Optical control of trimeric P2X receptors and acid-sensing ion channels.

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis-trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

Ectodomain movements of an ATP-gated ion channel (P2X2 receptor) probed by disulfide locking.

The ectodomain of the P2X receptor is formed mainly from two- or three-stranded ß-sheets provided symmetrically by each of the three subunits. These enclose a central cavity that is closed off furthest from the plasma membrane (the turret) and that joins with the transmembrane helices to form the ion permeation pathway. Comparison of closed and open crystal structures indicates that ATP binds in a pocket positioned between strands provided by different subunits and that this flexes the ß-sheets of the lower body and enlarges the central cavity: this pulls apart the outer ends of the transmembrane helices and thereby opens an aperture, or gate, where they intersect within the membrane bilayer. In the present work, we examined this opening model by introducing pairs of cysteines into the rat P2X2 receptor that might form disulfide bonds within or between subunits. Receptors were expressed in human embryonic kidney cells, and disulfide formation was assessed by observing the effect of dithiothreitol on currents evoked by ATP. Substitutions in the turret (P90C, P89C/S97C), body wall (S65C/S190C, S65C/D315C) and the transmembrane domains (V48C/I328C, V51C/I328C, S54C/I328C) strongly inhibited ATP-evoked currents prior to reduction with dithiothreitol. Western blotting showed that these channels also formed predominately as dimers and/or trimers rather than monomers. The results strongly support the channel opening mechanism proposed on the basis of available crystal structures.

P2X receptor intermediate activation states have altered nucleotide selectivity.

Purinergic P2X receptors are widely distributed in the nervous system and are known to play roles in primary afferent transmission and central respiratory regulation. They are trimeric membrane proteins, with the extracellular domain that provides three intersubunit ATP binding sites. We expressed the rat P2X7 receptor in human embryonic kidney cells and measured membrane currents before and after photo-affinity labeling with the agonist 2'(3')-O-(4-benzoylbenzoyl)-ATP (BzATP). After tethering BzATP with ultraviolet light, a persistent current remained after washing out the agonist. Additional current could now be elicited by other nucleotides (CTP and ADP) that are not normally effective as P2X receptor agonists. Similar results were obtained at P2X2 receptors even without previous agonist tethering: exposure to low concentrations of ATP caused the receptor to become sensitive to activation by CTP and ADP. The results show that ATP binding to the first of the three binding sites causes a conformational change to an intermediate closed state that shows increased effectiveness of pyrimidine and diphosphate nucleotide analogs.

P2X7 receptor channels allow direct permeation of nanometer-sized dyes.

P2X receptors are widely distributed in the nervous system, and P2X7 receptors have roles in neuropathic pain and in the release of cytokines from microglia. They are trimeric membrane proteins, which open an integral ion channel when ligated by extracellular ATP. This channel is preferentially permeable to small cations (sodium, potassium, calcium) but also allows permeation of larger cations such as N-methyl-d-glucamine. ATP also leads to entry of fluorescent dyes in many cells expressing P2X7 receptors, but controversy persists as to whether such large molecules pass directly through the open ion channel or enter the cell by a different route. We measured cellular fluorescence and membrane currents in individual human embryonic kidney cells expressing rat P2X7 receptors. Introduction of positive side chains by mutagenesis into the inner half of the pore-forming second transmembrane domain of the receptor (T348K, D352N, D352K) increased relative permeability of chloride ions. It also promoted entry of the large (>1 nm) negative dye fluorescein-5-isothiocyanate while decreasing entry of the structurally similar but positive dye ethidium. Furthermore, larger cysteine-reactive methanethiosulfonates [sulforhodamine-methanethiosulfonate and 2-((biotinoyl)amino)ethyl methanethiosulfonate] reduced both ATP-evoked currents and dye entry when applied to open P2X7[G345C] receptors. The results demonstrate that the open channel of the P2X7 receptor can allow passage of molecules with sizes up to 1.4 nm.

Scanned optogenetic control of mammalian somatosensory input to map input-specific behavioral outputs.

Somatosensory stimuli guide and shape behavior, from immediate protective reflexes to longer-term learning and higher-order processes related to pain and touch. However, somatosensory inputs are challenging to control in awake mammals due to the diversity and nature of contact stimuli. Application of cutaneous stimuli is currently limited to relatively imprecise methods as well as subjective behavioral measures. The strategy we present here overcomes these difficulties, achieving 'remote touch' with spatiotemporally precise and dynamic optogenetic stimulation by projecting light to a small defined area of skin. We mapped behavioral responses in freely behaving mice with specific nociceptor and low-threshold mechanoreceptor inputs. In nociceptors, sparse recruitment of single-action potentials shapes rapid protective pain-related behaviors, including coordinated head orientation and body repositioning that depend on the initial body pose. In contrast, activation of low-threshold mechanoreceptors elicited slow-onset behaviors and more subtle whole-body behaviors. The strategy can be used to define specific behavioral repertoires, examine the timing and nature of reflexes, and dissect sensory, motor, cognitive, and motivational processes guiding behavior.

To safely navigate their world, animals need to be able to tell apart a gentle touch from an eye-watering pinch, detect cold water or sense the throbbing pain stemming from an infected cut. These 'somatic' sensations are relayed through thousands of nerve endings embedded in the skin and other tissues. Yet the neurological mechanisms that underpin these abilities are complex and still poorly understood. Indeed, these nerve endings can be stimulated by extreme temperatures, harmful chemicals, friction or even internal signals such as inflammation. One event can also recruit many different types of endings: a cut for example, will involve responses to mechanical pressure, tissue damage and local immune response. To disentangle these different actors and how they affect behavior, scientists need to develop approaches that allow them to deliver specific stimuli with increased precision, and to monitor the impact on an animal. To achieve this goal, Schorscher-Petcu et al. used mice in which blue light could trigger specific types of nerve endings. For instance, depending on the genetic background of the animals, a laser could either activate nerve endings involved in pain or gentle touch. Crucially, this could be done from a distance by beaming light with exquisite precision onto the paws of the mice without physically touching or disturbing the animals. How the mice responded could then be observed without any interference. Their behavior was analyzed using a combination of high-speed videos, computer-driven recording systems, and machine learning. This revealed subtle changes in behavior that had not been detected before, spotting microscopic movements of the stimulated paw and mapping simultaneous whole-body movements such as changes in posture or head orientation. The approach therefore allows scientists to assess the impact of touch, pain or temperature sensation in freely behaving mice. It could also be harnessed to develop much needed treatments against chronic pain.

Epineural optogenetic activation of nociceptors initiates and amplifies inflammation.

Activation of nociceptor sensory neurons by noxious stimuli both triggers pain and increases capillary permeability and blood flow to produce neurogenic inflammation1,2, but whether nociceptors also interact with the immune system remains poorly understood. Here we report a neurotechnology for selective epineural optogenetic neuromodulation of nociceptors and demonstrate that nociceptor activation drives both protective pain behavior and inflammation. The wireless optoelectronic system consists of sub-millimeter-scale light-emitting diodes embedded in a soft, circumneural sciatic nerve implant, powered and driven by a miniaturized head-mounted control unit. Photostimulation of axons in freely moving mice that express channelrhodopsin only in nociceptors resulted in behaviors characteristic of pain, reflecting orthodromic input to the spinal cord. It also led to immune reactions in the skin in the absence of inflammation and potentiation of established inflammation, a consequence of the antidromic activation of nociceptor peripheral terminals. These results reveal a link between nociceptors and immune cells, which might have implications for the treatment of inflammation.

Controlling Engineered P2X Receptors with Light.

This chapter details methods to express and modify ATP-gated P2X receptor channels so that they can be controlled using light. Following expression in cells, a photoswitchable tool compound can be used to covalently modify mutant P2X receptors, as previously demonstrated for homomeric P2X2 and P2X3 receptors, and heteromeric P2X2/3 receptors. Engineered P2X receptors can be rapidly and reversibly opened and closed by different wavelengths of light. Light-activated P2X receptors can be mutated further to impart ATP-insensitivity if required. This method offers control of specific P2X receptor channels with high spatiotemporal precision to study their roles in physiology and pathophysiology.

Optical cuff for optogenetic control of the peripheral nervous system.

OBJECTIVE: Nerves in the peripheral nervous system (PNS) contain axons with specific motor, somatosensory and autonomic functions. Optogenetics offers an efficient approach to selectively activate axons within the nerve. However, the heterogeneous nature of nerves and their tortuous route through the body create a challenging environment to reliably implant a light delivery interface. APPROACH: Here, we propose an optical peripheral nerve interface-an optocuff-, so that optogenetic modulation of peripheral nerves become possible in freely behaving mice. MAIN RESULTS: Using this optocuff, we demonstrate orderly recruitment of motor units with epineural optical stimulation of genetically targeted sciatic nerve axons, both in anaesthetized and in awake, freely behaving animals. Behavioural experiments and histology show the optocuff does not damage the nerve thus is suitable for long-term experiments. SIGNIFICANCE: These results suggest that the soft optocuff might be a straightforward and efficient tool to support more extensive study of the PNS using optogenetics.

Time-Resolved Fast Mammalian Behavior Reveals the Complexity of Protective Pain Responses.

Potentially harmful stimuli are detected at the skin by nociceptor sensory neurons that drive rapid protective withdrawal reflexes and pain. We set out to define, at a millisecond timescale, the relationship between the activity of these sensory neurons and the resultant behavioral output. Brief optogenetic activation of cutaneous nociceptors was found to activate only a single action potential in each fiber. This minimal input was used to determine high-speed behavioral responses in freely behaving mice. The localized stimulus generated widespread dynamic repositioning and alerting sub-second behaviors whose nature and timing depended on the context of the animal and its position, activity, and alertness. Our findings show that the primary response to injurious stimuli is not limited, fixed, or localized, but is dynamic, and that it involves recruitment and gating of multiple circuits distributed throughout the central nervous system at a sub-second timescale to effectively both alert to the presence of danger and minimize risk of harm.

Pharmacological properties of the rhesus macaque monkey P2X7 receptor.

BACKGROUND AND PURPOSE: The human P2X7 (hP2X7) receptor exhibits striking pharmacological differences from its rodent counterparts, particularly in terms of its antagonist profile. Here, we characterized the functional and pharmacological properties of the rhesus macaque monkey P2X7 (rmP2X7) receptor in comparison with the hP2X7 receptor. EXPERIMENTAL APPROACH: The rmP2X7 and hP2X7 receptors were heterologously expressed in HEK293 cells. The receptor surface and total expression levels were examined by biotin-labelling and Western blotting. The functional and pharmacological properties were characterized using patch-clamp recording and single-cell imaging. KEY RESULTS: The rmP2X7 receptor showed strong cell surface expression. Both ATP and 2'(3')-O-(4-benzoylbenzoyl) adenosine-5'-triphosphate (BzATP) were full agonists in activating the rmP2X7 receptor; the EC50 values were 802 µM for ATP and 58 µM for BzATP, respectively, in extracellular low divalent cation solution. Prolonged activation of the rmP2X7 receptors induced detectable but low level YO-PRO-1 uptake. KN-62, AZ11645373 and A-438079, three hP2X7 selective antagonists, all potently inhibited the rmP2X7 receptor-mediated currents; the IC50 values were 86, 23 and 297 nM respectively. CONCLUSION AND IMPLICATIONS: The rmP2X7 receptor exhibits similar pharmacological properties to the hP2X7 receptor. The rhesus macaque monkey thus may represent a valuable model species in elucidating the mechanisms and pharmacological interventions of hP2X7 receptor-related diseases.

P2X receptor channels show threefold symmetry in ionic charge selectivity and unitary conductance.

In the closed structure of the P2X cation channel, three α-helical transmembrane domains cross the membrane obliquely. In rat P2X2 receptors, these intersect at Thr(339). Replacing Thr(339) by lysine in one, two or three subunits progressively increased chloride permeability and reduced unitary conductance. This implies that the closed-open transition involves a symmetrical separation of the three subunits and that Thr(339) from each subunit contributes symmetrically to the open channel permeation pathway.

New structure enlivens interest in P2X receptors.

P2X receptors are ATP-gated membrane ion channels with multifarious roles, including afferent sensation, autocrine feedback loops, and inflammation. Their molecular operation has been less well elucidated compared with other ligand-gated channels (nicotinic acetylcholine receptors, ionotropic glutamate receptors). This will change with the recent publication of the crystal structure of a closed P2X receptor. Here we re-interpret results from 15 years of experiments using site-directed mutagenesis with a model based on the new structure. Previous predictions of receptor stoichiometry, the extracellular ATP binding site, inter-subunit contacts, and many details of the permeation pathway fall into place in three dimensions. We can therefore quickly understand how the channel operates at the molecular level. This is important not only for ion- channel aficionados, but also those engaged in developing effective antagonists at P2X receptors for potential therapeutic use.

Functional and pharmacological properties of human and rat NaV1.8 channels.

The aim of this work is to characterise the functional properties of human and rat Na(V)1.8 channels and to investigate the action of anti-nociceptive agents. Na(V)1.8 alpha-subunits were expressed in mammalian sensory neuron-derived ND7/23 cells, and sodium currents were recorded using whole-cell patch clamp. The current-voltage curves for activation were similar for human and rat Na(V)1.8 channels. However, for inactivation, human Na(V)1.8 showed more hyperpolarised voltage-dependence than for the rat channel, faster development of inactivation, slower recovery from the fast component of inactivation, and faster recovery from the slow component. Thus, this would imply that the human channel is more inactivated at normal resting potentials. Compounds 227c89, A-803467, V102862, ralfinamide and tetracaine all showed greater affinity for the inactivated state than for the resting state. Compounds A-803467 and V102862 were the most potent, and A-803467 showed greater inactivated state affinity for human than for rat channels. Surprisingly, during recovery from inactivation, an increase in current was observed for V102862 and A-803467, probably due to disinhibition of resting block. Rather than the use-dependent inhibition normally seen with inactivated state blockers, for A-803467 this disinhibition led to an increase in current during repetitive stimulation, while V102862 showed less inhibition than otherwise expected at lower frequencies. Thus the data supports the suggestion that, while both V102862 and A-803467 are potent inhibitors of Na(V)1.8, the compound V102862, rather than A-803467, may be useful as an analgesic where physiological firing frequencies are higher.

Structural determinants of drugs acting on the Nav1.8 channel.

The aim of this work is to study the role of pore residues on drug binding in the Na(V)1.8 channel. Alanine mutations were made in the S6 segments, chosen on the basis of their roles in other Na(V) subtypes; whole cell patch clamp recordings were made from mammalian ND7/23 cells. Mutations of some residues caused shifts in voltage dependence of activation and inactivation, and gave faster time course of inactivation, indicating that the residues mutated play important roles in both activation and inactivation in the Na(V)1.8 channel. The resting and inactivated state affinities of tetracaine for the channel were reduced by mutations I381A, F1710A, and Y1717A (for the latter only inactivated state affinity was measured), and by mutation F1710A for the Na(V)1.8-selective compound A-803467, showing the involvement of these residues for each compound, respectively. For both compounds, mutation L1410A caused the unexpected appearance of a complete resting block even at extremely low concentrations. Resting block of native channels by compound A-803467 could be partially removed ("disinhibition") by repetitive stimulation or by a test pulse after recovery from inactivation; the magnitude of the latter effect was increased for all the mutants studied. Tetracaine did not show this effect for native channels, but disinhibition was seen particularly for mutants L1410A and F1710A. The data suggest differing, but partially overlapping, areas of binding of A-803467 and tetracaine. Docking of the ligands into a three-dimensional model of the Na(V)1.8 channel gave interesting insight as to how the ligands may interact with pore residues.