The AMIGO1 adhesion protein modulates movement of Kv2.1 voltage sensors

Kv2 voltage-gated potassium channels are modulated by 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 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. Statement of SignificanceKv2 potassium channels activate a potassium conductance that shapes neuronal action potentials. The AMIGO family of adhesion proteins modulate activation of Kv2 conductances, yet, which activation steps are modified is unknown. This study finds that AMIGO1 destabilizes the earliest resting conformation of the Kv2.1 voltage sensors to promote activation of channel conductance.

biophysics↗

bioRxiv2019

Molecular Determinants of μ-Conotoxin KIIIA interaction with the 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 -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.

biophysics↗

bioRxiv2019

An imaging method to measure conformational changes of voltage sensors of endogenous ion channels in tissue

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. 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 labeling intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to labeling intensity. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. This imaging method enabled identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

biophysics↗

bioRxiv2018

Facilitation of hERG channels by blockers: a mechanism predicted to reduce lethal cardiac arrhythmias

Fatal cardiac arrhythmias are caused by some, but not all, drugs that inhibit the cardiac rapid delayed-rectifier current (IKr) by blocking hERG channels. Here, we propose a novel mechanism that could make certain hERG blockers less proarrhythmic. Several drugs that block hERG channels, yet have favorable cardiac safety profiles, also evoke another effect; they increase the current amplitude upon low-voltage depolarization (facilitation). Voltage-clamp recordings of hERG block and facilitation by nifekalant, a Class III antiarrhythmic agent, constrained a model of human cardiac IKr. In human ventricular action potential simulations, nifekalant showed its therapeutic ability to suppress ectopic excitations, with or without facilitation. Without facilitation, excessive IKr block evoked early afterdepolarizations, which cause lethal arrhythmias. Facilitation prevented early afterdepolarizations at the same degree of block by increasing IKr during repolarization phase of action potentials. Thus, facilitation is proposed to reduce the arrhythmogenic risk of hERG blockers.\n\nAbbreviationsAP: action potential; APD: action potential duration; APD90: action potential duration measured at 90% repolarization; EAD: early afterdepolarization; hERG: human ether-ago-go-related gene; ICaL: L-type Ca2+ channel current; Inet: net ionic current; IK1: inward-rectifier potassium current; IKr: rapid component of the delayed-rectifier potassium current; INa: sodium current; ORd: OHara-Rudy dynamic

pharmacology and toxicology↗

bioRxiv2018

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

Endoplasmic reticulum (ER) and plasma membrane (PM) form junctions crucial to ion and lipid signaling and homeostasis. The Kv2.1 ion channel is unique among PM proteins in organizing ER-PM junctions. Here, we show that this organizing function is conserved between Kv2 family members that differ in their biophysical properties, modulation and cellular expression. Manipulation of actin cytoskeleton surrounding Kv2 ER-PM junctions affects their spatial organization. Kv2-containing ER-PM junctions overlap with those formed by canonical ER-PM tethers. ER-PM junction organization by Kv2 channels is unchanged by point mutations that eliminate ion conduction, but abolished by those that eliminate PM clustering without impacting ion channel function. Kv2.2 is distinct in lacking the reversible modulation of junction organization present in Kv2.1. Brain neurons in Kv2 double knockout mice have altered ER-PM junctions, demonstrating a conserved in vivo function for Kv2 family members distinct from their canonical role as ion-conducting channels shaping neuronal excitability.

cell biology↗