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γ-aminobutyric acid or GABA is an amino acid that functionally acts as a neurotransmitter and is critical to neurotransmission. GABA is also a metabolite in the Krebs cycle. It is therefore unsurprising that GABA and its receptors are also present outside of the central nervous system, including in immune cells. This observation suggests that GABAergic signaling impacts events beyond brain function and possibly human health beyond neurological disorders. Indeed, GABA receptor subunits are expressed in pathological disease states, including in disparate cancers. The role that GABA and its receptors may play in cancer development and progression remains unclear. If, however, those cancers have functional GABA receptors that participate in GABAergic signaling, it raises an important question whether these signaling pathways might be targetable for therapeutic benefit. Herein we summarize the effects of modulating Type-A GABA receptor signaling in various cancers and highlight how Type-A GABA receptors could emerge as a novel therapeutic target in cancer.

Competent antitumor immune cells are fundamental for tumor surveillance and combating active cancers. Once established, tumors generate a tumor microenvironment (TME) consisting of complex cellular and metabolic elements that serve to suppress the function of antitumor immune cells. T lymphocytes are key cellular elements of the TME. In this review, we explore the role of ion channels, particularly K+ channels, in mediating the suppressive effects of the TME on T cells. First, we will review the complex network of ion channels that mediate Ca2+ influx and control effector functions in T cells. Then, we will discuss how multiple features of the TME influence the antitumor capabilities of T cells via ion channels. We will focus on hypoxia, adenosine, and ionic imbalances in the TME, as well as overexpression of programmed cell death ligand 1 by cancer cells that either suppress K+ channels in T cells and/or benefit from regulating these channels’ activity, ultimately shaping the immune response. Finally, we will review some of the cancer treatment implications related to ion channels. A better understanding of the effects of the TME on ion channels in T lymphocytes could promote the development of more effective immunotherapies, especially for resistant solid malignancies.

Plasmalogens (Pls) are a class of membrane phospholipids which serve a number of essential biological functions. Deficiency of Pls is associated with common disorders such as Alzheimer's disease or ischemic heart disease. A complete lack of Pls due to genetically determined defective biosynthesis gives rise to rhizomelic chondrodysplasia punctata (RCDP), characterized by a number of severe disabling pathologic features and death in early childhood. Frequent cardiac manifestations of RCDP include septal defects, mitral valve prolapse, and patent ductus arteriosus. In a mouse model of RCDP, reduced nerve conduction velocity was partially rescued by dietary oral supplementation of the Pls precursor batyl alcohol (BA). Here, we examine the impact of Pls deficiency on cardiac impulse conduction in a similar mouse model (Gnpat KO). In‐vivo electrocardiographic recordings showed that the duration of the QRS complex was significantly longer in Gnpat KO mice than in age‐ and sex‐matched wild‐type animals, indicative of reduced cardiac conduction velocity. Oral supplementation of BA for 2 months resulted in normalization of cardiac Pls levels and of the QRS duration in Gnpat KO mice but not in untreated animals. BA treatment had no effect on the QRS duration in age‐matched wild‐type mice. These data suggest that Pls deficiency is associated with increased ventricular conduction time which can be rescued by oral BA supplementation.

Glioblastoma multiforme (GBM) is a highly malignant primary brain tumor. The current standard of care for GBM is the Stupp protocol which includes surgical resection, followed by radiotherapy concomitant with the DNA alkylator temozolomide; however, survival under this treatment regimen is an abysmal 12–18 months. New and emerging treatments include the application of a physical device, non-invasive ‘tumor treating fields’ (TTFs), including its concomitant use with standard of care; and varied vaccines and immunotherapeutics being trialed. Some of these approaches have extended life by a few months over standard of care, but in some cases are only available for a minority of GBM patients. Extensive activity is also underway to repurpose and reposition therapeutics for GBM, either alone or in combination with the standard of care. In this review, we present select molecules that target different pathways and are at various stages of clinical translation as case studies to illustrate the rationale for their repurposing-repositioning and potential clinical use.

Severe COVID-19 is characterized by cytokine storm, an excessive production of pro-inflammatory cytokines, which results in acute lung damage and death. Dexamethasone (Dex), a corticosteroid administered as a standard of care for severe COVID-19, has been shown to reduce the onset of severe complications in these patients. However, the mechanisms underlying the beneficial effects of Dex are poorly understood. We conducted a transcriptomic analysis (NanoString) of peripheral blood mononuclear cells (PBMCs) from COVID-19 patients with mild (no hospitalization) and severe (on ventilator in the ICU) disease. The latter group included patients ± Dex. Treatment of severe COVID-19 patients with Dex inhibited pro-inflammatory pathways, circulating cytotoxic and Th1 cells, interferon (IFN) signaling and the genes involved in cytokine storm (IL-1, IL-6 and IFN-γ), which were altered in severe vs mild disease. Pro-inflammatory immune cell functions are regulated by calcium signaling, which in turn is under the control of Kv1.3 potassium channels, but their role in the complications associated with COVID-19 remains elusive. RT-qPCR experiments in PBMCs from COVID-19 patient cohorts showed an increase in Kv1.3 expression in severe patients that was significantly reduced by Dex treatment. In agreement with these findings, in vitro treatment with Dex reduced Kv1.3 protein abundance in CD3+, CD4+, CD8+ T cell and CD56dim NK cells in healthy donor (HD) PBMC, while NanoString showed inhibition of pro-inflammatory genes and cytokines. Functional studies showed that Dex significantly reduced Kv1.3 activity and IFN-γ in HD T cells. Thus, our findings suggest that Kv1.3 inhibition contributes to the immunosuppression by Dex. This work was supported by Senior Pilot Grant, University of Cincinnati, Department of Internal Medicine to LC, NIH (R38HL155775 to MC) and Biospecimens and associated data were provided by the Cincinnati COVID-19 Repository at the University of Cincinnati and Cincinnati Children’s Hospital Medical Center.

Background Immunotherapy has emerged as a promising treatment modality for head and neck squamous cell carcinoma (HNSCC). Pembrolizumab, an anti-programmed death 1 antibody, is an immunotherapy agent currently approved for metastatic HNSCC and curative intent clinical trials. Although clinical responses to pembrolizumab are promising, many patients fail to respond. However, it is well known that T cell cytotoxicity and chemotaxis are critically important in the elimination of HNSCC tumors. These functions depend on ion channel activity and downstream Ca2+ fluxing abilities, which are defective in patients with HNSCC. The purpose of this study was to elucidate the effects of pembrolizumab on potassium (K+) channel (KCa3.1 and Kv1.3) activity, Ca2+ fluxes, and chemotaxis in the cytotoxic T cells of patients with HNSCC and to determine their correlation with treatment response. Methods Functional studies were conducted in CD8+ peripheral blood T cells (PBTs) and tumor infiltrating lymphocytes (TILs) from patients with HNSCC treated with pembrolizumab. Untreated patients with HNSCC were used as controls. The ion channel activity of CD8+ T cells was measured by patch-clamp electrophysiology; single-cell Ca2+ fluxing abilities were measured by live microscopy. Chemotaxis experiments were conducted in a three-dimensional collagen matrix. Pembrolizumab patients were stratified as responders or non-responders based on pathological response (percent of viable tumor remaining at resection; responders: ≤80% viable tumor; non-responders: >80% viable tumor). Results Pembrolizumab increased K+ channel activity and Ca2+ fluxes in TILs independently of treatment response. However, in PBTs from responder patients there was an increased KCa3.1 activity immediately after pembrolizumab treatment that was accompanied by a characteristic increase in Kv1.3 and Ca2+ fluxes as compared with PBTs from non-responder patients. The effects on Kv1.3 and Ca2+ were prolonged and persisted after tumor resection. Chemotaxis was also improved in responder patients’ PBTs. Unlike non-responders’ PBTs, pembrolizumab increased their ability to chemotax in a tumor-like, adenosine-rich microenvironment immediately after treatment, and additionally they maintained an efficient chemotaxis after tumor resection. Conclusions Pembrolizumab enhanced K+ channel activity, Ca2+ fluxes and chemotaxis of CD8+ T cells in patients with HNSCC, with a unique pattern of response in responder patients that is conducive to the heightened functionality of their cytotoxic T cells.

BackgroundImmunotherapy has emerged as a promising treatment modality for head and neck squamous cell carcinoma (HNSCC). Pembrolizumab, an anti-programmed death 1 antibody, is an immunotherapy agent currently approved for metastatic HNSCC and curative intent clinical trials. Although clinical responses to pembrolizumab are promising, many patients fail to respond. However, it is well known that T cell cytotoxicity and chemotaxis are critically important in the elimination of HNSCC tumors. These functions depend on ion channel activity and downstream Ca2+fluxing abilities, which are defective in patients with HNSCC. The purpose of this study was to elucidate the effects of pembrolizumab on potassium (K+) channel (KCa3.1 and Kv1.3) activity, Ca2+fluxes, and chemotaxis in the cytotoxic T cells of patients with HNSCC and to determine their correlation with treatment response.MethodsFunctional studies were conducted in CD8+peripheral blood T cells (PBTs) and tumor infiltrating lymphocytes (TILs) from patients with HNSCC treated with pembrolizumab. Untreated patients with HNSCC were used as controls. The ion channel activity of CD8+T cells was measured by patch-clamp electrophysiology; single-cell Ca2+fluxing abilities were measured by live microscopy. Chemotaxis experiments were conducted in a three-dimensional collagen matrix. Pembrolizumab patients were stratified as responders or non-responders based on pathological response (percent of viable tumor remaining at resection; responders: ≤80% viable tumor; non-responders: >80% viable tumor).ResultsPembrolizumab increased K+channel activity and Ca2+fluxes in TILs independently of treatment response. However, in PBTs from responder patients there was an increased KCa3.1 activity immediately after pembrolizumab treatment that was accompanied by a characteristic increase in Kv1.3 and Ca2+fluxes as compared with PBTs from non-responder patients. The effects on Kv1.3 and Ca2+were prolonged and persisted after tumor resection. Chemotaxis was also improved in responder patients’ PBTs. Unlike non-responders’ PBTs, pembrolizumab increased their ability to chemotax in a tumor-like, adenosine-rich microenvironment immediately after treatment, and additionally they maintained an efficient chemotaxis after tumor resection.ConclusionsPembrolizumab enhanced K+channel activity, Ca2+fluxes and chemotaxis of CD8+T cells in patients with HNSCC, with a unique pattern of response in responder patients that is conducive to the heightened functionality of their cytotoxic T cells.

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein, we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore, the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFNγ production, also Ca2+- and CaM-dependent, was instead not reduced in HNSCC T cells, which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity, but not IFNγ production, and reduced their chemotaxis in the presence of adenosine, thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally, 1-EBIO enhanced INFγ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore, they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers.

The lupin alkaloid sparteine is a well-known chiral diamine with a range of applications in asymmetric synthesis, as well as a blocker of voltage-gated sodium channels (VGSCs). However, there is only scarce information on the VGSC-blocking activity of sparteine derivatives where the structure of the parent alkaloid is retained. Building on the recent renewed availability of sparteine and derivatives we report herein how modification of sparteine at position 2 produces irreversible blockers of VGSCs. These compounds could be clinically envisaged as long-lasting local anesthetics.

The limited ability of cytotoxic CD8+ T cells to infiltrate solid tumors and function within the tumor microenvironment presents a major roadblock to effective immunotherapy. Ion channels and Ca2+-dependent signaling events control the activity of T cells and are implicated in the failure of immune surveillance in cancer. Reduced KCa3.1 channel activity mediates the heightened inhibitory effect of adenosine on the chemotaxis of circulating T cells from head and neck squamous cell carcinoma (HNSCC) patients. Herein, we conducted experiments that elucidate the mechanisms of KCa3.1 dysfunction and impaired chemotaxis in HNSCC CD8+ T cells. The Ca2+ sensor calmodulin (CaM) controls multiple cellular functions including KCa3.1 activation. Our data showed that CaM expression is lower in HNSCC than healthy donor (HD) T cells. This reduction was due to an intrinsic decrease in the genes encoding CaM combined to the failure of HNSCC T cells to upregulate CaM upon activation. Furthermore, the reduction in CaM was confined to the plasma membrane and resulted in decreased CaM-KCa3.1 association and KCa3.1 activity (which was rescued by the delivery of CaM). IFNγ production, also Ca2+- and CaM-dependent, was instead not reduced in HNSCC T cells, which maintained intact cytoplasmic CaM and Ca2+ fluxing ability. Knockdown of CaM in HD T cells decreased KCa3.1 activity, but not IFNγ production, and reduced their chemotaxis in the presence of adenosine, thus recapitulating HNSCC T cell dysfunction. Activation of KCa3.1 with 1-EBIO restored the ability of CaM knockdown HD T cells to chemotax in the presence of adenosine. Additionally, 1-EBIO enhanced INFγ production. Our data showed a localized downregulation of membrane-proximal CaM that suppressed KCa3.1 activity in HNSCC circulating T cells and limited their ability to infiltrate adenosine-rich tumor-like microenvironments. Furthermore, they indicate that KCa3.1 activators could be used as positive CD8+ T cell modulators in cancers.

Medulloblastoma (MB) is the most common malignant (WHO Grade IV) primary brain cancer in children, adolescents, and young adults. Radiotherapy (RT) is a mainstay of MB treatment, as it is for most childhood and adult cancers. RT dose and frequency needed to achieve efficacy in MB patients severely impacts survival outcomes and is the cause of long-term cognitive deficits. To improve on short-term side effects and long-term complications, scanning beam proton therapy is employed, when available. While this recent technological advance significantly reduces damage to surrounding healthy brain tissue, survivors continue to experience induced radiation damage, including neurocognitive sequelae. To impact survivors’ health-related quality of life and caregivers’ emotional and financial burden, it is critical to identify approaches that reduce RT dose to mitigate side-effects without impacting RT effectiveness. We are investigating targeting a unique MB electrochemical vulnerability as a means to sensitize MB tumor cells to RT. There are four MB molecular subgroups: wingless, sonic-hedgehog, Group 3, and Group 4. Our analysis of 763 MB tumor transcriptomes reveals that all Group 3 MB tumors share an enhanced expression of genes-coding for subunits of the Type-A GABA receptor (GABAAR), a chloride channel. Using patch-clamp electrophysiology, we found that GABAARs conduct Cl− in MB cells and that a brain-penetrant benzodiazepine (BZ) enhances this effect and triggers cytotoxic responses commensurate with mitochondrial depolarization. We find that BZ combined with RT, even at a sub-lethal dose, is highly effective in impairing the viability of MB tumor cells, greater than RT alone. Our BZis capable of penetrating the blood-brain barrier in minutes, is metabolically stable, and showed no toxicity in a primate model. We are investigating its suitability to be used concomitant with proton beam radiotherapy, replacing standard of care vincristine, to reduce radiation-induced brain toxicity experienced by MB patients and survivors while not decreasing RT effectiveness. Citation Format: Daniel Pomeranz Krummel, Laura Kallay, Debanjan Bhattacharya, Vaibhavkumar Gawali, Kamdem T. Donatien, Taukir Ahmed, James M. Cook, Michael Lamba, Susanne Wells, Ralph E. Vatner, Mathieu Sertorio, Dan T. Ionascu, Soma Sengupta. Targeting a unique electrochemical vulnerability in a pediatric brain tumor to potentiate proton beam radiotherapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr PO-009.

Head and neck cancer (HNC) is the sixth most common cancer worldwide with a five-year survival of 50%. Immunotherapy is emerging as a promising treatment modality in HNC. Pembrolizumab, an anti-PD-1 antibody, is an immunotherapy currently approved in metastatic HNC and in clinical trials for curative intent. Although there are promising responses to Pembrolizumab, its effect on cytotoxic CD8+ T cell function is not understood. T cell cytotoxicity is critically important in the elimination of tumors and in the tumor microenvironment, cytotoxicity depends on ion channel activity which is defective in HNC patients. Therefore, to elucidate mechanisms of Pembrolizumab, we studied potassium channel (KCa3.1 and Kv1.3) functionality and Ca2+ fluxes in CD8+ peripheral blood T cells (PBTs) of naïve HNC patients before/after Pembrolizumab treatment (n=22) as well as tumor infiltrating lymphocytes (TILs) functionality. Untreated HNC patients were controls (n= 13). Pembrolizumab-treated patients were categorized as responders (R) or non-responders (NR) based on pathological response. We then performed electrophysiological and Ca2+ flux experiments. We observed that Pembrolizumab increased KCa3.1 currents in PBTs (29.8%, p=0.026). Moreover, after treatment, R PBTs also had increases in Kv1.3 currents and Ca2+ fluxes (22.1%, p=0.047; 16.5%, p

The clinically important suppression of high-frequency discharges of excitable cells by local anesthetics (LA) is largely determined by drug-induced prolongation of the time course of repriming (recovery from inactivation) of voltage-gated Na(+) channels. This prolongation may result from periodic drug-binding to a high-affinity binding site during the action potentials and subsequent slow dissociation from the site between action potentials ("dissociation hypothesis"). For many drugs it has been suggested that the fast inactivated state represents the high-affinity binding state. Alternatively, LAs may bind with high affinity to a native slow-inactivated state, thereby accelerating the development of this state during action potentials ("stabilization hypothesis"). In this case, slow recovery between action potentials occurs from enhanced native slow inactivation. To test these two hypotheses we produced serial cysteine mutations of domain IV segment 6 in rNav1.4 that resulted in constructs with varying propensities to enter fast- and slow-inactivated states. We tested the effect of the LA lidocaine on the time course of recovery from short and long depolarizing prepulses, which, under drug-free conditions, recruited mainly fast- and slow-inactivated states, respectively. Among the tested constructs the mutation-induced changes in native slow recovery induced by long depolarizations were not correlated with the respective lidocaine-induced slow recovery after short depolarizations. On the other hand, for long depolarizations the mutation-induced alterations in native slow recovery were significantly correlated with the kinetics of lidocaine-induced slow recovery. These results favor the "dissociation hypothesis" for short depolarizations but the "stabilization hypothesis" for long depolarizations.

The aim of present study was to evaluate effect of lyophilized extract of Tridax procumbens Linn. in rodent models of inflammatory and neuropathic pain. The antiallodynic and antihyperalgesic activity of Tridax procumbens was assessed in Chronic Constriction Injury of Sciatic Nerve in rats (CCI) and Scald pain model in rats (burning pain) respectively at 100,200 and 400 mg/kg, p.o. In CCI model, the extract shows the dose dependent antiallodynic activity 60 min. post dose. In Scald pain model, extract shows the significant antihyperalgesic activity at 200 and 400 mg/kg at 60 and 90 min. post dose. The lyophilized extract of Tridax procumbens was found to be effective in neuropathic and inflammatory pain, suggesting its possible action via peripheral mechanisms. The activity may be attributed to the presence of flavonoids such as Quercetin.

Voltage-gated Na(+) channels (VGSCs) initiate action potentials thereby giving rise to rapid transmission of electrical signals along cell membranes and between cells. Depolarization of the cell membrane causes VGSCs to open but also gives rise to a nonconducting state termed inactivation. Inactivation of VGSCs serves a critical physiologic function as it determines the extent of excitability of neurons and of muscle cells. Depending on the time course of development and removal of inactivation both "fast-" and "slow"-inactivated states have been described. Evidence from mutagenesis studies suggests that fast inactivation is produced by a block of the internal vestibule by a tethered inactivation particle that has been mapped to the internal linker between domains III and IV. The motion of this linker may be regulated by parts of the internal C-terminus. The molecular mechanism of slow inactivation is less clear. However, aside from a high number of mutagenesis studies, the recent availability of 3D structures of crystallized prokaryotic VGSCs offers insights into the molecular motions associated with slow inactivation. One possible scenario is that slow movements of the voltage sensors are transmitted to the external vestibule giving rise to a conformational change of this region. This molecular rearrangement is transmitted to the S6 segments giving rise to collapse of the internal vestibule.

The limited ability of the cytotoxic CD8+ T cells to infiltrate solid tumors presents a major roadblock to developing effective immunotherapy. Adenosine (Ado) accumulates in high concentrations in solid tumors where it contributes to the suppression of T cell function. We have previously shown that Ado reduces the chemotactic ability of peripheral blood CD8+ T cells (PBTs) from head and neck squamous cell carcinoma (HNSCC) patients by suppressing KCa3.1 channel function. Herein, we conducted experiments to elucidate the mechanism of KCa3.1 dysregulation in HNSCC PBTs. KCa3.1 channels are calcium-activated and require binding of calmodulin (CAM). PBTs were isolated from HNSCC patients and healthy donors (HD), and activated with CD3/CD28 antibodies. CAM levels decreased post-activation in HNSCC PBTs (by ~24%, n=7) while they increased in HD PBTs (by ~37%, n=6, p=0.001). To study whether CAM downregulation contributes to KCa3.1 dysfunction, we transfected HD PBTs with siRNA against CAM (siCam). siCam transfection decreased CAM expression and KCa3.1 currents, but not KCa3.1 expression. We then studied whether CAM downregulation affects the T cell chemotactic response to Ado in HD PBTs. Control (scrRNA-transfected) HD PBTs migrated towards CXCL12 in the presence of Ado, but downregulation of CAM abrogated their chemotactic ability in the presence of Ado (~71% inhibition, n=8, p

The cardiac late Na+ current is generated by a small fraction of voltage-dependent Na+ channels that undergo a conformational change to a burst-gating mode, with repeated openings and closures during the action potential (AP) plateau. Its magnitude can be augmented by inactivation-defective mutations, myocardial ischemia, or prolonged exposure to chemical compounds leading to drug-induced (di)-long QT syndrome, and results in an increased susceptibility to cardiac arrhythmias. Using CytoPatch™ 2 automated patch-clamp equipment, we performed whole-cell recordings in HEK293 cells stably expressing human Nav1.5, and measured the late Na+ component as average current over the last 100 ms of 300 ms depolarizing pulses to -10 mV from a holding potential of -100 mV, with a repetition frequency of 0.33 Hz. Averaged values in different steady-state experimental conditions were further corrected by the subtraction of current average during the application of tetrodotoxin (TTX) 30 μM. We show that ranolazine at 10 and 30 μM in 3 min applications reduced the late Na+ current to 75.0 ± 2.7% (mean ± SEM, n = 17) and 58.4 ± 3.5% (n = 18) of initial levels, respectively, while a 5 min application of veratridine 1 μM resulted in a reversible current increase to 269.1 ± 16.1% (n = 28) of initial values. Using fluctuation analysis, we observed that ranolazine 30 μM decreased mean open probability p from 0.6 to 0.38 without modifying the number of active channels n, while veratridine 1 μM increased n 2.5-fold without changing p. In human iPSC-derived cardiomyocytes, veratridine 1 μM reversibly increased APD90 2.12 ± 0.41-fold (mean ± SEM, n = 6). This effect is attributable to inactivation removal in Nav1.5 channels, since significant inhibitory effects on hERG current were detected at higher concentrations in hERG-expressing HEK293 cells, with a 28.9 ± 6.0% inhibition (mean ± SD, n = 10) with 50 μM veratridine.

Despite the availability of several crystal structures of bacterial voltage-gated Na(+) channels, the structure of eukaryotic Na(+) channels is still undefined. We used predictions from available homology models and crystal structures to modulate an external access pathway for the membrane-impermeant local anesthetic derivative QX-222 into the internal vestibule of the mammalian rNaV1.4 channel. Potassium channel-based homology models predict amino acid Ile-1575 in domain IV segment 6 to be in close proximity to Lys-1237 of the domain III pore-loop selectivity filter. The mutation K1237E has been shown previously to increase the diameter of the selectivity filter. We found that an access pathway for external QX-222 created by mutations of Ile-1575 was abolished by the additional mutation K1237E, supporting the notion of a close spatial relationship between sites 1237 and 1575. Crystal structures of bacterial voltage-gated Na(+) channels predict that the side chain of rNaV1.4 Trp-1531 of the domain IV pore-loop projects into the space between domain IV segment 6 and domain III pore-loop and, therefore, should obstruct the putative external access pathway. Indeed, mutations W1531A and W1531G allowed for exceptionally rapid access of QX-222. In addition, W1531G created a second non-selective ion-conducting pore, bypassing the outer vestibule but probably merging into the internal vestibule, allowing for control by the activation gate. These data suggest a strong structural similarity between bacterial and eukaryotic voltage-gated Na(+) channels.

Local anaesthetics (LA) exert their action by prolongation of the time course of repriming of voltage-gated Na channels after repolarization of the action potential. This may result from slow drug-dissociation from fast inactivated states or, alternatively, from stabilization of a native slow inactivated state (IM, Chen ZH et al. J Physiol. 524:37). The C-terminal part of transmembrane S6 segment of domain IV (DIV-S6) is an important determinant of inactivation gating and LA block. We performed serial cysteine scanning mutagenesis of positions 1575-1586 in DIV-S6 of rNav1.4 channels and investigated the relationship between changes in mutation-induced alterations of IM and respective changes in LA-induced prolongation of recovery. The constructs were expressed in TsA201 cells and studied by means of whole-cell patch-clamp technique. We examined the time course of recovery from fast and slow inactivation produced by 50 ms and by 10 s conditioning pulses to −20 mV, respectively. The LA Lidocaine (500 µM; LIDO) significantly slowed recovery from fast inactivation in all tested constructs except F1579C. The time constant of LIDO - induced slow recovery was ∼ 150 ms. Under LIDO-free conditions a similar time constant of slow recovery was found most constructs following 10 s depolarization, presumably reflecting recovery from native IM. However, the fraction of channels recovering from IM (F- IM) varied greatly among the tested constructs. LIDO significantly increased F- IM in most constructs. Interestingly, there was a significant negative correlation between F-IM without LIDO and the LIDO-induced increase in F- IM(R2 = 0.91; P < 0.0001). Furthermore, in M1585C F- IM was 0 under LIDO-free conditions but increased to 0.73±0.02 with LIDO. The data suggest that in the tested constructs slow recovery with LIDO reflects slow dissociation from fast inactivation rather than recovery from IM. Funding support: Austrian Science Fund W1232-B11.

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.

The frequently used local anaesthetic lidocaine is believed to reach its binding site in the intracellular vestibule of the voltage-gated sodium channel (NaV)via the cell membrane. QX-222 is a permanently charged, quaternary amine analogue of lidocaine that can access this binding site via a hydrophilic route across the channel protein. This pathway exists in the wild-type heart sodium channel (NaV1.5). In addition, mutations at a site in the upper part of the S6 segment in domain IV have also been shown to open such an external access pathway (EAP; positions 1760 in rNaV1.2, 1575 in rNaV1.4; Ragsdale et al. Science 265:1724,Sunami et al. Mol. Pharmacol. 59:684).In the first crystal structure of a NaV (NaVAb, Payandeh et al. Nature 475:353) a tryptophan (W179, homologous to W1531 in rNav1.4) of the P-loop in domain IV is positioned in close proximity to I202 (homologous to I1575 of rNav1.4). Therefore, we tested the hypothesis that mutations at site W1531 may modulate the EAP.Whole-cell patch clamp measurements were performed on tsA201 cells transiently transfected with rNaV1.4 constructs. Development of block was assessed by by application of 25 ms pulses to 0 mV at 2 Hz stimulation frequency from a holding potential of −120 mV.QX-222 blocked currents through W1531A and W1531G by 21±3% and 15±2%, respectively. In both constructs block development was extremely fast (time constants: ∼3s and ∼2s for W1531A and W1531G), i.e. ∼10-20 fold more rapid than I1575A (∼40s). Thus, mutations at site 1531 open an access pathway allowing for rapid block by QX-222, as predicted from the crystal structure of NaVAb.Funded by Austrian Science Fund (FWF, P210006-B11 and W1232-B11).

Background The plant alkaloid ibogaine has shown promising antiaddictive properties in animals and humans. Although not licensed as a therapeutic drug, and despite evidence that ibogaine may disturb the rhythm of the heart, this alkaloid is used as an anti-addiction drug in alternative medicine. We have recently reported that therapeutic concentrations of ibogaine inhibit human ERG (hERG) potassium channels, and thereby uncovered a mechanism by which the drug may induce life-threatening cardiac arrhythmias.

Background Some blockers of voltage-gated Na and Ca channels are assumed to pass through the membrane and then bind to amino acids in the internal vestibule by access from the internal side of the membrane. However, in the heart isoform of the voltage-gated Na channel, in L-type calcium channels and in T-type calcium channels an additional external access pathway (EAP) through the protein has been suggested. Furthermore, in voltage-gated Na channels (NaV) mutations at a specific site in the middle of the domain IV transmembrane segment 6 (site 1575 in rNaV1.4, 1760 in rNaV1.4) open an EAP for QX-222, a permanently charged, hydrophilic lidocaine analogue. Recently, the first crystal structure of a NaV was published [1]. In this bacterial channel structure (NaVAb) the side chain homologous to rNaV1.4 I1575 (I202 in NaVAb) is in close contact with a pore-loop sidechain, homologous to rNaV1.4 W1531 (W179 in NaVAb). In contrast, in all currently available structural homology models of NaV, W1531 is not in contact with I1575. If W1531 were positioned as suggested in the NaVAb structure then a reduction in the length of the side chain at this site would be predicted to open the EAP. To test this hypothesis we generated the mutations W1531A and W1531G and tested these constructs for block by external QX-222.