Non-specific Ion Channel Research Articles

Progesterone interacts with the mutational hot-spot of TRPV4 and acts as a ligand relevant for fast Ca2+-signalling

Steroids are also known to induce immediate physiological and cellular response which occurs within minutes to seconds, or even faster. Such non-genomic actions of steroids are rapid and are proposed to be mediated by different ion channels. Transient receptor potential vanilloid sub-type 4 (TRPV4), is a non-specific polymodal ion channel which is involved in several physiological and cellular processes. In this work, we explored the possibilities of Progesterone (P4) as an endogenous ligand for TRPV4. We demonstrate that P4 docks as well as physically interacts with the TM4-loop-TM5 region of TRPV4, a region which is a mutational hotspot for different diseases. Live cell imaging experiments with a genetically encoded Ca2+-sensor suggests that P4 causes quick influx of Ca2+ specifically in the TRPV4 expressing cells, which can be partially blocked by TRPV4-specific inhibitor, suggesting that P4 can act as a ligand for TRPV4. Such P4-mediated Ca2+-influx is altered in cells expressing disease causing TRPV4 mutants, namely in L596P, R616Q, and also in embryonic lethal mutant L618P. P4 dampens, both in terms of “extent” as well as the “pattern” of the Ca2+-influx by other stimulus too in cells expressing TRPV4-Wt, suggesting that P4 crosstalk with the TRPV4-mediated Ca2+-signalling, both in quick and long-term manner. We propose that P4 crosstalk with TRPV4 might be relevant for both acute and chronic pain as well as for other health-related functions.

Assessment of Respiratory Enzymes in Intact Cells by Permeabilization with Alamethicin.

We here describe measurements of respiratory enzymes in situ, which can be done on very small cell samples and make mitochondrial isolation unnecessary. The method is based on the ability of the fungal peptide alamethicin to permeate biological membranes from the net positively charged side, and form nonspecific ion channels. These channels allow rapid transport of substrates and products across the plasma membrane, the inner mitochondrial membrane, and the inner plastid envelope. In this way, mitochondrial enzyme activities can be studied without disrupting the cells. The enzymes can be investigated in their natural proteinaceous environment and the activity of enzymes, also those sensitive to detergents or to dilution, can be quantified on a whole cell basis. We here present protocols for in situ measurement of two mitochondrial enzymatic activities: malate oxidation measured as oxygen consumption by the electron transport chain, which is sensitive to detergents, and NAD+-isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme that dissociates upon dilution.

Influence of Mechanical Unloading on Articular Chondrocyte Dedifferentiation.

Due to the limited self-repair capacity of articular cartilage, the surgical restoration of defective cartilage remains a major clinical challenge. The cell-based approach, which is known as autologous chondrocyte transplantation (ACT), has limited success, presumably because the chondrocytes acquire a fibroblast-like phenotype in monolayer culture. This unwanted dedifferentiation process is typically addressed by using three-dimensional scaffolds, pellet culture, and/or the application of exogenous factors. Alternative mechanical unloading approaches are suggested to be beneficial in preserving the chondrocyte phenotype. In this study, we examined if the random positioning machine (RPM) could be used to expand chondrocytes in vitro such that they maintain their phenotype. Bovine chondrocytes were exposed to (a) eight days in static monolayer culture; (b) two days in static monolayer culture, followed by six days of RPM exposure; and, (c) eight days of RPM exposure. Furthermore, the experiment was also conducted with the application of 20 mM gadolinium, which is a nonspecific ion-channel blocker. The results revealed that the chondrocyte phenotype is preserved when chondrocytes go into suspension and aggregate to cell clusters. Exposure to RPM rotation alone does not preserve the chondrocyte phenotype. Interestingly, the gene expression (mRNA) of the mechanosensitive ion channel TRPV4 decreased with progressing dedifferentiation. In contrast, the gene expression (mRNA) of the mechanosensitive ion channel TRPC1 was reduced around fivefold to 10-fold in all of the conditions. The application of gadolinium had only a minor influence on the results. This and previous studies suggest that the chondrocyte phenotype is preserved if cells maintain a round morphology and that the ion channel TRPV4 could play a key role in the dedifferentiation process.

Mathematical modelling of ICAN-mediated persistent firing in hippocampal neurons

Persistent neural activity has been the focus of neuroscientific research since it was first associated with complex cognitive behaviours. In particular, persistent firing has long been thought to be the neural mechanisms underlying short-term memory encoding and storage [1]. This activity is often elicited by short transient stimuli that have to be retained in memory for long delay periods, in the order of several seconds, after the original stimulus disappeared. In this scenario, the brain stores information for future execution of action depending on that information. Persistent activity elicited in a recurrent network comprising strong excitation in the local circuit has been extensively described in the literature (for a review see [2]). However, recent findings have shown that memory can also be encoded in brain regions which do not display such recurrent connection topology, including the CA1 hippocampal area [3]. Emerging studies are pointing towards intrinsic neural mechanisms independent of synaptic connections, as a complementary mechanism for the maintenance of persistent activity in the hippocampus [4]. These include various cytoplasmic currents flowing through the membrane, characterised by slow ion channel kinetics, and particular neurotransmitter modulation. Our work investigates persistent firing activity in networks of hippocampal neurons, elicited by leveraging intrinsic currents rather than network dynamics. Building on previous findings [5], we model a hippocampal pyramidal neuron using the Hodgkin-Huxley model, with low-threshold Ca2+ currents governing the inward flow of calcium ions in the cell membrane. The intracellular calcium concentration mediates the opening of calcium-activated non-specific (CAN) ion channels [6], which causes an increase in the ionic flow through the cell membrane. Therefore the CAN channels equip the neuron with an after-spike depolarisation mechanism, which enables it to emit action potentials in the absence of external stimulation. Given a transient 250ms 200pA current injection, the neuron is capable of eliciting and maintaining persistent activity with a firing rate of 6Hz for long delay periods (~30s). Moreover, this behaviour is in accord with that displayed in neural recordings of hippocampal slice preparations [4]. Connecting these persistent firing neurons in a network comprising strong local excitation yields a wide range of behaviours depending on the interaction between CAN and synaptic currents. Indeed, our network model is capable of displaying rhythmic behaviour in the form of short synchronised bursts with intra-burst frequencies of 20-40Hz and inter-burst frequencies of 3Hz. These results hint towards a possible mechanism for the generation of memory-related oscillatory activity in the hippocampus.

Ginseng pharmacology: a new paradigm based on gintonin-lysophosphatidic acid receptor interactions.

Ginseng, the root of Panax ginseng, is used as a traditional medicine. Despite the long history of the use of ginseng, there is no specific scientific or clinical rationale for ginseng pharmacology besides its application as a general tonic. The ambiguous description of ginseng pharmacology might be due to the absence of a predominant active ingredient that represents ginseng pharmacology. Recent studies show that ginseng abundantly contains lysophosphatidic acids (LPAs), which are phospholipid-derived growth factor with diverse biological functions including those claimed to be exhibited by ginseng. LPAs in ginseng form a complex with ginseng proteins, which can bind and deliver LPA to its cognate receptors with a high affinity. As a first messenger, gintonin produces second messenger Ca2+ via G protein-coupled LPA receptors. Ca2+ is an intracellular mediator of gintonin and initiates a cascade of amplifications for further intercellular communications by activation of Ca2+-dependent kinases, receptors, gliotransmitter, and neurotransmitter release. Ginsenosides, which have been regarded as primary ingredients of ginseng, cannot elicit intracellular [Ca2+]i transients, since they lack specific cell surface receptor. However, ginsenosides exhibit non-specific ion channel and receptor regulations. This is the key characteristic that distinguishes gintonin from ginsenosides. Although the current discourse on ginseng pharmacology is focused on ginsenosides, gintonin can definitely provide a mode of action for ginseng pharmacology that ginsenosides cannot. This review article introduces a novel concept of ginseng ligand-LPA receptor interaction and proposes to establish a paradigm that shifts the focus from ginsenosides to gintonin as a major ingredient representing ginseng pharmacology.

Mechanosensitive ion channel MscL controls ionic fluxes during cold and heat stress in Synechocystis.

Calcium plays an essential role in a variety of stress responses of eukaryotic cells; however, its function in prokaryotes is obscure. Bacterial ion channels that transport Ca(2+) are barely known. We investigated temperature-induced changes in intracellular concentration of Ca(2+), Na(+) and K(+) in the cyanobacterium Synechocystis sp. strain PCC 6803 and its mutant that is defective in mechanosensitive ion channel MscL. Concentration of cations rapidly and transiently increased in wild-type cells in response to cold and heat treatments. These changes in ionic concentrations correlated with the changes in cytoplasmic volume that transiently decreased in response to temperature treatments. However, no increase in ionic concentrations was observed in the MscL-mutant cells. It implies that MscL functions as a non-specific ion channel, and it participates in regulation of cell volume under temperature-stress conditions.

Role of β-hydroxybutyrate, its polymer poly-β-hydroxybutyrate and inorganic polyphosphate in mammalian health and disease.

We provide a comprehensive review of the role of β-hydroxybutyrate (β-OHB), its linear polymer poly-β-hydroxybutyrate (PHB), and inorganic polyphosphate (polyP) in mammalian health and disease. β-OHB is a metabolic intermediate that constitutes 70% of ketone bodies produced during ketosis. Although ketosis has been generally considered as an unfavorable pathological state (e.g., diabetic ketoacidosis in type-1 diabetes mellitus), it has been suggested that induction of mild hyperketonemia may have certain therapeutic benefits. β-OHB is synthesized in the liver from acetyl-CoA by β-OHB dehydrogenase and can be used as alternative energy source. Elevated levels of PHB are associated with pathological states. In humans, short-chain, complexed PHB (cPHB) is found in a wide variety of tissues and in atherosclerotic plaques. Plasma cPHB concentrations correlate strongly with atherogenic lipid profiles, and PHB tissue levels are elevated in type-1 diabetic animals. However, little is known about mechanisms of PHB action especially in the heart. In contrast to β-OHB, PHB is a water-insoluble, amphiphilic polymer that has high intrinsic viscosity and salt-solvating properties. cPHB can form non-specific ion channels in planar lipid bilayers and liposomes. PHB can form complexes with polyP and Ca2+ which increases membrane permeability. The biological roles played by polyP, a ubiquitous phosphate polymer with ATP-like bonds, have been most extensively studied in prokaryotes, however polyP has recently been linked to a variety of functions in mammalian cells, including blood coagulation, regulation of enzyme activity in cancer cells, cell proliferation, apoptosis and mitochondrial ion transport and energy metabolism. Recent evidence suggests that polyP is a potent activator of the mitochondrial permeability transition pore in cardiomyocytes and may represent a hitherto unrecognized key structural and functional component of the mitochondrial membrane system.

Lipid charge regulation of non-specific biological ion channels

Ion channels are specialized proteins that enable the movement of charges through otherwise impermeable lipidic membranes. Their action is essential in living organisms facilitating electric signaling, muscle contraction or osmotic stress response among other effects. The protein and the lipid charges configure a polarized interface that yields local ionic concentrations and electric potentials that are very different from those of the bulk electrolyte. The combined effect of gradients of ionic concentration and electric potential causes the transport of ions through channels. Here we analyze charge regulation effects in different protein-lipid conformations, stressing how important is the role of electrostatic interactions in the ion channel function that traditionally has been rationalized paying attention mainly to changes in pore size. Tuning lipid charge combined with conductance and selectivity measurements is shown to be a complementary method to evidence lipid involvement in the structure of a biological ion channel.

Targeting Autonomic Neurons to Discover the Triggers of Atrial Fibrillation

Atrial fibrillation (AF) is the most prevalent and persistent cardiac rhythm disorder worldwide but current management strategies are not optimal. Drug therapy targets non-specific ion channels with limited efficacy, side effects and eventual recurrence. Catheter ablation has received much attention but success rates are variable while the procedure itself carries significant risks. These limitations provide the impetus to identify novel, specific therapies. The target of this study is to visualize autonomic ganglia cells that trigger AF using a promoter driven construct directed towards parasympathetic ganglia located in the retro-cardiac fat pad that envelopes the left atrium (LAFP).

Dielectric characterization of costal cartilage chondrocytes

BackgroundChondrocytes respond to biomechanical and bioelectrochemical stimuli by secreting appropriate extracellular matrix proteins that enable the tissue to withstand the large forces it experiences. Although biomechanical aspects of cartilage are well described, little is known of the bioelectrochemical responses. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using dielectric spectroscopy. MethodsDielectric spectroscopy allows non-invasive probing of biological cells. An in house computer program is developed to extract dielectric properties of human costal cartilage cells from raw cell suspension impedance data measured by a microfluidic device. The dielectric properties of chondrocytes are compared with other cell types in order to comparatively assess the electrical nature of chondrocytes. ResultsThe results suggest that electrical cell membrane characteristics of chondrocyte cells are close to cardiomyoblast cells, cells known to possess an array of active ion channels. The blocking effect of the non-specific ion channel blocker gadolinium is tested on chondrocytes with a significant reduction in both membrane capacitance and conductance. ConclusionsWe have utilized a microfluidic chamber to mimic biomechanical events through changes in bioelectrochemistry and described the dielectric properties of chondrocytes to be closer to cells derived from electrically excitably tissues. General significanceThe study describes dielectric characterization of human costal chondrocyte cells using physical tools, where results and methodology can be used to identify potential anomalies in bioelectrochemical responses that may lead to cartilage disorders.

Structural and functional characterization of annexin 1 from Medicago truncatula

Annexins are calcium- and membrane-binding proteins that have been shown to have diverse properties such as actin, integrin and GTP binding, both in animals and plants. Recently, Medicago truncatula annexin 1 (AnnMt1) has been suggested to participate in nodulation (Nod factor signaling) and mycorrhization in legume plants. In this report we demonstrate for the first time that recombinant AnnMt1 (rec-AnnMt1) mediates membrane permeabilization to cations with conductance ranging from 16 pS to 329 pS. In agreement with other structurally determined annexins, homology modeling of AnnMt1 suggests that most of the functional determinants are found on the convex surface of the modeled structure. In conclusion, we propose a potential constitutive role of AnnMt1 in Nod factor signaling as a non-specific ion channel.

Relationship between heat-induced fibrillogenicity and hemolytic activity of thermostable direct hemolysin and a related hemolysin of Vibrio parahaemolyticus

The formation of nonspecific ion channels by small oligomeric amyloid intermediates is toxic to the host's cellular membranes. Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are major virulence factors of Vibrio parahaemolyticus. We have previously reported the crystal structure of TDH tetramer with the central channel. We have also identified the molecular mechanism underlying the paradoxical responses to heat treatment of TDH, known as the Arrhenius effect, which is the reversible amyloidogenic property. In the present report, we describe the biophysical properties of TRH, which displays 67% amino acid similarity with TDH. Molecular modeling provided a good fit of the overall structure of TDH and TRH. Size-exclusion chromatography, ultracentrifugation, and transmission electron microscopy revealed that TRH formed tetramer in solution. These toxins showed similar hemolytic activity on red blood cells. However, TRH had less amyloid-like structure than TDH analyzed by thioflavin T-binding assay and far-UV circular dichroism spectra. These data indicated that amyloidogenicity upon heating is not essential for the membrane disruption of erythrocytes, but the maintenance of tetrameric structure is indispensable for the hemolytic activity of the TDH and TRH.

Interactions of Mitochondrial Presequence Peptides with the Mitochondrial Outer Membrane Preprotein Translocase TOM

TOM protein-conducting channels serve as the main entry sites into mitochondria for virtually all mitochondrial proteins. When incorporated into lipid bilayers, they form large, relatively nonspecific ion channels that are blocked by peptides derived from mitochondrial precursor proteins. Using single-channel electrical recordings, we analyzed the interactions of mitochondrial presequence peptides with single TOM pores. The largest conductance state of the translocon represents the likely protein-conducting conformation of the channel. The frequency (but not the duration) of the polypeptide-induced blockage is strongly modulated by the substrate concentration. Structural differences between substrates are reflected in characteristic blockage frequencies and duration of blockage. To our knowledge, this study provides first quantitative data regarding the kinetics of polypeptide interaction with the mitochondrial TOM machinery.

Pharmacological separation of hEAG and hERG K+ channel function in the human mammary carcinoma cell line MCF-7

Pharmacological inhibitors of the human ether-a-go-go (hEAG) potassium channel, astemizole and imipramine, have been used to demonstrate that hEAG plays a role in cancer cell proliferation. Astemizole and imipramine are, however, relatively non-specific ion channel blockers, as astemizole can also block the related potassium channel, human ether-a-go-go-related (hERG). Therefore, we aimed to determine the molecular target of astemizole, in the human mammary carcinoma cell line MCF-7. We initially confirmed the expression of KCNH1 and KCNH2 mRNA and hEAG and hERG channel protein in MCF-7 cells. Using a [3H]-thymidine incorporation assay we determined that astemizole inhibited MCF-7 cell proliferation, whereas the hERG-specific channel blocker E-4031 had no effect. We then determined that E-4031 inhibited the regulatory volume decrease (RVD) observed in these cells following exposure to hypotonic solutions, confirming that functional hERG channels are present and may be important for cell volume regulation in MCF-7 cells. Our results suggest, for the first time, that hERG is involved in cell volume regulation. In addition, the function of hEAG and hERG in MCF-7 cell proliferation can be separated pharmacologically by utilizing the channel inhibitors astemizole and E-4031. The hEAG channel function in MCF-7 cells appears to be involved in the regulation of cell proliferation, whereas hERG is involved in cell volume regulation.

TRPV1 at nerve endings regulates growth cone morphology and movement through cytoskeleton reorganization

While the importance of Ca(2+) channel activity in axonal path finding is established, the underlying mechanisms are not clear. Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones. These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N-arachidonoyl-dopamine (NADA), and affect the integrity of microtubule cytoskeleton. Using TRPV1-transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retraction, and collapse and formation of varicosities along neurites. These changes were due to TRPV1-activation-mediated disassembly of microtubules and are partly Ca(2+)-independent. Prolonged activation with very low doses (1 nM) of NADA results in shortening of neurites in the majority of isolectin B4-positive dorsal root ganglia neurones. We postulate that TRPV1 activation plays an inhibitory role in sensory neuronal extension and motility by regulating the disassembly of microtubules. This might have a role in the chronification of pain.

Effect of ultrasound-activated microbubbles on the cell electrophysiological properties

New clinical applications of ultrasound contrast microbubbles extend beyond imaging and diagnosis toward therapeutic applications. Cell membrane permeability and the uptake of substances have been shown to be enhanced by microbubbles under ultrasound stimulation. However, the mechanisms of action of ultrasound-activated microbubbles are still unknown. The aim of our study was to examine how microbubbles and ultrasound interact with cells in an attempt to understand the sonoporation mechanism. The ruptured-patch-clamp whole-cell technique was used to measure membrane potential variations of a single cell. SonoVue microbubbles and mammary breast cancer cell line MDA-MB-231 were used. Ultrasound was applied using single-element transducers of 1 MHz. Microbubbles and cells were simultaneously video monitored during ultrasound exposure. Our results showed that, during sonoporation, a marked cell membrane hyperpolarization occurs ( n = 6 cells) at negative pressures above 150 kPa, indicating the activation of specific ion channels while the cell and the microbubbles remain viable. The hyperpolarization was sustained for as long as the microbubbles are in a direct contact with the cell and the ultrasound waves are transmitted. Smaller acoustic amplitudes induced only mild hyperpolarization, whereas shutting off the ultrasound brings the cell membrane potential to its resting value. However, ultrasound alone did not affect the cell membrane potential. A similar hyperpolarization of the cell membrane was observed when a mechanical pressure was applied on the cell through a glass probe. In conclusion, the results demonstrate that microbubbles’ oscillations under ultrasound activation entail modifications of the electrophysiologic cell activities by triggering the modulation of ionic transports through the plasmic cell membrane. However, only cells in direct contact with the microbubbles are impacted. The mechanisms involved are likely related to activation of specific channels sensitive to mechanical stresses (stretch-activated channels) and possibly nonspecific ion channels. (E-mail: bouakaz@med.univ-tours.fr)

H + Pump Provides an Early Asymmetry Signal

Adams et al. identified the H + -V-ATPase, which is a vacuolar and plasma membrane H + transporter, in a pharmacological screen of Xenopus embryos in which various transporters were inhibited and defects in left-right asymmetry (heterotaxia) were scored. Inhibition of the H + -V-ATPase with drugs such as concanamycin or expression of a dominant-negative H + -V-ATPase subunit resulted in heterotaxia and loss of asymmetric expression of one of the first genes with asymmetric expression, Nodal , suggesting that H + -V-ATPase provides a very early asymmetry signal. H + -V-ATPase subunits were more abundant on the right side of the embryo as early as the two-cell stages. Proton efflux was greater on the right side of the embryo and was inhibited by concanamycin. In addition, the right side of the embryo was hyperpolarized relative to the left side, which is expected from the electrogenic nature of the H + -V-ATPase. Elimination of asymmetric H + flux by expression of a nonasymmetrically localized plasma membrane H + pump or exposure of the embryos to low pH (pH 5 to 6), or elimination of the hyperpolarization of the membrane by incubating the embryos with the drug palytoxin that converts the Na + , K + -ATPase into a nonspecific ion channel produced heterotaxia. This suggests that the activity of the H + -V-ATPase produces asymmetry through a combination of an effect on pH and membrane potential. A role for H + -V-ATPase in asymmetry was also noted for chick embryos and zebrafish embryos and appeared to serve as one of the earliest signals for asymmetry. Disruption of H + -V-ATPase activity randomized the expression of the genes encoding nodal and sonic hedgehog in chicks, and in zebrafish H + -V-ATPase activity was required for asymmetric expression of southpaw and prior to formation of the Kuppfer's vesicle, a ciliated organ involved in organ asymmetry. D. S. Adams, K. R. Robinson, T. Fukumoto, S. Yuan, R. C. Albertson, P. Yelick, L. Kuo, M. McSweeney, M. Levin, Early, H + -V-ATPase-dependent proton flux is necessary for consistent left-right patterning of non-mammalian vertebrates. Development 133 , 1657-1671 (2006). [Abstract] [Full Text]

ATP dependence of the non-specific ion channel in Torpedo synaptic vesicles

Synaptic vesicles of Torpedo electromotor neurons contain a high amount of ATP. The concentration of total ATP is around 120 mM, whereas the free [ATP] is about 5-6 mM. We examined the effect of intravesicular ATP on the non-specific ion channel in Torpedo-fused synaptic vesicles. It was found that this channel is closed when the ATP concentration is above 2 mM, but it is very frequently open at lower ATP concentrations. Unmasking this ion channel at a low ATP concentration may be significant for post-fusion control of transmitter release by the 'kiss and run' mechanism in normal conditions, while during metabolic stress it may underlie dissipation of important gradients across the vesicle membrane.

Peptide Blockers of the Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Amyloid β

Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid beta peptide (Abeta) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by Abeta has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking Abeta-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to Abeta. Using this approach, we identified a heptapeptide denoted IQ, which binds with nanomolar affinity to Abeta and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that Abeta inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by Abeta. These results indicate that the Abeta binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for Abeta neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble Abeta in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD.

Changes in transient receptor potential cation channel superfamily V (TRPV) mRNA expression in the mouse inner ear ganglia after kanamycin challenge

The transient receptor potential cation channel subfamily V (TRPV) is a non-specific cation ion channel receptor family that is gated by heat, protons, low extracellular osmolarity and arachidonic acid derivatives. Since some of these endogenous agonists of TRPV receptors are reactive oxygen intermediates produced by lipoxygenases, it has been hypothesized that some members of the TRPV family may respond to challenges by reactive oxygen species. This study used real-time PCR to quantitatively track changes in TRPV1-4 mRNA expression in the spiral, vestibular, and trigeminal ganglia and the kidney from kanamycin (KM)-treated mice. TRPV1, TRPV2, TRPV3 and TRPV4 mRNAs were expressed in spiral and vestibular ganglia, and TRPV2 and TRPV1 mRNAs were most predominant in control mice. After KM (700 mg/kg s.c. b.i.d., 14 days), TRPV1 mRNA and protein expression were significantly up-regulated both in the spiral and vestibular ganglia, but expression was unaffected in the trigeminal ganglion and kidney. Real-time PCR also demonstrated a significant down-regulation in TRPV4 mRNA expression in the inner ear ganglia and kidney after KM treatment. All these mRNA and protein expression changes were eliminated by simultaneous administration of dihydroxybenzoate (300 mg/kg s.c. b.i.d., 14 days), an anti-oxidant that blocks KM ototoxicity. It is proposed that up-regulated TRPV1 expression during KM exposure may promote ganglion cell survival by contributing to neuronal depolarization, with KM-induced tinnitus and dizziness as consequences.