Voltage-gated sodium (Na_V) channels are critical molecular determinants of action potential generation and propagation in excitable cells. Normal Na_V channel function disruption can affect physiological neuronal signaling and lead to increased sensitivity to pain, congenital indifference to pain, uncoordinated movement, seizures, or paralysis. Human genetic studies have identified human Na_V1.7 (hNa_V1.7), hNa_V1.8, and hNa_V1.9 channel subtypes as crucial players in pain signaling. The premise that subtype selective Na_V inhibitors can reduce pain has been reinforced through intensive target validation and therapeutic development efforts. However, an ideal therapeutic has yet to emerge. This review is focused on recent progress, current challenges, and future opportunities to develop Na_V channel targeting small molecules and peptides as non-addictive therapeutics to treat pain.

The human voltage-gated sodium channel, hNa_V1.5, is responsible for the rapid upstroke of the cardiac action potential and is target for antiarrhythmic therapy. Despite the clinical relevance of hNa_V1.5-targeting drugs, structure-based molecular mechanisms of promising or problematic drugs have not been investigated at atomic scale to inform drug design. Here, we used Rosetta structural modeling and docking as well as molecular dynamics simulations to study the interactions of antiarrhythmic and local anesthetic drugs with hNa_V1.5. These calculations revealed several key drug binding sites formed within the pore lumen that can simultaneously accommodate up to two drug molecules. Molecular dynamics simulations identified a hydrophilic access pathway through the intracellular gate and a hydrophobic access pathway through a fenestration between DIII and DIV. Our results advance the understanding of molecular mechanisms of antiarrhythmic and local anesthetic drug interactions with hNa_V1.5 and will be useful for rational design of novel therapeutics.

Capsaicin receptor TRPV1 is a nociceptor for vanilloid molecules, such as capsaicin and resiniferatoxin (RTX). Even though cryo-EM structures of TRPV1 in complex with these molecules are available, how their binding energetically favors the open conformation is not known. Here, we report an approach to control the number of bound RTX molecules (0-4) in functional rat TRPV1. The approach allowed direct measurements of each of the intermediate open states under equilibrium conditions at both macroscopic and single-molecule levels. We found that RTX binding to each of the four subunits contributes virtually the same activation energy, which we estimated to be 1.70 to 1.86 kcal/mol and found to arise predominately from destabilizing the closed conformation. We further showed that sequential bindings of RTX increase open probability without altering single-channel conductance, confirming that there is likely a single open-pore conformation for TRPV1 activated by RTX.

The voltage-gated sodium (Na_V) channel subtype Na_V1.7 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 Na_V1.7 channel (hNa_V1.7). We developed a structural model of hNa_V1.7 using Rosetta computational modeling and performed in silico docking of KIIIA using RosettaDock to predict residues forming specific pairwise contacts between KIIIA and hNa_V1.7. We experimentally validated these contacts using mutant cycle analysis. Comparison between our KIIIA-hNa_V1.7 model and the cryo-EM structure of KIIIA-hNa_V1.2 revealed key similarities and differences between Na_V 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 Na_V channels.

Ion channels are critical drug targets for a range of pathologies, such as epilepsy, pain, itch, autoimmunity, and cardiac arrhythmias. To develop effective and safe therapeutics, it is necessary to design small molecules with high potency and selectivity for specific ion channel subtypes. There has been increasing implementation of structure-guided drug design for the development of small molecules targeting ion channels. We evaluated the performance of two RosettaLigand docking methods, RosettaLigand and GALigandDock, on the structures of known ligand-cation channel complexes. Ligands were docked to voltage-gated sodium (Na_V), voltage-gated calcium (Ca_V), and transient receptor potential vanilloid (TRPV) channel families. For each test case, RosettaLigand and GALigandDock methods frequently sampled a ligand-binding pose within a root mean square deviation (RMSD) of 1-2 Å relative to the experimental ligand coordinates. However, RosettaLigand and GALigandDock scoring functions cannot consistently identify experimental ligand coordinates as top-scoring models. Our study reveals that the proper scoring criteria for RosettaLigand and GALigandDock modeling of ligand-ion channel complexes should be assessed on a case-by-case basis using sufficient ligand and receptor interface sampling, knowledge about state-specific interactions of the ion channel, and inherent receptor site flexibility that could influence ligand binding.

Background

To the authors' knowledge, there are no other published clinical studies that have employed either systemic or local biologic response modifiers in the treatment of patients with pancreatic carcinoma. The purpose of this study was to determine the feasibility and safety of allogeneic mixed lymphocyte culture (cytoimplant) delivered by endoscopic ultrasound (EUS)-guided fine-needle injection (FNI) in patients with advanced pancreatic carcinoma.

Methods

Eight patients with unresectable adenocarcinoma of the pancreas were enrolled: 4 patients in Stage II, 3 in Stage III, and 1 in Stage IV. Cytoimplants were delivered locally into the tumor using a novel EUS-guided FNI technique. Escalating doses of 3, 6, or 9 billion cells were implanted into the pancreatic tumor by a single EUS-guided FNI. Toxicity (modified National Cancer Institute criteria) was assessed at Day 1, Week 1, and Months 1 and 3. Clinical endpoints included Karnofsky performance status (KPS), CA 19-9, tumor response (computed tomography and/or EUS), and survival with follow-up examinations and imaging tests on months 3, 6, 9, 12, and 24.

Results

There were no bone marrow, hemorrhagic, infectious, renal, cardiac, or pulmonary toxicities. There were 3 transient Grade 3 gastrointestinal toxicities, and 3 patients had transient episodes of hyperbilirubinemia that were reversed by replacement of biliary stents. Seven of 8 patients (86%) experienced low grade fever that responded to acetaminophen, and all fever was resolved within the first 4 weeks. There were no procedure-related complications. There were 2 partial responses and 1 minor response, with a median survival of 13.2 months.

Conclusions

A single injection of cytoimplant immunotherapy by EUS-guided FNI appears to be feasible and is not associated with substantial toxicity.

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gα_t), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gα_t the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s^-1 Analyzing swelling as osmotically driven water influx, we find the H₂O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10^-5 cm⋅s^-1, comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H₂O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

Dominant mutations in voltage-gated sodium channel Na_V1.7 cause inherited erythromelalgia, a debilitating pain disorder characterized by severe burning pain and redness of the distal extremities. Na_V1.7 is preferentially expressed within peripheral sensory and sympathetic neurons. Here, we describe a novel Na_V1.7 mutation in an 11-year-old male with underdevelopment of the limbs, recurrent attacks of burning pain with erythema, and swelling in his feet and hands. Frequency and duration of the episodes gradually increased with age, and relief by cooling became less effective. The patient's sister had short stature and reported similar complaints of erythema and burning pain, but with less intensity. Genetic analysis revealed a novel missense mutation in Na_V1.7 (2567G>C; p.Gly856Arg) in both siblings. The G856R mutation, located within the DII/S4-S5 linker of the channel, substitutes a highly conserved non-polar glycine by a positively charged arginine. Voltage-clamp analysis of G856R currents revealed that the mutation hyperpolarized (-11.2 mV) voltage dependence of activation and slowed deactivation but did not affect fast inactivation, compared with wild-type channels. A mutation of Gly-856 to aspartic acid was previously found in a family with limb pain and limb underdevelopment, and its functional assessment showed hyperpolarized activation, depolarized fast inactivation, and increased ramp current. Structural modeling using the Rosetta computational modeling suite provided structural clues to the divergent effects of the substitution of Gly-856 by arginine and aspartic acid. Although the proexcitatory changes in gating properties of G856R contribute to the pathophysiology of inherited erythromelalgia, the link to limb underdevelopment is not well understood.

Transient receptor potential vanilloid 2 (TRPV2) is involved in many critical physiological and pathophysiological processes, making it a promising drug target. Here we present cryo-electron microscopy (cryo-EM) structures of rat TRPV2 in lipid nanodiscs activated by 2-aminoethoxydiphenyl borate (2-APB) and propose a TRPV2-specific 2-ABP binding site at the interface of S5 of one monomer and the S4-S5 linker of the adjacent monomer. In silico docking and electrophysiological studies confirm the key role of His521 and Arg539 in 2-APB activation of TRPV2. Additionally, electrophysiological experiments show that the combination of 2-APB and cannabidiol has a synergetic effect on TRPV2 activation, and cryo-EM structures demonstrate that both drugs were able to bind simultaneously. Together, our cryo-EM structures represent multiple functional states of the channel, providing a native picture of TRPV2 activation by small molecules and a structural framework for the development of TRPV2-specific activators.