Grant Higerd-Rusli MD, PhD

All Publications

• Sculpting excitable membranes: voltage-gated ion channel delivery and distribution. Nature reviews. Neuroscience Tyagi, S., Higerd-Rusli, G. P., Akin, E. J., Waxman, S. G., Dib-Hajj, S. D. 2025

  Abstract

  The polarized and domain-specific distribution of membrane ion channels is essential for neuronal homeostasis, but delivery of these proteins to distal neuronal compartments (such as the axonal ends of peripheral sensory neurons) presents a logistical challenge. Recent developments have enabled the real-time imaging of single protein trafficking and the investigation of the life cycle of ion channels across neuronal compartments. These studies have revealed a highly regulated process involving post-translational modifications, vesicular sorting, motor protein-driven transport and targeted membrane insertion. Emerging evidence suggests that neuronal activity and disease states can dynamically modulate ion channel localization, directly influencing excitability. This Review synthesizes current knowledge on the spatiotemporal regulation of ion channel trafficking in both central and peripheral nervous system neurons. Understanding these processes not only advances our fundamental knowledge of neuronal excitability, but also reveals potential therapeutic targets for disorders involving aberrant ion channel distribution, such as chronic pain and neurodegenerative diseases.

  View details for DOI 10.1038/s41583-025-00917-2

  View details for PubMedID 40175736

  View details for PubMedCentralID 1392413

• Expanding testing early in the H5N1 outbreak. Lancet (London, England) Karan, A., Higerd-Rusli, G., Hernandez, M., Dhillon, R., Pinsky, B. A. 2025; 405 (10481): 779-780

  View details for DOI 10.1016/S0140-6736(25)00090-X

  View details for PubMedID 40057336

• Targeted ubiquitination of NaV1.8 reduces sensory neuronal excitability. bioRxiv : the preprint server for biology Tyagi, S., Ghovanloo, M., Alsaloum, M., Effraim, P., Higerd-Rusli, G. P., Dib-Hajj, F., Zhao, P., Liu, S., Waxman, S. G., Dib-Hajj, S. D. 2025

  Abstract

  Chronic pain and addiction are a significant global health challenge. Voltage-gated sodium channel Na V 1.8, a pivotal driver of pain signaling, is a clinically validated target for the development of novel, non-addictive pain therapeutics. Small molecule inhibitors against Na V 1.8 have shown promise in acute pain indications, but large clinical effect sizes have not yet been demonstrated and efficacy in chronic pain indications are lacking. An alternative strategy to target Na V 1.8 channels for analgesia is to reduce the number of channels that are present on nociceptor membranes. We generated a therapeutic heterobifunctional protein, named UbiquiNa V , that contains a Na V 1.8-selective binding module and the catalytic subunit of the NEDD4 E3 Ubiquitin ligase. We show that UbiquiNav significantly reduces channel expression in the plasma membrane and reduces Na V 1.8 currents in rodent sensory neurons. We demonstrate that UbiquiNa V is selective for Na V 1.8 over other Na V isoforms and other components of the sensory neuronal electrogenisome. We then show that UbiquiNa V normalizes the distribution of Na V 1.8 protein to distal axons, and that UbiquiNa V normalizes the neuronal hyperexcitability in in vitro models of inflammatory and chemotherapy-induced neuropathic pain. Our results serve as a blueprint for the design of therapeutics that leverage the selective ubiquitination of Na V 1.8 channels for analgesia.

  View details for DOI 10.1101/2025.02.04.636451

  View details for PubMedID 39975312

• Real-time imaging of axonal membrane protein life cycles NATURE PROTOCOLS Tyagi, S., Higerd-Rusli, G. P., Akin, E. J., Baker, C. A., Liu, S., Dib-Hajj, F. B., Waxman, S. G., Dib-Hajj, S. D. 2024; 19 (9): 2771-2802

  Abstract

  The construction of neuronal membranes is a dynamic process involving the biogenesis, vesicular packaging, transport, insertion and recycling of membrane proteins. Optical imaging is well suited for the study of protein spatial organization and transport. However, various shortcomings of existing imaging techniques have prevented the study of specific types of proteins and cellular processes. Here we describe strategies for protein tagging and labeling, cell culture and microscopy that enable the real-time imaging of axonal membrane protein trafficking and subcellular distribution as they progress through some stages of their life cycle. First, we describe a process for engineering membrane proteins with extracellular self-labeling tags (either HaloTag or SNAPTag), which can be labeled with fluorescent ligands of various colors and cell permeability, providing flexibility for investigating the trafficking and spatiotemporal regulation of multiple membrane proteins in neuronal compartments. Next, we detail the dissection, transfection and culture of dorsal root ganglion sensory neurons in microfluidic chambers, which physically compartmentalizes cell bodies and distal axons. Finally, we describe four labeling and imaging procedures that utilize these enzymatically tagged proteins, flexible fluorescent labels and compartmentalized neuronal cultures to study axonal membrane protein anterograde and retrograde transport, the cotransport of multiple proteins, protein subcellular localization, exocytosis and endocytosis. Additionally, we generated open-source software for analyzing the imaging data in a high throughput manner. The experimental and analysis workflows provide an approach for studying the dynamics of neuronal membrane protein homeostasis, addressing longstanding challenges in this area. The protocol requires 5-7 days and expertise in cell culture and microscopy.

  View details for DOI 10.1038/s41596-024-00997-x

  View details for Web of Science ID 001237759400001

  View details for PubMedID 38831222

  View details for PubMedCentralID PMC11721981

• Compartment-specific regulation of NaV1.7 in sensory neurons after acute exposure to TNF-a CELL REPORTS Tyagi, S., Higerd-Rusli, G. P., Ghovanloo, M., Dib-Hajj, F., Zhao, P., Liu, S., Kim, D., Shim, J., Park, K., Waxman, S. G., Choi, J., Dib-Hajj, S. D. 2024; 43 (2): 113685

  Abstract

  Tumor necrosis factor α (TNF-α) is a major pro-inflammatory cytokine, important in many diseases, that sensitizes nociceptors through its action on a variety of ion channels, including voltage-gated sodium (NaV) channels. We show here that TNF-α acutely upregulates sensory neuron excitability and current density of threshold channel NaV1.7. Using electrophysiological recordings and live imaging, we demonstrate that this effect on NaV1.7 is mediated by p38 MAPK and identify serine 110 in the channel's N terminus as the phospho-acceptor site, which triggers NaV1.7 channel insertion into the somatic membrane. We also show that the N terminus of NaV1.7 is sufficient to mediate this effect. Although acute TNF-α treatment increases NaV1.7-carrying vesicle accumulation at axonal endings, we did not observe increased channel insertion into the axonal membrane. These results identify molecular determinants of TNF-α-mediated regulation of NaV1.7 in sensory neurons and demonstrate compartment-specific effects of TNF-α on channel insertion in the neuronal plasma membrane.

  View details for DOI 10.1016/j.celrep.2024.113685

  View details for Web of Science ID 001175292900001

  View details for PubMedID 38261513

  View details for PubMedCentralID PMC10947185

• Conserved but not critical: Trafficking and function of Na_V1.7 are independent of highly conserved polybasic motifs FRONTIERS IN MOLECULAR NEUROSCIENCE Tyagi, S., Sarveswaran, N., Higerd-Rusli, G. P., Liu, S., Dib-Hajj, F. B., Waxman, S. G., Dib-Hajj, S. D. 2023; 16: 1161028

  Abstract

  Non-addictive treatment of chronic pain represents a major unmet clinical need. Peripheral voltage-gated sodium (NaV) channels are an attractive target for pain therapy because they initiate and propagate action potentials in primary afferents that detect and transduce noxious stimuli. NaV1.7 sets the gain on peripheral pain-signaling neurons and is the best validated peripheral ion channel involved in human pain, and previous work has shown that it is transported in vesicles in sensory axons which also carry Rab6a, a small GTPase known to be involved in vesicular packaging and axonal transport. Understanding the mechanism of the association between Rab6a and NaV1.7 could inform therapeutic modalities to decrease trafficking of NaV1.7 to the distal axonal membrane. Polybasic motifs (PBM) have been shown to regulate Rab-protein interactions in a variety of contexts. In this study, we explored whether two PBMs in the cytoplasmic loop that joins domains I and II of human NaV1.7 were responsible for association with Rab6a and regulate axonal trafficking of the channel. Using site-directed mutagenesis we generated NaV1.7 constructs with alanine substitutions in the two PBMs. Voltage-clamp recordings showed that the constructs retain wild-type like gating properties. Optical Pulse-chase Axonal Long-distance (OPAL) imaging in live sensory axons shows that mutations of these PBMs do not affect co-trafficking of Rab6a and NaV1.7, or the accumulation of the channel at the distal axonal surface. Thus, these polybasic motifs are not required for interaction of NaV1.7 with the Rab6a GTPase, or for trafficking of the channel to the plasma membrane.

  View details for DOI 10.3389/fnmol.2023.1161028

  View details for Web of Science ID 000961288000001

  View details for PubMedID 37008789

  View details for PubMedCentralID PMC10060856

• Inflammation differentially controls transport of depolarizing Nav versus hyperpolarizing Kv channels to drive rat nociceptor activity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Higerd-Rusli, G. P., Tyagi, S., Baker, C. A., Liu, S., Dib-Hajj, F. B., Dib-Hajj, S. D., Waxman, S. G. 2023; 120 (11): e2215417120

  Abstract

  Inflammation causes pain by shifting the balance of ionic currents in nociceptors toward depolarization, leading to hyperexcitability. The ensemble of ion channels within the plasma membrane is regulated by processes including biogenesis, transport, and degradation. Thus, alterations in ion channel trafficking may influence excitability. Sodium channel NaV1.7 and potassium channel KV7.2 promote and oppose excitability in nociceptors, respectively. We used live-cell imaging to investigate mechanisms by which inflammatory mediators (IM) modulate the abundance of these channels at axonal surfaces through transcription, vesicular loading, axonal transport, exocytosis, and endocytosis. Inflammatory mediators induced a NaV1.7-dependent increase in activity in distal axons. Further, inflammation increased the abundance of NaV1.7, but not of KV7.2, at axonal surfaces by selectively increasing channel loading into anterograde transport vesicles and insertion at the membrane, without affecting retrograde transport. These results uncover a cell biological mechanism for inflammatory pain and suggest NaV1.7 trafficking as a potential therapeutic target.

  View details for DOI 10.1073/pnas.2215417120

  View details for Web of Science ID 000980558900007

  View details for PubMedID 36897973

  View details for PubMedCentralID PMC10089179

• Paclitaxel effects on axonal localization and vesicular trafficking of Na_V1.8 FRONTIERS IN MOLECULAR NEUROSCIENCE Baker, C. A., Tyagi, S., Higerd-Rusli, G. P., Liu, S., Zhao, P., Dib-Hajj, F. B., Waxman, S. G., Dib-Hajj, S. D. 2023; 16: 1130123

  Abstract

  Patients treated with paclitaxel (PTX) or other antineoplastic agents can experience chemotherapy-induced peripheral neuropathy (CIPN), a debilitating side effect characterized by numbness and pain. PTX interferes with microtubule-based transport, which inhibits tumor growth via cell cycle arrest but can also affect other cellular functions including trafficking of ion channels critical to transduction of stimuli by sensory neurons of the dorsal root ganglia (DRG). We examined the effects of PTX on voltage-gated sodium channel NaV1.8, which is preferentially expressed in DRG neurons, using a microfluidic chamber culture system and chemigenetic labeling to observe anterograde channel transport to the endings of DRG axons in real time. PTX treatment increased the numbers of NaV1.8-containing vesicles traversing the axons. Vesicles in PTX-treated cells exhibited greater average velocity, along with shorter and less frequent pauses along their trajectories. These events were paralleled by greater surface accumulation of NaV1.8 channels at the distal ends of DRG axons. These results were consistent with observations that NaV1.8 is trafficked in the same vesicles containing NaV1.7 channels, which are also involved in pain syndromes in humans and are similarly affected by PTX treatment. However, unlike Nav1.7, we did not detect increased NaV1.8 current density measured at the neuronal soma, suggesting a differential effect of PTX on trafficking of NaV1.8 in soma versus axonal compartments. Therapeutic targeting of axonal vesicular traffic would affect both Nav1.7 and Nav1.8 channels and increase the possibilities of alleviating pain associated with CIPN.

  View details for DOI 10.3389/fnmol.2023.1130123

  View details for Web of Science ID 000939686600001

  View details for PubMedID 36860665

  View details for PubMedCentralID PMC9970094

• The fates of internalized NaV1.7 channels in sensory neurons: Retrograde cotransport with other ion channels, axon-specific recycling, and degradation JOURNAL OF BIOLOGICAL CHEMISTRY Higerd-Rusli, G. P., Tyagi, S., Liu, S., Dib-Hajj, F. B., Waxman, S. G., Dib-Hajj, S. D. 2023; 299 (1): 102816

  Abstract

  Neuronal function relies on the maintenance of appropriate levels of various ion channels at the cell membrane, which is accomplished by balancing secretory, degradative, and recycling pathways. Neuronal function further depends on membrane specialization through polarized distribution of specific proteins to distinct neuronal compartments such as axons. Voltage-gated sodium channel NaV1.7, a threshold channel for firing action potentials in nociceptors, plays a major role in human pain, and its abundance in the plasma membrane is tightly regulated. We have recently characterized the anterograde axonal trafficking of NaV1.7 channels in Rab6A-positive vesicles, but the fate of internalized channels is not known. Membrane proteins that have undergone endocytosis can be directed into multiple pathways including those for degradation, recycling to the membrane, and transcytosis. Here, we demonstrate NaV1.7 endocytosis and dynein-dependent retrograde trafficking in Rab7-containing late endosomes together with other axonal membrane proteins using real-time imaging of live neurons. We show that some internalized NaV1.7 channels are delivered to lysosomes within the cell body, and that there is no evidence for NaV1.7 transcytosis. In addition, we show that NaV1.7 is recycled specifically to the axonal membrane as opposed to the soma membrane, suggesting a novel mechanism for the development of neuronal polarity. Together, these results shed light on the mechanisms by which neurons maintain excitable membranes and may inform efforts to target ion channel trafficking for the treatment of disorders of excitability.

  View details for DOI 10.1016/j.jbc.2022.102816

  View details for Web of Science ID 001012061200001

  View details for PubMedID 36539035

  View details for PubMedCentralID PMC9843449

• Depolarizing Na_v and Hyperpolarizing K_vChannels Are Co-Trafficked in Sensory Neurons JOURNAL OF NEUROSCIENCE Higerd-Rusli, G. P., Alsaloum, M., Tyagi, S., Sarveswaran, N., Estacion, M., Akin, E. J., Dib-Hajj, F. B., Liu, S., Sosniak, D., Zhao, P., Dib-Hajj, S. D., Waxman, S. G. 2022; 42 (24): 4794-4811

  Abstract

  Neuronal excitability relies on coordinated action of functionally distinction channels. Voltage-gated sodium (NaV) and potassium (KV) channels have distinct but complementary roles in firing action potentials: NaV channels provide depolarizing current while KV channels provide hyperpolarizing current. Mutations and dysfunction of multiple NaV and KV channels underlie disorders of excitability, including pain and epilepsy. Modulating ion channel trafficking may offer a potential therapeutic strategy for these diseases. A fundamental question, however, is whether these channels with distinct functional roles are transported independently or packaged together in the same vesicles in sensory axons. We have used Optical Pulse-Chase Axonal Long-distance imaging to investigate trafficking of NaV and KV channels and other axonal proteins from distinct functional classes in live rodent sensory neurons (from male and female rats). We show that, similar to NaV1.7 channels, NaV1.8 and KV7.2 channels are transported in Rab6a-positive vesicles, and that each of the NaV channel isoforms expressed in healthy, mature sensory neurons (NaV1.6, NaV1.7, NaV1.8, and NaV1.9) is cotransported in the same vesicles. Further, we show that multiple axonal membrane proteins with different physiological functions (NaV1.7, KV7.2, and TNFR1) are cotransported in the same vesicles. However, vesicular packaging of axonal membrane proteins is not indiscriminate, since another axonal membrane protein (NCX2) is transported in separate vesicles. These results shed new light on the development and organization of sensory neuron membranes, revealing complex sorting of axonal proteins with diverse physiological functions into specific transport vesicles.SIGNIFICANCE STATEMENT Normal neuronal excitability is dependent on precise regulation of membrane proteins, including NaV and KV channels, and imbalance in the level of these channels at the plasma membrane could lead to excitability disorders. Ion channel trafficking could potentially be targeted therapeutically, which would require better understanding of the mechanisms underlying trafficking of functionally diverse channels. Optical Pulse-chase Axonal Long-distance imaging in live neurons permitted examination of the specificity of ion channel trafficking, revealing co-packaging of axonal proteins with opposing physiological functions into the same transport vesicles. This suggests that additional trafficking mechanisms are necessary to regulate levels of surface channels, and reveals an important consideration for therapeutic strategies that target ion channel trafficking for the treatment of excitability disorders.

  View details for DOI 10.1523/JNEUROSCI.0058-22.2022

  View details for Web of Science ID 000817218600003

  View details for PubMedID 35589395

  View details for PubMedCentralID PMC9188389

• Inhibition of sodium conductance by cannabigerol contributes to a reduction of dorsal root ganglion neuron excitability BRITISH JOURNAL OF PHARMACOLOGY Ghovanloo, M., Estacion, M., Higerd-Rusli, G. P., Zhao, P., Dib-Hajj, S., Waxman, S. G. 2022; 179 (15): 4010-4030

  Abstract

  Cannabigerol (CBG), a non-psychotropic phytocannabinoid and a precursor of ∆9 -tetrahydrocannabinol and cannabidiol, has been suggested to act as an analgesic. A previous study reported that CBG (10 μM) blocks voltage-gated sodium (Nav ) currents in CNS neurons, although the underlying mechanism is not well understood. Genetic and functional studies have validated Nav 1.7 channels as an opportune target for analgesic drug development. The effects of CBG on Nav 1.7 channels, which may contribute to its analgesic properties, have not been previously investigated.To determine the effects of CBG on Nav channels, we used stably transfected HEK cells and primary dorsal root ganglion (DRG) neurons to characterize compound effects using experimental and computational techniques. These included patch-clamp, multielectrode array, and action potential modelling.CBG is a ~10-fold state-dependent Nav channel inhibitor (KI -KR : ~2-20 μM) with an average Hill-slope of ~2. We determined that, at lower concentrations, CBG predominantly blocks sodium Gmax and slows recovery from inactivation. However, as the concentration is increased, CBG also induces a hyperpolarizing shift in the half-voltage of inactivation. Our modelling and multielectrode array recordings suggest that CBG attenuates DRG excitability.Inhibition of Nav 1.7 channels in DRG neurons may underlie CBG-induced neuronal hypoexcitability. As most Nav 1.7 channels are inactivated at the resting membrane potential of DRG neurons, they are more likely to be inhibited by lower CBG concentrations, suggesting functional selectivity against Nav 1.7 channels, compared with other Nav channels (via Gmax block).

  View details for DOI 10.1111/bph.15833

  View details for Web of Science ID 000784617100001

  View details for PubMedID 35297036

• Paclitaxel increases axonal localization and vesicular trafficking of Na_v1.7 BRAIN Akin, E. J., Alsaloum, M., Higerd, G. P., Liu, S., Zhao, P., Dib-Hajj, F. B., Waxman, S. G., Dib-Hajj, S. D. 2021; 144: 1727-1737

  Abstract

  The microtubule-stabilizing chemotherapy drug paclitaxel (PTX) causes dose-limiting chemotherapy-induced peripheral neuropathy (CIPN), which is often accompanied by pain. Among the multifaceted effects of PTX is an increased expression of sodium channel Nav1.7 in rat and human sensory neurons, enhancing their excitability. However, the mechanisms underlying this increased Nav1.7 expression have not been explored, and the effects of PTX treatment on the dynamics of trafficking and localization of Nav1.7 channels in sensory axons have not been possible to investigate to date. In this study we used a recently developed live imaging approach that allows visualization of Nav1.7 surface channels and long-distance axonal vesicular transport in sensory neurons to fill this basic knowledge gap. We demonstrate concentration and time-dependent effects of PTX on vesicular trafficking and membrane localization of Nav1.7 in real-time in sensory axons. Low concentrations of PTX increase surface channel expression and vesicular flux (number of vesicles per axon). By contrast, treatment with a higher concentration of PTX decreases vesicular flux. Interestingly, vesicular velocity is increased for both concentrations of PTX. Treatment with PTX increased levels of endogenous Nav1.7 mRNA and current density in dorsal root ganglion neurons. However, the current produced by transfection of dorsal root ganglion neurons with Halo-tag Nav1.7 was not increased after exposure to PTX. Taken together, this suggests that the increased trafficking and surface localization of Halo-Nav1.7 that we observed by live imaging in transfected dorsal root ganglion neurons after treatment with PTX might be independent of an increased pool of Nav1.7 channels. After exposure to inflammatory mediators to mimic the inflammatory condition seen during chemotherapy, both Nav1.7 surface levels and vesicular transport are increased for both low and high concentrations of PTX. Overall, our results show that PTX treatment increases levels of functional endogenous Nav1.7 channels in dorsal root ganglion neurons and enhances trafficking and surface distribution of Nav1.7 in sensory axons, with outcomes that depend on the presence of an inflammatory milieu, providing a mechanistic explanation for increased excitability of primary afferents and pain in CIPN.

  View details for DOI 10.1093/brain/awab113

  View details for Web of Science ID 000710929200020

  View details for PubMedID 33734317

  View details for PubMedCentralID PMC8320304

• Status of peripheral sodium channel blockers for non-addictive pain treatment NATURE REVIEWS NEUROLOGY Alsaloum, M., Higerd, G. P., Effraim, P. R., Waxman, S. G. 2020; 16 (12): 689-705

  Abstract

  The effective and safe treatment of pain is an unmet health-care need. Current medications used for pain management are often only partially effective, carry dose-limiting adverse effects and are potentially addictive, highlighting the need for improved therapeutic agents. Most common pain conditions originate in the periphery, where dorsal root ganglion and trigeminal ganglion neurons feed pain information into the CNS. Voltage-gated sodium (NaV) channels drive neuronal excitability and three subtypes - NaV1.7, NaV1.8 and NaV1.9 - are preferentially expressed in the peripheral nervous system, suggesting that their inhibition might treat pain while avoiding central and cardiac adverse effects. Genetic and functional studies of human pain disorders have identified NaV1.7, NaV1.8 and NaV1.9 as mediators of pain and validated them as targets for pain treatment. Consequently, multiple NaV1.7-specific and NaV1.8-specific blockers have undergone clinical trials, with others in preclinical development, and the targeting of NaV1.9, although hampered by technical constraints, might also be moving ahead. In this Review, we summarize the clinical and preclinical literature describing compounds that target peripheral NaV channels and discuss the challenges and future prospects for the field. Although the potential of peripheral NaV channel inhibition for the treatment of pain has yet to be realized, this remains a promising strategy to achieve non-addictive analgesia for multiple pain conditions.

  View details for DOI 10.1038/s41582-020-00415-2

  View details for Web of Science ID 000584595100001

  View details for PubMedID 33110213

  View details for PubMedCentralID 4932255

• Building sensory axons: Delivery and distribution of Na_v1.7 channels and effects of inflammatory mediators SCIENCE ADVANCES Akin, E. J., Higerd, G. P., Mis, M. A., Tanaka, B. S., Adi, T., Liu, S., Dib-Hajj, F. B., Waxman, S. G., Dib-Hajj, S. D. 2019; 5 (10): eaax4755

  Abstract

  Sodium channel NaV1.7 controls firing of nociceptors, and its role in human pain has been validated by genetic and functional studies. However, little is known about NaV1.7 trafficking or membrane distribution along sensory axons, which can be a meter or more in length. We show here with single-molecule resolution the first live visualization of NaV1.7 channels in dorsal root ganglia neurons, including long-distance microtubule-dependent vesicular transport in Rab6A-containing vesicles. We demonstrate nanoclusters that contain a median of 12.5 channels at the plasma membrane on axon termini. We also demonstrate that inflammatory mediators trigger an increase in the number of NaV1.7-carrying vesicles per axon, a threefold increase in the median number of NaV1.7 channels per vesicle and a ~50% increase in forward velocity. This remarkable enhancement of NaV1.7 vesicular trafficking and surface delivery under conditions that mimic a disease state provides new insights into the contribution of NaV1.7 to inflammatory pain.

  View details for DOI 10.1126/sciadv.aax4755

  View details for Web of Science ID 000493059800018

  View details for PubMedID 31681845

  View details for PubMedCentralID PMC6810356