Proton-activated Ion Channels Research Articles

Physiological and Pharmacological Characteristics of Native Proton-Activated Ion Channels

Proton-activated ion channels (ASICs) are widely distributed in the CNS and take part in many physiological processes. We report here studies characterizing pH-induced currents in various neuron types. Overall, the data provide evidence that currents in all these neurons are mediated mainly by heterodimeric ASICs; interneurons in the striatum radiatum of the hippocampus, pyramidal neurons in layer 2 of the medial prefrontal cortex, and interneurons in the striatum have receptors with weak pH sensitivity and pharmacological profiles combining the features of both ASIC1a and ASIC2a subunits. Pyramidal cells in layer 3 of the medial prefrontal cortex also have the same pharmacological profile but greater sensitivity to protons. In cerebellar Purkinje cells, heteromeric receptors are formed mainly from ASIC1a and ASIC2b subunits, as there is no sensitivity to ASIC2a-specific ligands though currents are induced by weak acidification.

The Chimeric Approach Reveals That Differences in the TRPV1 Pore Domain Determine Species-specific Sensitivity to Block of Heat Activation

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.

Ligand-Gated Pentameric ion Channels, from Binding to Gating

X-ray structures of molluscan acetylcholine-binding proteins and procaryotic proton-activated ion channels (ELIC and GLIC) enable us to model the ligand binding and activation mechanism of ligand-gated pentameric ion channels. Common versus distinct features can be deduced from the binding of agonists, antagonists and allosteric modulators in subunit interfaces of nicotinic acetylcholine, A-type gamma-aminobutyric acid, glycine and 5-HT(3)-type serotonin receptors. Ligand interactions in subunit interfaces elicit conformational waves from the closure of the agonist-binding cavity through binding loops, beta-strands and transmembrane helices to pore gating.

Ligand-Gated Pentameric ion Channels, from Binding to Gating

X-ray structures of molluscan acetylcholine-binding proteins and procaryotic proton-activated ion channels (ELIC and GLIC) enable us to model the ligand binding and activation mechanism of ligand-gated pentameric ion channels. Common versus distinct features can be deduced from the binding of agonists, antagonists and allosteric modulators in subunit interfaces of nicotinic acetylcholine, A-type γ-aminobutyric acid, glycine and 5-HT3-type serotonin receptors. Ligand interactions in subunit interfaces elicit conformational waves from the closure of the agonist-binding cavity through binding loops, ß-strands and transmembrane helices to pore gating. Keywords: AChBP, pLGIC, nicotinic receptors, GABAA/C receptors, glycine receptors, 5-HT3 receptors, interface binding cavity, binding-gating coupling, allostery, homology modeling

Interaction of the Aromatics Tyr-72/Trp-288 in the Interface of the Extracellular and Transmembrane Domains Is Essential for Proton Gating of Acid-sensing Ion Channels

Acid-sensing ion channels are proton-activated ion channels expressed in the nervous system. They belong to the family of ENaC/Degenerins whose members share a conserved structure but are activated by widely diverse stimuli. We show that interaction of two aromatic residues, Tyr-72, located immediately after the first transmembrane segment, and Trp-288, located at the tip of a loop of the extracellular domain directed toward the first transmembrane segment, is essential for proton activation of the acid-sensing ion channels. The subdomain containing Trp-288 is a module tethered to the rest of the extracellular domain by short linkers and intrasubunit interactions between residues in the putative “proton sensor.” Mutations in these two areas shift the apparent affinity of protons toward a more acidic range and change the kinetics of activation and desensitization. These results are consisting with displacement of the module relative to the rest of the extracellular domain to allow interaction of Trp-288 with Tyr-72 during gating. We propose that such interaction may provide functional coupling between the extracellular domain and the pore domain.

Projected pain from noxious heat stimulation of an exposed peripheral nerve – A case report

Distinct sensory properties of unmyelinated axons in the isolated rat sciatic nerve have previously been revealed by measuring stimulated neuropeptide (CGRP) release in response to noxious stimuli. Axonal sensitization to heat by inflammatory mediators has been demonstrated and shown to depend on the heat- and proton-activated ion channel TRPV1. Recently, we have demonstrated in vitro that heat stimulation of nociceptive axons generates ectopic action potential discharge which resembles the heat response of the corresponding cutaneous nerve endings. It remained however, to be established whether adequate axonal stimulation could also generate projected sensations in a conscious human subject. In a singular human trial, the superficial radial nerve (SR) was exposed and stimulated mechanically as well as with noxious cold (3 °C). These stimuli were unable to induce any conscious local or projected sensations. However, controlled radiant heat applied to the nerve resulted in intense slowly adapting burning pain sensations projected into the center of the SR innervation area. No local sensation was reported. Thus, presumably activated nervi nevorum in the sheath of a healthy nerve do not cause conscious sensations, while axons of passage in mid-nerve exhibit a sensory transduction capacity for noxious heat though not for mechanical and cold stimulation. Axonal heat transduction may therefore become a source of ectopic discharge and neuropathic pain when heat threshold drops to body temperature as is the case with peripheral nerve endings in inflamed skin.

Sensory Transduction in Peripheral Nerve Axons Elicits Ectopic Action Potentials

Sensory properties of unmyelinated axons in the isolated rat sciatic nerve have been revealed previously by measuring stimulated neuropeptide release in response to noxious stimuli. In addition, axonal sensitization by inflammatory mediators has been demonstrated and shown to depend on the heat- and proton-activated ion channel transient receptor potential vanilloid receptor-1. It was unclear whether this responsiveness is accompanied by ectopic generation of action potentials, which may play a crucial role in painful neuropathies. We explored this hypothesis using the isolated mouse skin-nerve preparation. This method enabled us to directly compare the sensory properties of axons in the peripheral nerve with their characterized cutaneous terminals in the receptive field using propagated action potentials as an index of axonal activation. Single-fiber recordings from 51 mechanosensitive mouse C-fibers revealed that a majority of the polymodal nociceptors responded with an encoding discharge rate to graded heating of the cutaneous receptive field (n = 38) as well as of the saphenous nerve carrying the fiber under investigation (n = 25; 66%). Axonal heat responses paralleled those of the receptive fields with regard to thresholds and discharge rates (41.5 +/- 4.3 degrees C; 7.7 +/- 9.6 spikes in a 20 s 32-48 degrees C ranged stimulation). In contrast, axonal mechanosensitivity was poor and noxious cold sensitivity more rarely encountered. In conclusion, peripheral nerve axons exhibit sensory transduction capacities similar to their nociceptive terminals in the skin with respect to noxious heat, although not to mechanical and cold sensitivity. This may become a source of ectopic discharge and pain if axonal heat threshold drops to body temperature, as may be the case during inflammation-like processes in peripheral nerves.

Dynamics of desensitization and recovery of proton-activated ion channels in pheochromocytoma cells

Proton-activated ion channels of the ASIC 1a subtype in pheochromocytoma PC12 cells were inactivated under the long-lasting influence of an acidic milieu (pH 6.0) because of desensitization developing in a monoexponential mode with τ = 1.7 ± 0.3 sec. The recovery of currents after desensitization was also monoexponential, with τ = 4.9 ± 0.8 sec. Approximation of the dose-effect curve by the Hill equation showed that activation of a channel of this type needs, most probably, binding of three hydrogen ions.

Algogenic Peripheral Effects of RFa Peptides

Earlier, we showed on a nerve-skin preparation that FMRFa and other RFa-like peptides activate a considerable proportion of somatosensory nociceptive afferent units (C fibers). To check the algogenic effect of RFa peptides in vivo, we recorded the parameters of a few behavioral phenomena after subcutaneous injections of solutions of FMRFa and KNFLRFa into the hindlimb of mice. Introduction of the above peptides evoked intensive pain reactions of the animals manifested in long-lasting licking of the injected limb and an increase in the number of the cycles of licking of this limb. We hypothesize that interaction of RFa peptides with proton-activated ion channels (ASICs) in the membranes of sensory nociceptive neurons is one of the possible mechanisms of this nociceptive reaction.

Responses Evoked in Afferent Fibers by Mechanostimulation of the Skin in vitro: Modulation by RFa-Like Peptides

In experiments in vitro on a skin patch-n. saphenous preparation, we studied the effects of RFa-like peptides on the responses of single slow-and fast-conducting afferent fibers evoked by mechanostimulation of the skin. RFa-like peptides were shown to exert modulatory influences on the mechanostimulation-evoked activity in a significant proportion of such units; a considerable proportion of the effects in the fibers of different types were of opposite direction. In most nociceptive thin C fibers, responses evoked by mechanostimulation of the skin were facilitated by the peptides, while those in A fibers were suppressed. It is supposed that RFa-like peptides are involved in the formation of the hyperalgesia state. The effect of these peptides on proton-activated ion channels (ASICs) is one of the possible mechanisms of an increase in the sensitivity of afferent fibers to mechanical stimuli. This modulatory effect was observed both in an acidified (pH 5.2) and in a normal (pH 7.4) media; this is why another mechanism exists that is not related to ASICs. A suppressory effect of RFa peptides on the mechanostimulation-evoked activity of primary afferents developed with a significant delay; probably, it is mediated by the effects of peptides on G proteins.

Excitatory amino acid receptor-channels in Purkinje cells in thin cerebellar slices

Glutamate receptors of the N-methyl-D-aspartate (NMDA) and non-NMDA type serve different functions during excitatory synaptic transmission. Although many central neurons bear both types of receptor, the evidence concerning the sensitivity of cerebellar Purkinje cells to NMDA is contradictory. To investigate the receptor types present in Purkinje cells, we have used whole-cell and outside-out patch-clamp methods to record from cells in thin cerebellar slices from young rats. At a holding potential of -70 mV (in nominally Mg(2+)-free medium, with added glycine) NMDA caused a whole-cell current response which consisted of a dramatic increase in the frequency of synaptic currents. In the presence of tetrodotoxin (TTX) and the gamma-aminobutyric acidA (GABAA) receptor antagonist bicuculline, spontaneous synaptic currents and responses to NMDA were inhibited. In a proportion of cells a small polysynaptic response to NMDA persisted, which was further reduced by the non-NMDA receptor antagonist 6-cyano-2,3-dihydro-7-nitroquinoxalinedione (CNQX). The non-NMDA glutamate receptor agonists kainate (KA), quisqualate (QA) and s-alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (s-AMPA), evoked large inward currents due to the direct activation of receptors in Purkinje cells. NMDA applied to excised membrane patches failed to evoke any single-channel currents, whereas s-AMPA and QA caused small inward currents accompanied by marked increases in current noise. Spectral analysis of the s-AMPA noise in patches gave an estimated mean channel conductance of approximately 4 pS.(ABSTRACT TRUNCATED AT 250 WORDS)