Search

Animals requiring purposeful movement for survival are endowed with mechanoreceptors, called proprioceptors, that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we identified nonredundant roles for two voltage-gated sodium channels (Na V s), Na V 1.1 and Na V 1.6, in mammalian proprioception. Deletion of Na V 1.6 in somatosensory neurons (Na V 1.6 cKO mice) causes severe motor deficits accompanied by loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target Na V 1.1 (Na V 1.1 cKO ). In Na V 1.6 cKO animals, we observed impairments in proprioceptor end-organ structure and a marked reduction in skeletal muscle myofiber size that were absent in Na V 1.1 cKO mice. We attribute the differential contributions of Na V 1.1 and Na V 1.6 to distinct cellular localization patterns. Collectively, we provide evidence that Na V s uniquely shape neural signaling within a somatosensory modality.

Thermosensation requires the activation of a unique collection of ion channels and receptors that work in concert to transmit thermal information. It is widely accepted that transient receptor potential melastatin 8 (TRPM8) activation is required for normal cold sensing; however, recent studies have illuminated major roles for other ion channels in this important somatic sensation. In addition to TRPM8, other TRP channels have been reported to contribute to cold transduction mechanisms in diverse sensory neuron populations, with both leak- and voltage-gated channels being identified for their role in the transmission of cold signals. Whether the same channels that contribute to physiological cold sensing also mediate noxious cold signaling remains unclear; however, recent work has found a conserved role for the kainite receptor, GluK2, in noxious cold sensing across species. Additionally, cold-sensing neurons likely engage in functional crosstalk with nociceptors to give rise to cold pain. This Review will provide an update on our understanding of the relationship between various ion channels in the transduction and transmission of cold and highlight areas where further investigation is required., (© 2024 Lewis and Griffith.)

Animals that require purposeful movement for survival are endowed with mechanosensory neurons called proprioceptors that provide essential sensory feedback from muscles and joints to spinal cord circuits, which modulates motor output. Despite the essential nature of proprioceptive signaling in daily life, the mechanisms governing proprioceptor activity are poorly understood. Here, we have identified distinct and nonredundant roles for two voltage-gated sodium channels (Na V s), Na V 1.1 and Na V 1.6, in mammalian proprioception. Deletion of Na V 1.6 in somatosensory neurons (Na V 1.6 cKO mice) causes severe motor deficits accompanied by complete loss of proprioceptive transmission, which contrasts with our previous findings using similar mouse models to target Na V 1.1 (Na V 1.1 cKO ). In Na V 1.6 cKO animals, loss of proprioceptive feedback caused non-cell-autonomous impairments in proprioceptor end-organs and skeletal muscle that were absent in Na V 1.1 cKO mice. We attribute the differential contribution of Na V 1.1 and Na V 1.6 in proprioceptor function to distinct cellular localization patterns. Collectively, these data provide the first evidence that Na V subtypes uniquely shape neurotransmission within a somatosensory modality., Competing Interests: Competing interests: All other authors declare they have no competing interests

The analysis of action potentials and other membrane voltage fluctuations provides a powerful approach for interrogating the function of excitable cells. However, a major bottleneck in the interpretation of this critical data is the lack of intuitive, agreed-upon software tools for its analysis. Here, we present SanPy, an open-source and freely available software package for the analysis and exploration of whole-cell current-clamp recordings written in Python. SanPy provides a robust computational engine with an application programming interface. Using this, we have developed a cross-platform desktop application with a graphical user interface that does not require programming. SanPy is designed to extract common parameters from action potentials, including threshold time and voltage, peak, half-width, and interval statistics. In addition, several cardiac parameters are measured, including the early diastolic duration and rate. SanPy is built to be fully extensible by providing a plugin architecture for the addition of new file loaders, analysis, and visualizations. A key feature of SanPy is its focus on quality control and data exploration. In the desktop interface, all plots of the data and analysis are linked, allowing simultaneous data visualization from different dimensions with the goal of obtaining ground-truth analysis. We provide documentation for all aspects of SanPy, including several use cases and examples. To test SanPy, we performed analysis on current-clamp recordings from heart and brain cells. Taken together, SanPy is a powerful tool for whole-cell current-clamp analysis and lays the foundation for future extension by the scientific community., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Thermosensation, the ability to detect and estimate temperature, is an evolutionarily conserved process that is essential for survival. Thermosensing is impaired in various pain syndromes, resulting in thermal allodynia, the perception of an innocuous temperature as painful, or thermal hyperalgesia, an exacerbated perception of a painful thermal stimulus. Several behavioral assays exist to study thermosensation and thermal pain in rodents, however, most rely on reflexive withdrawal responses or the subjective quantification of spontaneous nocifensive behaviors. Here, we created a new apparatus, the thermal escape box, which can be attached to temperature-controlled plates and used to assess temperature-dependent effort-based decision-making. The apparatus consists of a light chamber with an opening that fits around temperature-controlled plates, and a small entryway into a dark chamber. A mouse must choose to stay in a brightly lit aversive area or traverse the plates to escape to the enclosed dark chamber. We quantified escape latencies of adult C57Bl/6 mice at different plate temperatures from video recordings and found they were significantly longer at 5 °C, 18 °C, and 52 °C, compared to 30 °C, a mouse's preferred ambient temperature. Differences in escape latencies were abolished in male Trpm8 -/- mice and in male Trpv1 -/- animals. Finally, we show that chronic constriction injury procedures or oxaliplatin treatement significantly increased escape latencies at cold temperatures compared to controls, the later of which was prevented by the analgesic meloxicam. This demonstrates the utility of this assay in detecting cold pain. Collectively, our study has identified a new and effective tool that uses cost-benefit valuations to study thermosensation and thermal pain., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Theanne N. Griffith has assigned a provisional patent to the University of California related to the described behavioral assay. Other authors report no conflicts of interest., (© 2024 The Authors. Published by Elsevier Inc.)

The analysis of action potentials and other membrane voltage fluctuations provide a powerful approach for interrogating the function of excitable cells. Yet, a major bottleneck in the interpretation of this critical data is the lack of intuitive, agreed upon software tools for its analysis. Here, we present SanPy, a Python-based open-source and freely available software pipeline for the analysis and exploration of whole-cell current-clamp recordings. SanPy provides a robust computational engine with an application programming interface. Using this, we have developed a cross-platform graphical user interface that does not require programming. SanPy is designed to extract common parameters from action potentials including threshold time and voltage, peak, half-width, and interval statistics. In addition, several cardiac parameters are measured including the early diastolic duration and rate. SanPy is built to be fully extensible by providing a plugin architecture for the addition of new file loaders, analysis, and visualizations. A key feature of SanPy is its focus on quality control and data exploration. In the desktop interface, all plots of the data and analysis are linked allowing simultaneous data visualization from different dimensions with the goal of obtaining ground truth analysis. We provide documentation for all aspects of SanPy including several use cases and examples. To test SanPy, we have performed analysis on current-clamp recordings from heart and brain cells. Taken together, SanPy is a powerful tool for whole-cell current-clamp analysis and lays the foundation for future extension by the scientific community.

We are 52 Black scientists. Here, we establish the context of Juneteenth in STEMM and discuss the barriers Black scientists face, the struggles they endure, and the lack of recognition they receive. We review racism's history in science and provide institutional-level solutions to reduce the burdens on Black scientists., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The voltage-gated sodium Na V 1.7 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 Na V 1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human Na V 1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC 50 s of 7 nM and 4 nM for hNa V 1.7 and more than 1000-fold selectivity over human Na V 1.1, Na V 1.3, Na V 1.4, Na V 1.5, Na V 1.8, and Na V 1.9 channels. PTx2-3127 inhibits Na V 1.7 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 Na V 1.7 channels have transformative potential to define a new class of biologics to treat pain., Competing Interests: PN, HN, KW, BH, HW, VY is named inventor on a patent application entitled 'Peptides targeting sodium channels to treat pain' based on this research, filed by the University of California. (U.S. provisional application no. 63/358,684, filed July 6, 2022), RS, VS, PT, YC, ML, TG No competing interests declared, AT, RK, AG is affiliated with AnaBios Corporation. The author has no financial interests to declare, MP is affiliated with Ambiopharm Inc. The author has no financial interests to declare, JS Reviewing editor, eLife, (© 2022, Nguyen, Nguyen et al.)

The voltage-gated sodium channel (Na V ), Na V 1.1, is well-studied in the central nervous system; conversely, its contribution to peripheral sensory neuron function is more enigmatic. Here, we identify a new role for Na V 1.1 in mammalian proprioception. RNAscope analysis and in vitro patch-clamp recordings in genetically identified mouse proprioceptors show ubiquitous channel expression and significant contributions to intrinsic excitability. Notably, genetic deletion of Na V 1.1 in sensory neurons caused profound and visible motor coordination deficits in conditional knockout mice of both sexes, similar to conditional Piezo2-knockout animals, suggesting that this channel is a major contributor to sensory proprioceptive transmission. Ex vivo muscle afferent recordings from conditional knockout mice found that loss of Na V 1.1 leads to inconsistent and unreliable proprioceptor firing characterized by action potential failures during static muscle stretch; conversely, afferent responses to dynamic vibrations were unaffected. This suggests that while a combination of Piezo2 and other Na V isoforms is sufficient to elicit activity in response to transient stimuli, Na V 1.1 is required for transmission of receptor potentials generated during sustained muscle stretch. Impressively, recordings from afferents of heterozygous conditional knockout animals were similarly impaired, and heterozygous conditional knockout mice also exhibited motor behavioral deficits. Thus, Na V 1.1 haploinsufficiency in sensory neurons impairs both proprioceptor function and motor behaviors. Importantly, human patients harboring Na V 1.1 loss-of-function mutations often present with motor delays and ataxia; therefore, our data suggest that sensory neuron dysfunction contributes to the clinical manifestations of neurological disorders in which Na V 1.1 function is compromised. Collectively, we present the first evidence that Na V 1.1 is essential for mammalian proprioceptive signaling and behaviors., Competing Interests: CE, CL, SO, MD, SG, KW, DO, KW, TG No competing interests declared, (© 2022, Espino et al.)

Cold sensation is initiated in the periphery by a specialized population of cold-sensitive neurons, referred to as cold receptors, who transmit decreases in temperature with sub-degree resolution using a diverse assortment of ion channels and receptors. It is largely accepted that normal cold signaling is initiated through activation of transient receptor potential melastatin 8 (TRPM8) expressing neurons. Conversely, the mechanisms underlying cold-induced pain signaling are not as well defined. Interestingly, mounting evidence demonstrates functional interplay between cold signaling and other somatic sensations, such as itch and warmth; thus, cold-sensing pathways also engage in sensory crosstalk and population coding mechanisms. In this review, we will discuss recent advances in our understanding of cold sensation and address major gaps in knowledge that require more investigation., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Pain is a physiological response to bodily damage and serves as a warning of potential threat. Pain can also transform from an acute response to noxious stimuli to a chronic condition with notable emotional and psychological components that requires treatment. Indeed, the management of chronic pain is currently an important unmet societal need. Several reports have implicated the release of the neurotransmitter adenosine triphosphate (ATP) and subsequent activation of purinergic receptors in distinct pain etiologies. Purinergic receptors are broadly expressed in peripheral neurons and the spinal cord; thus, purinergic signaling in sensory neurons or in spinal circuits may be critical for pain processing. Nevertheless, an outstanding question remains: what are the mechanisms of ATP release that initiate nociceptive signaling? Connexin and pannexin channels are established conduits of ATP release and have been suggested to play important roles in a variety of pathologies, including several models of pain. As such, these large-pore channels represent a new and exciting putative pharmacological target for pain treatment. Herein, we will review the current evidence for a role of connexin and pannexin channels in ATP release during nociceptive signaling, such as neuropathic and inflammatory pain. Collectively, these studies provide compelling evidence for an important role of connexins and pannexins in pain processing., (© 2021. The Author(s).)

Small-diameter vesicular glutamate transporter 3-lineage (Vglut3 lineage ) dorsal root ganglion (DRG) neurons play an important role in mechanosensation and thermal hypersensitivity; however, little is known about their intrinsic electrical properties. We therefore set out to investigate mechanisms of excitability within this population. Calcium microfluorimetry analysis of male and female mouse DRG neurons demonstrated that the cooling compound menthol selectively activates a subset of Vglut3 lineage neurons. Whole-cell recordings showed that small-diameter Vglut3 lineage DRG neurons fire menthol-evoked action potentials and exhibited robust, transient receptor potential melastatin 8 (TRPM8)-dependent discharges at room temperature. This heightened excitability was confirmed by current-clamp and action potential phase-plot analyses, which showed menthol-sensitive Vglut3 lineage neurons to have more depolarized membrane potentials, lower firing thresholds, and higher evoked firing frequencies compared with menthol-insensitive Vglut3 lineage neurons. A biophysical analysis revealed voltage-gated sodium channel (Na V ) currents in menthol-sensitive Vglut3 lineage neurons were resistant to entry into slow inactivation compared with menthol-insensitive neurons. Multiplex in situ hybridization showed similar distributions of tetrodotoxin (TTX)-sensitive Na V transcripts between TRPM8-positive and -negative Vglut3 lineage neurons; however, Na V 1.8 transcripts, which encode TTX-resistant channels, were more prevalent in TRPM8-negative neurons. Conversely, pharmacological analyses identified distinct functional contributions of Na V subunits, with Na V 1.1 driving firing in menthol-sensitive neurons, whereas other small-diameter Vglut3 lineage neurons rely primarily on TTX-resistant Na V channels. Additionally, when Na V 1.1 channels were blocked, the remaining Na V current readily entered into slow inactivation in menthol-sensitive Vglut3 lineage neurons. Thus, these data demonstrate that TTX-sensitive Na V s drive action potential firing in menthol-sensitive sensory neurons and contribute to their heightened excitability. SIGNIFICANCE STATEMENT Somatosensory neurons encode various sensory modalities including thermoreception, mechanoreception, nociception, and itch. This report identifies a previously unknown requirement for tetrodotoxin-sensitive sodium channels in action potential firing in a discrete subpopulation of small-diameter sensory neurons that are activated by the cooling agent menthol. Together, our results provide a mechanistic understanding of factors that control intrinsic excitability in functionally distinct subsets of peripheral neurons. Furthermore, as menthol has been used for centuries as an analgesic and anti-pruritic, these findings support the viability of Na V 1.1 as a therapeutic target for sensory disorders., (Copyright © 2019 the authors.)

Epithelial-neuronal signaling is essential for sensory encoding in touch, itch, and nociception; however, little is known about the release mechanisms and neurotransmitter receptors through which skin cells govern neuronal excitability. Merkel cells are mechanosensory epidermal cells that have long been proposed to activate neuronal afferents through chemical synaptic transmission. We employed a set of classical criteria for chemical neurotransmission as a framework to test this hypothesis. RNA sequencing of adult mouse Merkel cells demonstrated that they express presynaptic molecules and biosynthetic machinery for adrenergic transmission. Moreover, live-cell imaging directly demonstrated that Merkel cells mediate activity- and VMAT-dependent release of fluorescent catecholamine neurotransmitter analogs. Touch-evoked firing in Merkel-cell afferents was inhibited either by pre-synaptic silencing of SNARE-mediated vesicle release from Merkel cells or by neuronal deletion of β 2 -adrenergic receptors. Together, these results identify both pre- and postsynaptic mechanisms through which Merkel cells excite mechanosensory afferents to encode gentle touch. VIDEO ABSTRACT., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Key Points: Kainate receptors (KARs) are ionotropic glutamate receptors (iGluRs) that modulate synaptic transmission and intrinsic neuronal excitability. KARs associate with the auxiliary proteins neuropilin- and tolloid-like 1 and 2 (Neto1 and Neto2), which act as allosteric modulators of receptor function impacting all biophysical properties of these receptors studied to date. M3-S2 linkers play a critical role in KAR gating; we found that individual residues in these linkers bidirectionally influence Neto2 modulation of KAR desensitization in an agonist specific manner. We also identify the D1 dimer interface as a novel site of Neto2 modulation and functionally correlate the actions of Neto2 modulation of desensitization with modulation of cation sensitivity. We identify these domains as determinants of Neto2 modulation. Thus, our work contributes to the understanding of auxiliary subunit modulation of KAR function and could aid the development of KAR-specific modulators to alter receptor function., Abstract: Kainate receptors (KARs) are important modulators of synaptic transmission and intrinsic neuronal excitability in the CNS. Their activity is shaped by the auxiliary proteins Neto1 and Neto2, which impact KAR gating in a receptor subunit- and Neto isoform-specific manner. The structural basis for Neto modulation of KAR gating is unknown. Here we identify the M3-S2 gating linker as a critical determinant contributing to Neto2 modulation of KARs. M3-S2 linkers control both the valence and magnitude of Neto2 modulation of homomeric GluK2 receptors. Furthermore, a single mutation in this domain abolishes Neto2 modulation of heteromeric receptor desensitization. Additionally, we found that cation sensitivity of KAR gating is altered by Neto2 association, suggesting that stability of the D1 dimer interface in the ligand-binding domain (LBD) is an important determinant of Neto2 actions. Moreover, modulation of cation sensitivity was eliminated by mutations in the M3-S2 linkers, thereby correlating the action of Neto2 at these structurally discrete sites on receptor subunits. These results demonstrate that the KAR M3-S2 linkers and LBD dimer interface are critical determinants for Neto2 modulation of receptor function and identify these domains as potential sites of action for the targeted development of KAR-specific modulators that alter the function of auxiliary proteins in native receptors., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

The ability of a neuron to transduce extracellular signals into long lasting changes in neuronal morphology is central to its normal function. Increasing evidence shows that coordinated regulation of synaptic and nuclear signaling in response to NMDA receptor activation is crucial for long term memory, synaptic tagging, and epigenetic signaling. Although mechanisms have been proposed for synapse-to-nuclear communication, it is unclear how signaling is coordinated at both subcompartments. Here, we show that activation of NMDA receptors induces the bi-directional and concomitant shuttling of the scaffold protein afadin from the cytosol to the nucleus and synapses. Activity-dependent afadin nuclear translocation peaked 2 h post-stimulation, was independent of protein synthesis, and occurred concurrently with dendritic spine remodeling. Moreover, activity-dependent afadin nuclear translocation coincides with phosphorylation of histone H3 at serine 10 (H3S10p), a marker of epigenetic modification. Critically, blocking afadin nuclear accumulation attenuated activity-dependent dendritic spine remodeling and H3 phosphorylation. Collectively, these data support a novel model of neuronal nuclear signaling whereby dual-residency proteins undergo activity-dependent bi-directional shuttling from the cytosol to synapses and the nucleus, coordinately regulating dendritic spine remodeling and histone modifications.

GABA(A) receptors meet all the pharmacological criteria required to be considered important general anaesthetic targets. In the following study, the modulatory effects of various commercially available and novel cyclohexanols were investigated on recombinant human γ-aminobutyric acid (GABA(A), α(1)β(2)γ(2s)) receptors expressed in Xenopus oocytes, and compared to the modulatory effects on GABA currents observed with exposures to the intravenous anaesthetic agent, propofol. Submaximal EC(20) GABA currents were typically enhanced by co-applications of 3-300 μM cyclohexanols. For instance, at 30 μM 2,6-diisopropylcyclohexanol (a novel compound) GABA responses were increased ~3-fold (although similar enhancements were achieved at 3 μM propofol). As regards rank order for modulation by the cyclohexanol analogues at 30 μM, the % enhancements for 2,6-dimethylcyclohexanol~2,6-diethylcyclohexanol~2,6-diisopropylcyclohexanol~2,6-di-sec-butylcyclohexanol ≫2,6-di-tert-butylcyclohexanol~4-tert-butylcyclohexanol>cyclohexanol~cyclopentanol~2-methylcyclohexanol. We further tested the potencies of the cyclohexanol analogues as general anaesthetics using a tadpole in vivo assay. Both 2,6-diisopropylcyclohexanol and 2,6-dimethylcyclohexanol were effective as anaesthetics with EC(50)s of 14.0 μM and 13.1 μM respectively, while other cyclohexanols with bulkier side chains were less potent. In conclusion, our data indicate that cyclohexanols are both positive modulators of GABA(A) receptors currents and anaesthetics. The positioning and size of the alkyl groups at the 2 and 6 positions on the cyclohexanol ring were critical determinants of activity., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deterioration of cognitive abilities, amyloid-β peptide (Aβ) accumulation and synaptic alterations. Previous studies indicated that hyperforin, a component of the St John's Wort, prevents Aβ neurotoxicity and some behavioral impairments in a rat model of AD. In this study we examined the ability of tetrahydrohyperforin (IDN5607), a stable hyperforin derivative, to prevent the cognitive deficit and synaptic impairment in an in vivo model of AD. In double transgenic APPswe/PSEN1ΔE9 mice, IDN5706 improves memory and prevents the impairment of synaptic plasticity in a dose-dependent manner, inducing a recovery of long-term potentiation. In agreement with these findings, IDN5706 prevented the decrease in synaptic proteins in hippocampus and cortex. In addition, decreased levels of tau hyperphosphorylation, astrogliosis, and total fibrillar and oligomeric forms of Aβ were determined in double transgenic mice treated with IDN5706. In cultured cells, IDN5706 decreased the proteolytic processing of the amyloid precursor protein that leads to Aβ peptide generation. These findings indicate that IDN5706 ameliorates AD neuropathology and could be considered of therapeutic relevance in AD treatment.

St. John's Wort (SJW) has been used medicinally for over 5,000 years. Relatively recently, one of its phloroglucinol derivatives, hyperforin, has emerged as a compound of interest. Hyperforin first gained attention as the constituent of SJW responsible for its antidepressant effects. Since then, several of its neurobiological effects have been described, including neurotransmitter re-uptake inhibition, the ability to increase intracellular sodium and calcium levels, canonical transient receptor potential 6 (TRPC6) activation, N-methyl-D-aspartic acid (NMDA) receptor antagonism as well as antioxidant and anti-inflammatory properties. Until recently, its pharmacological actions outside of depression had not been investigated. However, hyperforin has been shown to have cognitive enhancing and memory facilitating properties. Importantly, it has been shown to have neuroprotective effects against Alzheimer's disease (AD) neuropathology, including the ability to disassemble amyloid-beta (Abeta) aggregates in vitro, decrease astrogliosis and microglia activation, as well as improve spatial memory in vivo. This review will examine some of the early studies involving hyperforin and its effects in the central nervous system (CNS), with an emphasis on its potential use in AD therapy. With further investigation, hyperforin could emerge to be a likely therapeutical candidate in the treatment of this disease.

Menthol and related compounds were investigated for modulation of recombinant human gamma-aminobutyric acid type A (GABA(A), alpha(1)beta(2)gamma(2s)) receptor currents expressed in Xenopus oocytes. Sub-maximal (EC(20)) GABA currents were typically enhanced by co-applications of 3-300 microM (+)-menthol (e.g. by approximately 2-fold at 50 microM) > isopulegol > isomenthol> alpha-terpineol >> cyclohexanol. We studied menthol's actions on GABA(A) receptors compared to sedatives (benzodiazepines) and intravenous anesthetics (barbiturates, steroids, etomidate and propofol). Flumazenil (a benzodiazepine antagonist) did not inhibit menthol enhancements while currents directly activated by 50 microM propofol were significantly inhibited (by 26+/-3%) by 50 microM (+)-menthol. GABA(A) receptors containing beta(2) subunits with either a point mutation in a methionine residue to a tryptophan at the 286 position (in transmembrane domain 3, TM-3) or a tyrosine to a tryptophan at the 444 position (TM-4) are insensitive to modulation by propofol. Enhancements of GABA EC(20) currents by menthol were equally abolished in GABA(A) alpha(1)beta(2)(M286W)gamma(2s) and alpha(1)beta(2)(Y444W)gamma(2s) receptors while positive modulations by benzodiazepines, barbiturates and steroids were unaffected. Menthol may therefore exert its actions on GABA(A) receptors via sites distinct from benzodiazepines, steroids and barbiturates, and via sites important for modulation by propofol. Finally, using an in vivo tadpole assay, addition of (+)-menthol resulted in a loss of righting reflex with an EC(50) of 23.5+/-4.7 microM (approximately10-fold less potent anesthesia than propofol). Thus, menthol and analogs share general anesthetic action with propofol, possibly via action at similar sites on the GABA(A) receptor.