Viktorie Vlachova

Temperature coefficient of membrane currents induced by noxious heat in sensory neurones in the rat

1 Membrane currents induced by noxious heat (Iheat) were studied in cultured dorsal root ganglion (DRG) neurones from newborn rats using ramps of increasing temperature of superfusing solutions. 2 I heat was observed in about 70 % of small (< 25 μm) DRG neurones. At ‐60 mV, Iheat exhibited a threshold at about 43 °C and reached its maximum, sometimes exceeding 1 nA, at 52 °C (716 ± 121 pA; n= 39). 3 I heat exhibited a strong temperature sensitivity (temperature coefficient over a 10 °C temperature range (Q10) = 17·8 ± 2·1, mean ± s.d., in the range 47‐51 °C; n= 41), distinguishing it from the currents induced by capsaicin (1 μM), bradykinin (5 μM) and weak acid (pH 6·1 or 6·3), which exhibited Q10 values of 1·6‐2·8 over the whole temperature range (23‐52 °C). Repeated heat ramps resulted in a decrease of the maximum Iheat and the current was evoked at lower temperatures. 4 A single ramp exceeding 57 °C resulted in an irreversible change in Iheat. In a subsequent trial, maximum Iheat was decreased to less than 50 %, its threshold was lowered to a temperature just above that in the bath and its maximum Q10 was markedly lower (5·6 ± 0·8; n= 8). 5 DRG neurones that exhibited Iheat were sensitive to capsaicin. However, four capsaicin‐sensitive neurones out of 41 were insensitive to noxious heat. There was no correlation between the amplitude of capsaicin‐induced responses and Iheat. 6 In the absence of extracellular Ca2+, Q10 for Iheat was lowered from 25·3 ± 7·5 to 4·2 ± 0·4 (n= 7) in the range 41‐50 °C. The tachyphylaxis, however, was still observed. 7 A high Q10 of Iheat suggests a profound, rapid and reversible change in a protein structure in the plasma membrane of heat‐sensitive nociceptors. It is hypothesized that this protein complex possesses a high net free energy of stabilization (possibly due to ionic bonds) and undergoes disassembly when exposed to noxious heat. The liberated components activate distinct cationic channels to generate Iheat. Their affinity to form the complex at low temperatures irreversibly decreases after one exposure to excessive heat.

COPPER MODULATION OF NMDA RESPONSES IN MOUSE AND RAT CULTURED HIPPOCAMPAL NEURONS

The effect of Cu2+ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole‐cell patch‐clamp and a fast perfusion system. Analysis of the Cu2+ concentration‐response curve for inhibition of NMDA‐induced currents suggests that free Cu2+ directly inhibits NMDA receptors with an IC50 of 0.27 μM. Cu2+ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA‐Cu2+ and glycine‐Cu2+ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu2+ (10–100 μM) of responses to 10 μM NMDA was essentially voltage‐independent. The onset of inhibition by 100 μM Cu2+ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU2+ acts as a non‐competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu2+ to NMDA receptors, calculated from the rate of onset of block by Cu2+ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cuz+ interacts preferentially with agonist‐bound receptors. Our results show that Cu2+ is a potent inhibitor of NMDA receptor‐mediated responses.

Reducing and Oxidizing Agents Sensitize Heat-Activated Vanilloid Receptor (TRPV1) Current

We have previously reported that the reducing agent dithiothreitol (DTT) strongly increases thermally induced activity of the transient receptor potential vanilloid receptor-1 (TRPV1) channel. Here, we show that exposure to oxidizing agents also enhances the heat-induced activation of TRPV1. The actions of sulfhydryl modifiers on heat-evoked whole-cell membrane currents were examined in TRPV1-transfected human embryonic kidney 293T cells. The sensitizing effects of the membrane-permeable oxidizing agents diamide (1 mM), chloramine-T (1 mM), and the copper-o-complex (100:400 μM) were not reversed by washout, consistent with the stable nature of covalently modified sulfhydryl groups. In contrast, the membrane-impermeable cysteine-specific oxidant 5,5′-dithio-bis-(2-nitrobenzoic acid) (0.5 mM) was ineffective. The alkylating agent N-ethylmaleimide (1 mM) strongly and irreversibly affected heat-evoked responses in a manner that depended on DTT pretreatment. Extracellular application of the membrane-impermeable reducing agent glutathione (10 mM) mimicked the effects of 10 mM DTT in potentiating the heat-induced and voltage-induced membrane currents. Using site-directed mutagenesis, we identified Cys621 as the residue responsible for the extracellular modulation of TRPV1 by reducing agents. These data suggest that the vanilloid receptor is targeted by redox-active substances that directly modulate channel activity at sites located extracellularly as well as within the cytoplasmic domains. The results obtained demonstrate that an optimal redox state is crucial for the proper functioning of the TRPV1 channel and both its reduced and oxidized states can result in an increase in responsiveness to thermal stimuli.

A technique for fast application of heated solutions of different composition to cultured neurones

A technique is described that allows the application of fast temperature changes (time constant approximately 300 ms) of solutions superfusing cultured neurones under whole-cell mode of membrane current recording. Its principle is in heating the common outlet of the manifold which consists of 12 tubes connected to barrels containing test solutions of different composition. The outlet is made from a glass capillary (25 mm length, 620/350 microns outer/inner diameter) coated on the outside wall with platinum for a length of 12 mm. The heating element, a platinum layer, is electrically connected to the probe fixed to the micromanipulator used for positioning the manifold. The solutions, driven by gravity, are applied by opening electronic valves controlled either manually or in programmed sequences. The DC current for heating is controlled either manually or by external voltage command. The advantage of the technique is that the same temperature pattern can be applied to 12 different solutions. The technique is used for classifying sensory neurones in culture with respect to their sensitivity to heat and algogens; however, it is applicable to any study of the effects of increased temperature on the activity of ion channels in cultured cells.

Ciguatoxins activate specific cold pain pathways to elicit burning pain from cooling

Ciguatoxins are sodium channel activator toxins that cause ciguatera, the most common form of ichthyosarcotoxism, which presents with peripheral sensory disturbances, including the pathognomonic symptom of cold allodynia which is characterized by intense stabbing and burning pain in response to mild cooling. We show that intraplantar injection of P‐CTX‐1 elicits cold allodynia in mice by targeting specific unmyelinated and myelinated primary sensory neurons. These include both tetrodotoxin‐resistant, TRPA1‐expressing peptidergic C‐fibres and tetrodotoxin‐sensitive A‐fibres. P‐CTX‐1 does not directly open heterologously expressed TRPA1, but when co‐expressed with Nav channels, sodium channel activation by P‐CTX‐1 is sufficient to drive TRPA1‐dependent calcium influx that is responsible for the development of cold allodynia, as evidenced by a large reduction of excitatory effect of P‐CTX‐1 on TRPA1‐deficient nociceptive C‐fibres and of ciguatoxin‐induced cold allodynia in TRPA1‐null mutant mice. Functional MRI studies revealed that ciguatoxin‐induced cold allodynia enhanced the BOLD (Blood Oxygenation Level Dependent) signal, an effect that was blunted in TRPA1‐deficient mice, confirming an important role for TRPA1 in the pathogenesis of cold allodynia.

Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions

We have developed an improved technique for fast cooling and heating of solutions superfusing isolated cells under patch-clamp or calcium imaging conditions. The system meets the requirements for studying temperature dependency of all kinds of ion channels, in particular temperature-gated ion channels. It allows the application of temperature changes within a range of 5-60 degrees C at maximum rates of -40 degrees C/s to 60 degrees C/s. Barrels filled with different solutions are connected to a manifold consisting of seven silica capillaries (320 microm inner diameter, i.d.). A common outlet consists of a glass capillary through which the solutions are applied onto the cell surface. The upper part of this capillary is embedded in a temperature exchanger driven by a miniature Peltier device which preconditions the temperature of the passing solution. The lower part of the capillary carries an insulated copper wire, densely coiled over a length of 7 mm, and connected to a dc current source for resistive heating. The Peltier device and the heating element are electrically connected to the headstage probe which is fixed on to a micromanipulator for positioning of the manifold. The temperature of the flowing solution is measured by a miniature thermocouple inserted into the common outlet capillary near to its orifice which is placed at a distance of less than 100 microm from the surface of the examined cell. The temperature is either manually controlled by voltage commands or adjusted via the digital-to-analog converter of a conventional data acquisition interface. Examples are given of using the device in patch-clamp studies on heterologously expressed TRPV1, TRPM8, and on cultured rat sensory neurons.

Ethanol inhibits cold‐menthol receptor TRPM8 by modulating its interaction with membrane phosphatidylinositol 4,5‐bisphosphate

Ethanol has opposite effects on two members of the transient receptor potential (TRP) family of ion channels: it inhibits the cold‐menthol receptor TRPM8, whereas it potentiates the activity of the heat‐ and capsaicin‐gated vanilloid receptor TRPV1. Both thermosensitive cation channels are critically regulated by the membrane lipid, phosphatidylinositol 4,5‐bisphosphate (PIP2). The effects of this phospholipid on TRPM8 and TRPV1 are also functionally opposite: PIP2 is necessary for the activation of TRPM8 but it constitutively inhibits TRPV1. This parallel led us to investigate the possible role of PIP2 in the ethanol‐induced modulation of rat TRPM8, heterologously expressed in HEK293T cells. In this study, we characterize the effects of ethanol (0.1–10%) on whole‐cell currents produced by menthol and by low temperature (< 17°C). We show that the inclusion of PIP2 in the intracellular solution results in a strong reduction in the ethanol‐induced inhibition of menthol‐evoked responses. Conversely, intracellular dialysis with anti‐PIP2 antibody or with the PIP2 scavenger, poly l‐lysine, enhanced the ethanol‐induced inhibition of TRPM8. A 20 min pre‐incubation with wortmannin caused a modest decrease in inhibition produced by 1% ethanol, indicating that the ethanol‐induced inhibition is not mediated by lipid kinases. These findings suggest that ethanol inhibits TRPM8 by weakening the PIP2–TRPM8 channel interaction; a similar mechanism may contribute to the ethanol‐mediated modulation of some other PIP2‐sensitive TRP channels.

Gadolinium activates and sensitizes the vanilloid receptor TRPV1 through the external protonation sites.

Gadolinium is a recognized blocker of many types of cation channels, including several channels of the transient receptor potential (TRP) superfamily. In this study, we demonstrate that Gd(3+), in addition to its blocking effects, activates and potentiates the recombinant vanilloid receptor TRPV1 expressed in HEK293T cells. Whole-cell currents through TRPV1 were induced by Gd(3+) with a half-maximal activation achieved at 72 microM at +40 mV. Gd(3+), at concentrations up to 100 microM, lowered the threshold for heat activation and potentiated the currents induced by capsaicin (1 microM) and low extracellular pH (6). Higher concentrations of Gd(3+) (>300 microM) blocked the TRPV1 channel. Neutralizations of the two acidic residues, Glu600 and Glu648, which are the key residues conferring the proton-sensitivity to TRPV1, resulted in a loss of Gd(3+)-induced activation and/or a reduction in its potentiating effects. A trivalent nonlanthanide, Al(3+), that possesses much a smaller atomic mass than Gd(3+) blocked but did not activate or sensitize the TRPV1 channel. These findings indicate that Gd(3+) activates and potentiates the TRPV1 by neutralizing two specific proton-sensitive sites on the extracellular side of the pore-forming loop.

Contribution of the Putative Inner-Pore Region to the Gating of the Transient Receptor Potential Vanilloid Subtype 1 Channel (TRPV1)

The transient receptor potential vanilloid receptor-1 (TRPV1) is a sensory neuron-specific nonselective cation channel that is gated in response to various noxious stimuli: pungent vanilloids, low pH, noxious heat, and depolarizing voltages. By its analogy to K+ channels, the S6 inner helix domain of TRPV1 (Y666-G683) is a prime candidate to form the most constricted region of the permeation pathway and might therefore encompass an as-yet-unmapped gate of the channel. Using alanine-scanning mutagenesis, we identified 16 of 17 residues, that when mutated affected the functionality of the TRPV1 channel with respect to at least one stimulus modality. T670A was the only substitution producing the wild-type channel phenotype, whereas Y666A and N676A were nonfunctional but present at the plasma membrane. The periodicity of the functional effects of mutations within the TRPV1 inner pore region is consistent with an α-helical structure in which T670 and A680 might play the roles of two bending “hinges.”

Functional changes in the vanilloid receptor subtype 1 channel during and after acute desensitization.

Agonist-induced desensitization of the transient receptor potential vanilloid receptor-1 (TRPV1) is one of the key strategies that offer a way to alleviate neuropathic and inflammatory pain. This process is initiated by TRPV1 receptor activation and the subsequent entry of extracellular Ca(2+) through the channel into sensory neurones. One of the prominent mechanisms responsible for TRPV1 desensitization is dephosphorylation of the TRPV1 protein by the Ca(2+)/calmodulin-dependent enzyme, phosphatase 2B (calcineurin). Of several consensus phosphorylation sites identified so far, the most notable are two sites for Ca(2+)/calmodulin dependent kinase II (CaMKII) at which the dynamic equilibrium between the phosphorylated and dephosphorylated states presumably regulates agonist binding. We examined the mechanisms of acute Ca(2+)-dependent desensitization using whole-cell patch-clamp techniques in human embryonic kidney (HEK) 293T cells expressing the wild type or CaMKII phosphorylation site mutants of rat TRPV1. The nonphosphorylatable mutant S502A/T704I was capsaicin-insensitive but the S502A/T704A construct was fully functional, indicating a requirement for a specific residue at position 704. A point mutation at the nearby conserved residue R701 strongly affected the heat, capsaicin and pH-evoked currents. As this residue constitutes a stringent CaMKII consensus site but is also predicted to be involved in the interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), these data suggest that in addition to dephosphorylation, or as its consequence, a short C-terminal juxtamembrane segment adjacent to the transient receptor potential box composed of R701 and T704 might be involved in the decelerated gating kinetics of the desensitized TRPV1 channel.