Ladislav Vyklicky

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.

Subtype-dependence of N-methyl-D-aspartate receptor modulation by pregnenolone sulfate.

N-methyl-D-aspartate receptors play a critical role in synaptogenesis, synaptic plasticity, and excitotoxicity. They are heteromeric complexes of NR1 combined with NR2A-D and/or NR3A-B subunits. The subunit composition determines the biophysical and pharmacological properties of the N-methyl-D-aspartate receptor channel complex. In this study, we report that responses mediated by recombinant rat N-methyl-D-aspartate receptors expressed in human embryonic kidney HEK293 cells are differentially affected by naturally occurring neurosteroid pregnenolone sulfate. We show that responses induced by 1mM glutamate in NR1-1a/NR2A and NR1-1a/NR2B receptors are potentiated five- to eight-fold more by pregnenolone sulfate than responses of NR1-1a/NR2C and NR1-1a/NR2D receptors with no differences in the concentration of pregnenolone sulfate that produced 50% potentiation. In addition to potentiation, pregnenolone sulfate also has an inhibitory effect at recombinant N-methyl-D-aspartate receptors, with values of the concentration of pregnenolone sulfate that produces 50% inhibition of NR1/NR2D=NR1/NR2C<NR1/NR2B<NR1/NR2A. In addition, we show that the structure of the extracellular loop between the third and fourth transmembrane domains of the NR2 subunit is critical for both the potentiating and inhibitory effects of pregnenolone sulfate. The modulatory effects of pregnenolone sulfate are consistent with a model in which this neurosteroid acts at two distinct binding sites on the N-methyl-D-aspartate receptor. These data provide insight into the mechanisms by which pregnenolone sulfate and related sulfated neurosteroids modulate activity of N-methyl-D-aspartate receptor channels.

Molecular Mechanism of Pregnenolone Sulfate Action at NR1/NR2B Receptors

NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission and synaptic plasticity as well as excitotoxicity. They have several binding sites for allosteric modulators, including neurosteroids, endogenous compounds synthesized by the nervous tissue and expected to act locally. Whole-cell patch-clamp recording from human embryonic kidney 293 cells expressing NR1-1a/NR2B receptors revealed that neurosteroid pregnenolone sulfate (PS) (300 μm), when applied to resting NMDA receptors, potentiates the amplitude of subsequent responses to 1 mm glutamate fivefold and slows their deactivation twofold. The same concentration of PS, when applied during NMDA receptor activation by 1 mm glutamate, has only a small effect. The association and dissociation rate constants of PS binding and unbinding from resting NMDA receptors are estimated to be 3.3 ± 2.0 mm-1sec-1 and 0.12 ± 0.02 sec-1, respectively, corresponding to an apparent affinity Kd of 37 μm. The results of experiments indicate that the molecular mechanism of PS potentiation of NMDA receptor responses is attributable to an increase in the peak channel open probability (Po). Responses to glutamate recorded in the continuous presence of PS exhibit marked time-dependent decline. Our results indicate that the decline is induced by a change of the NMDA receptor affinity for PS after receptor activation. These results suggest that the PS is a modulator of NMDA receptor Po, the effectiveness of which is lowered by glutamate binding. This modulation may have important consequences for the neuronal excitability.

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.

Temperature dependence of NR1/NR2B NMDA receptor channels

N-methyl-D-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9-46.5 degrees C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q(10)=10.3), resensitization (Q(10)=4.6) and unbinding (Q(10)=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (tau(fast): Q(10)=3.7; tau(slow): Q(10)=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.

Neurosteroid modulation of N-methyl-D-aspartate receptors: Molecular mechanism and behavioral effects

Glutamate is the main neurotransmitter released at synapses in the central nervous system of vertebrates. Its excitatory role is mediated through activation of specific glutamatergic ionotropic receptors, among which the N-methyl-D-aspartate (NMDA) receptor subtype has attracted considerable attention in recent years. Substantial progress has been made in elucidating the roles these receptors play under physiological and pathological conditions and in our understanding of the functional, structural, and pharmacological properties of NMDA receptors. Many pharmacological compounds have been identified that affect the activity of NMDA receptors, including neurosteroids. This review summarizes our knowledge about molecular mechanisms underlying the neurosteroid action at NMDA receptors as well as about the action of neurosteroids in animal models of human diseases.