Transient receptor potential ankyrin 1 channel: An evolutionarily tuned thermosensor.

The discovery of the role of the transient receptor potential ankyrin 1 (TRPA1) channel as a polymodal detector of cold and pain-producing stimuli almost two decades ago catalyzed the consequent identification of various vertebrate and invertebrate orthologues. In different species, the role of TRPA1 has been implicated in numerous physiological functions, indicating that the molecular structure of the channel exhibits evolutionary flexibility. Until very recently, information about the critical elements of the temperature-sensing molecular machinery of thermosensitive ion channels such as TRPA1 had lagged far behind information obtained from mutational and functional analysis. Current developments in single-particle cryo-electron microscopy are revealing precisely how the thermosensitive channels operate, how they might be targeted with drugs, and at which sites they can be critically regulated by membrane lipids. This means that it is now possible to resolve a huge number of very important pharmacological, biophysical and physiological questions in a way we have never had before. In this review, we aim at providing some of the recent knowledge on the molecular mechanisms underlying the temperature sensitivity of TRPA1. We also demonstrate how the search for differences in temperature and chemical sensitivity between human and mouse TRPA1 orthologues can be a useful approach to identifying important domains with a key role in channel activation.

Calcium-dependent desensitization of vanilloid receptor TRPV1: a mechanism possibly involved in analgesia induced by topical application of capsaicin.

The rationale for the topical application of capsaicin and other vanilloids in the treatment of pain is that such compounds selectively excite and subsequently desensitize nociceptive neurons. This desensitization is triggered by the activation of vanilloid receptors (TRPV1), which leads to an elevation in intracellular free Ca2+ levels. Depending on the vanilloid concentration and duration of exposure, the Ca2+ influx via TRPV1 desensitizes the channels themselves, which may represent not only a feedback mechanism protecting the cell from toxic Ca2+ overload, but also likely contributes to the analgesic effects of capsaicin. This review summarizes the current state of knowledge concerning the mechanisms that underlie the acute capsaicin-induced Ca2+-dependent desensitization of TRPV1 channels and explores to what extent they may contribute to capsaicin-induced analgesia. In view of the polymodal nature of TRPV1, we illustrate how the channels behave in their desensitized state when activated by other stimuli such as noxious heat or depolarizing voltages. We also show that the desensitized channel can be strongly reactivated by capsaicin at concentrations higher than those previously used to desensitize it. We provide a possible explanation for a high incidence of adverse effects of topical capsaicin and point to a need for more accurate clinical criteria for employing it as a reliable remedy.

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.

Molecular basis of TRPA1 regulation in nociceptive neurons. A review.

Transient receptor potential A1 (TRPA1) is an excitatory ion channel that functions as a cellular sensor, detecting a wide range of proalgesic agents such as environmental irritants and endogenous products of inflammation and oxidative stress. Topical application of TRPA1 agonists produces an acute nociceptive response through peripheral release of neuropeptides, purines and other transmitters from activated sensory nerve endings. This, in turn, further regulates TRPA1 activity downstream of G-protein and phospholipase C-coupled signaling cascades. Despite the important physiological relevance of such regulation leading to nociceptor sensitization and consequent pain hypersensitivity, the specific domains through which TRPA1 undergoes post-translational modifications that affect its activation properties are yet to be determined at a molecular level. This review aims at providing an account of our current knowledge on molecular basis of regulation by neuronal inflammatory signaling pathways that converge on the TRPA1 channel protein and through modification of its specific residues influence the extent to which this channel may contribute to pain.

Single K+ currents during differentiation of embryonic muscle cells in vitro.

After 3-7 days in culture, chicken myotubes possess five types of K+ channel: two high-conductance channels of 195 and 105 pS which are sensitive to tetraethylammonium (TEA), an ATP-sensitive channel of 64 pS and two low-conductance channels of 40 and 15 pS which are insensitive to TEA and ATP. The same population of channels is to be found in EGTA-treated muscle cells with blocked fusion and, with the exception of the ATP-sensitive channel, also in 1-day-old myoblasts. There are differences between myoblasts and myotubes in the percentage of incidence of individual channel types. High-conductance K+ channels are most frequently to be observed in myotubes, but they are rare in myoblasts and EGTA-treated cells where low-conductance K+ channels predominate.

The effects of excessive heat on heat-activated membrane currents in cultured dorsal root ganglia neurons from neonatal rat.

The effects of high temperature (53-61 degrees C) on membrane currents (I(heat)) or depolarization (V(heat)) induced by noxious heat were studied in cultured dorsal root ganglia neurons from neonatal rats using the whole cell patch clamp technique. I(heat) or V(heat) produced by 3 s ramps of increasing temperature between 43 and 50 degrees C exhibited a fast slope (Q10>10) that was similar both during rising and falling temperature (n=85). Temperatures exceeding 52 degrees C resulted in slowdown in the recovery of I(heat), and the threshold for inducing I(heat) was shifted to lower temperatures in successive trials. These high temperatures (54-60 degrees C) caused a linear and incomplete recovery of I(heat) (Q10 decreased to <5; 4.5 +/- 0.4; n=17) and in successive trials the threshold of I(heat) decreased to temperatures close to that in the bath. The neurons, however, remained sensitive to capsaicin and to decreased extracellular pH. It is suggested that exposure of nociceptive neurons to excessive noxious heat results in an irreversible decrease of the energy barrier between the resting and activated state of the protein structures responsible for generation of I(heat). This may explain the sensitization of nociceptors after heat injury.

Oxidizing reagent copper-o-phenanthroline is an open channel blocker of the vanilloid receptor TRPV1.

The TRPV1 channel plays an important role in generating nociceptive signals in mammalian primary sensory neurons. It consists of 838 amino acids with six transmembrane segments (TM1-TM6), a pore-forming loop between TM5 and TM6 and N- and C- terminals located intracellularly. It is a homotetramer and forms a nonselective cationic channel that can be opened by capsaicin, weak acids and noxious heat. There are 18 cysteines (Cys), three of which are located on the extracellular side of the receptor in and around the region of the pore-forming loop. We report that the TRPV1 channel in transfected HEK293T cells and in cultured rat DRG neurons is blocked in the open state by an oxidizing agent Cu-o-phenanthroline complex (Cu:Phe). The effects of Cu:Phe are concentration dependent ( IC50 = 5.2 : 20.8 microm ) and fully reversible. Cu:Phe applied immediately before exposure to an acidic solution, capsaicin or noxious heat is without effect. Substitutions of the extracellular Cys residues (616, 621, 634) by glycine individually or together do not alter the blocking effects of Cu:Phe suggesting that disulfide cross-linking does not represent the underlying mechanism. It is suggested that the complex Cu:Phe, a bulky, positively charged molecule, represents a very effective and reversible open channel blocker of TRPV1.

Membrane currents induced by L-homocysteic acid in mouse cultured hippocampal neurons.

The concentration-response relationship of membrane currents induced by L-homocysteic acid was studied on mouse embryonic hippocampal neurons in culture (n = 56). In the majority of neurons two phases in the dose-response relationship could be distinguished. The first was characterized by responses to 3-100 microM L-homocysteic acid which desensitized with a time-constant greater than 1 s in a concentration-dependent manner and were antagonized by 30 microM D-L-2-amino-5-phosphonovaleric acid indicating activation of the N-methyl-D-aspartate receptors. At higher concentrations of L-homocysteic acid this component was strongly depressed. The second phase was characterized by sustained responses that were concentration-dependent (1 mM L-homocysteic acid maximum concentration tested) and were not blocked by D-L-2-amino-5-phosphonovaleric acid indicating activation of non-N-methyl-D-aspartate receptors. Eight neurons did not exhibit these two-phase characteristics in the concentration-response relationship at the beginning of the recording. The magnitude of responses to L-homocysteic acid was positively related to concentration and the responses were partially blocked by D-L-2-amino-5-phosphonovaleric acid. In these neurons, however, repeated applications of L-homocysteic acid at concentrations 30 microM up to 300 microM resulted in a long-lasting, three- to four-fold increase of the membrane current. This increase was completely blocked by D-L-2-amino-5-phosphonovaleric acid (50-100 microM) suggesting that it was produced by activation of receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Axotomy-induced change in the properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor channels in rat motoneurons.

Properties of (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor channels were studied in fluorescence-labelled control and axotomized motoneurons in spinal cord slices using a patch-clamp technique. Axotomy performed on the third postnatal day resulted in motoneuron death. Application of AMPA or kainate induced large whole-cell currents, but outside-out patches isolated from control motoneurons were either unresponsive or displayed only single-channel activity in response to rapid application of AMPA. Measurement of AMPA receptor channel openings in outside-out patches revealed multiple single-channel conductance levels: 12.2+/-1.0, 21. 9+/-1.5 and 32.6+/-3.2pS. In control motoneurons dialysed with spermine, the current-voltage relationship of responses induced by activation of AMPA receptor channels exhibited various degrees of inward rectification. The rectification index, the ratio of responses at +40 and -60mV, was used to compare the degree of inward rectification. The mean values of rectification index of responses to focal application of AMPA and AMPA receptor-mediated excitatory postsynaptic currents induced by focal electric stimulation were 0. 64+/-0.17 and 0.50+/-0.27, respectively. In axotomized motoneurons, the degree of rectification was significantly less for both responses induced by application of AMPA and for excitatory postsynaptic currents (0.91+/-0.09 and 0.95+/-0.12, respectively). Deactivation of AMPA receptors assessed from motoneuron excitatory postsynaptic currents at -70 mV was independent of postnatal age, with tau(fast)=0.88+/-0.35ms (A(fast)=78.2+/-11.8%) and tau(slow)=6. 3+/-3.2ms. In axotomized motoneurons, the decay time constants of excitatory postsynaptic currents were similar, tau(fast)=0.91+/-0. 42ms (A(fast)=85.8+/-12.6%) and tau(slow)=5.9+/-3.4ms. However, the mean amplitude of excitatory postsynaptic currents was only 43% of the amplitude recorded in control motoneurons. The results show that the current induced by activation of AMPA receptors in neonatal motoneurons is mediated by opening of both Ca(2+)-permeable and Ca(2+)-impermeable channels. As a result of axotomy, an experimental model of neurodegeneration, AMPA receptor channels in injured motoneurons destined to die become predominantly Ca(2+) impermeable. These findings suggest phenotypic control of AMPA receptor channel properties, presumably by affecting their subunit composition.

Reducing agent dithiothreitol facilitates activity of the capsaicin receptor VR-1.

The vanilloid receptor subtype 1 (VR1) is expressed in a sub-population of small dorsal root ganglion (DRG) neurones in mammals and serves as the common transducer of the pain-producing signals, such as noxious heat, acids and capsaicin [Caterina et al., Nature 389 (1997) 816-824; Tominaga et al., Neuron 21 (1998) 531-543]. On the extracellular side, VR1 has three cysteine residues at positions 616, 621 and 634. Here we report that dithiothreitol (DTT) (2-60 mM), an agent that maintains -SH groups of cysteines in a reduced state, greatly facilitates membrane currents induced by noxious heat or capsaicin (1 microM) in cultured DRG neurones from the rat and in VR1-transfected HEK293 cells. The effects of DTT are concentration-dependent and fully reversible. We suggest that the ratio between free sulfhydryl groups and disulfide bonds of the cysteine residues of VR1 pre-sets sensitivity of primary nociceptors to algogens and may represent a new target for treating some pain states in humans.

Intracellular spermine decreases open probability of N-methyl-D-aspartate receptor channels.

Spermine and related polyamines have been shown to be endogenous regulators of several ion channel types including ionotropic glutamate receptors. The effect of spermine on N-methyl-d-aspartate (NMDA) receptors in cultured rat hippocampal neurons was studied using single-channel and whole-cell patch clamp recordings. Intracellular spermine resulted in the dose-dependent inhibition of NMDA-induced responses. Spermine reversibly inhibited the single NMDA receptor channel activity in inside-out patches suggesting a membrane-delimited mechanism of action. Open probability of NMDA receptor channels was decreased in a dose-dependent manner. Mechanism of spermine-induced inhibition of NMDA receptor was different from that of intracellular Ca(2+)-induced NMDA receptor inactivation. Both pharmacological studies and single channel analysis indicate that in contrast to the effect of extracellular spermine the intracellular spermine effect is not dependent on the NMDA receptor subunit composition. We propose that intracellular spermine has a direct inhibitory effect on NMDA receptors that is different from calcium-induced NMDA receptor inactivation and spermine-induced voltage-dependent inhibition of AMPA/kainate receptors. Spermine-induced tonic change in the open probability of NMDA receptor channels may play a role in mechanisms underlying short-term changes in the synaptic efficacy.

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.

Evidence that excitatory amino acids not only activate the receptor channel complex but also lead to use-dependent block.

The effects of fast application of excitatory amino acids N-methyl-D-aspartate (NMDA), L-aspartate (ASP), L-glutamate (GLU), quisqualate (QU) and kainate (KAIN) were studied in neurons from the embryonic spinal cord of the chick in monolayer cultures by employing the 'patch clamp' technique in the 'whole cell' mode. It was found that NMDA, ASP, GLU and QU, but not KAIN, induced responses that exhibited several components. The early component decayed with a time constant of 2 s to a lower level of membrane current and discontinuation of the application was followed by an after-current which returned to the base-line with a time constant of about 7 s. It is suggested that NMDA, ASP, GLU and QU, but not KAIN, not only activate the receptor channel complex but also induce use-dependent block.

Glutamine-induced membrane currents in cultured chick spinal cord neurons.

The effects of L-glutamine (GLN) on cultured spinal cord neurons from the chick were studied in the whole cell mode of the patch clamp technique. GLN induced membrane currents rectified at positive membrane potentials (m.p.) and reversed polarity close to zero m.p. The dose-response curve was nearly linear at a semilogarithmic scale for concentrations of 10(-5) M-10(-2) M. Summation of the responses evoked by GLN (10(-3) M) and glycine (10(-3) M) was observed when these two amino acids were applied together, while no significant increase of the responses was present when GLN was applied together with L-glutamate (10(-3) M) or kainate (10(-3) M). It is suggested that GLN binds to the glutamate receptors and activates the same type of ionic channels as glutamate and kainate.

Voltage-dependent chloride channels with several substates in excised patches from mouse neuroblastoma cells.

Single channels in mouse neuroblastoma cells with a high conductance of about 400 pS were described using the patch-clamp technique in the inside-out configuration. The channels were selective for Cl- as compared to cations and exhibited a linear I-V relationship between +40 and -40 mV. These Cl- channels were voltage-dependent and were activated by both depolarizing and hyperpolarizing potential steps from 0 mV to 10-40 mV. They closed, becoming inactivated, in tens of milliseconds (for depolarization) up to tens of seconds (for hyperpolarization) after each potential step. The typical feature of Cl- channels described was the dissipation of their conductance into several substates during the course of individual recordings.

Procaine excites nociceptors in cultures from dorsal root ganglion of the rat.

Procaine, a classical local anesthetic, produces, at low concentration (2-200 microM), excitation in a distinct population of small sensory neurons isolated from newborn rats (2D) and cultured for 1-5 days. The excitation or inward current (>50 pA) induced by procaine was observed in 59 out of 78 neurons. Nearly all these procaine-sensitive neurons (56 of 59) were also sensitive to capsaicin while 8 procaine-insensitive neurons responded to capsaicin (1 microM). In procaine-sensitive neurons tested for responsiveness to noxious heat, a 10 s temperature ramp from 24 to 48 degrees C induced an inward current of 413 +/- 47 pA (SEM, n = 27) and this current was enhanced, in the presence of procaine, about 3-fold (2.8 +/- 0.4, SEM, n = 27). The responses to procaine were concentration dependent and underwent pronounced tachyphylaxis after repeated applications. The voltage-current relationship exhibited outward rectification and the apparent reversal at 25 +/- 4.2 mV (SEM, n = 9) suggesting that the current is carried by cations including Ca2+. This procaine effect may offer an explanation for toxic consequence of the clinical use of local anesthetics.

Ionic currents in neuroblastoma clone E-7 cells.

Ionic currents were studied in exponentially growing neuroblastoma cells (clone E-7) derived from mouse neuroblastoma C-1300 with the patch-clamp technique in the whole cell mode (Pflügers Arch., 391 (1981) 85-100). In differentiated cells, with one or several processes, an early inactivating inward current approximately equal to 50 microA/cm2 was observed in response to depolarizing steps from the holding potential -60 to -70 mV, which was insensitive to 2 microM tetrodotoxin but readily blocked by Co2+ (6 mM). This inward current was followed by a delayed outward current which was eliminated by 12 mM tetraethylammonium. In the undifferentiated cells, only delayed outward current was observed. It is suggested that, in differentiated cells both Ca2+ and delayed rectifier K+ channels exist, while only the latter are present in undifferentiated cells.

Cobalt ions block L-glutamate and L-aspartate-induced currents in cultured neurons from embryonic chick spinal cord.

The effects of Co2+ on L-glutamate and L-aspartate responses wee studied in cultured spinal cord neurons of the embryonic spinal cord of the chick by employing the patch-clamp technique in whole cell mode [9]. It was found that Co2+ blocks the responses at negative membrane potentials for both amino acids, while only partial inhibition was observed at positive membrane potentials. Co2+ alone decreases the resting membrane current which exhibits reversal close to zero. It is suggested that the effects of Co2+ are produced by non-specific interaction with negative charges on the outer side of the membrane.

Dual effects of muscarinic M2 receptors on the synthesis of cyclic AMP in CHO cells: background and model.

It has been observed in several laboratories that muscarinic agonists have dual effects on the synthesis of cyclic AMP in cell lines expressing muscarinic M2 or M4 receptors, producing strong inhibition at low agonist concentrations and lesser inhibition or stimulation at high agonist concentrations. Data obtained on CHO cells (known to express adenylyl cyclases VI and VII) are best interpreted on the assumption that the upward phase of the concentration-response curves reflects simultaneous inhibition of adenylyl cyclase VI via the Gi proteins, with which the M2 and M4 receptors communicate with high affinity, and stimulation of adenyly cyclases VI and VII via the Gs proteins, with which the M2 and M4 receptors communicate with low affinity. A simplified model is described which permits one to predict how the shapes of the concentration-response curves will be affected by changes in the concentration of receptors, the affinities of activated receptors for Gi or Gs proteins, and other parameters.

Capsaicin-induced membrane currents in cultured sensory neurons of the rat.

Membrane currents induced by capsaicin (CAPS) in cultured sensory neurons from 1- to 2-day-old rats were studied. Responses to CAPS (10 microM) exceeding 1 nA at -50 mV were found in smaller, usually bipolar or tripolar neurons in which GABA (30 microM) induced small or no response. Large, unipolar neurons, which exhibited large responses to GABA, were completely insensitive to CAPS (10 microM). In contrast to GABA, responses to CAPS exhibited a slow rise and slow decay and a marked tachyphylaxis after repeated CAPS applications at high concentrations which made it difficult to study the concentration-response relationship. In partially run-down neurons, which exhibited quasi stable responses, the slope of the ascending phase was concentration-dependent with an apparent association rate constant K1 9 x 10(4) [M-1s-1]. The time constant of the decay was 3.5 s, and was concentration-independent. However, in 5 neurones the EC50 measured from the first series of CAPS applications at increasing concentrations was 0.31 +/- 0.5 microM with a Hill coefficient 1.66 +/- 0.35. The responses to CAPS reversed at +10.4 +/- 2.5 mV suggesting that the current is carried nonselectively by monovalent cations and Ca2+. The channel conductance of CAPS-gated channels at -50 mV calculated from the mean membrane current and variance of the current noise in outside-out patches or measured directly was 28 pS (n = 5). It is suggested that the CAPS-gated channels are either controlled by receptors with a very high affinity or that the channels are controlled by membrane-bound protein(s) which do not depend in their function on the supply of GTP or other intracellular metabolites.