Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.azobenzene photoswitch | purinergic receptors O ptogenetic approaches in neuroscience rely on the heterologous expression of engineered light-gated ion channels or pumps to trigger or inhibit electrical activity of selected neurons (1, 2). This powerful and revolutionizing technique provides an exquisite remote control of neuronal circuits that drive behavior in animals. Of special interest is the optogenetic pharmacology (also known as optochemical genetic) (3, 4), which allows the control of an ion channel or receptor function by a photoswitchable ligand that is irreversibly tethered to the genetically modified protein through cysteine substitution (3, 4). Ligands are pharmacologically active substances targeting either competitive (5-7), or noncompetitive binding sites (8-10), and light sensitivity is mostly conferred by substituting a photoisomerizable azobenzene derivative, which interconverts reversibly between a long trans-isomer and a short cis-isomer (11). However, this approach is technically demanding, as it requires the design of site-specific ligands for each target. The development of versatile methods with generic photoswitchable molecules would thus improve the optochemical strategy.Here, we present optogating, a unique method for the optical control of ATP-activated P2X channel gating. P2X receptors are a family of trimeric, cation-selective channels (12, 13) comprising seven mammalian subtypes that mediate a variety of physiological responses, including fast synaptic transmission, contraction of smooth muscle, modulation of neurotransmitter release, and pain sensation (12,14,15). P2X receptors are considered as emerging therapeutic targets because of their link to cancer (16), inflammatory (17), and neuronal diseases, including neuropathic pain (18). Inspired by previous works showing that chemical modification of the transmembrane (TM) pore region of the P2X2 receptor affects channel gating through labeling of single cysteine mutants by p...

Section: Discussionmentioning

confidence: 74%

“…2C). These persistent effects demonstrate bistability of the optogating method, a key feature that was already observed for other receptors (6,24); it allows tethered azobenzenes to remain in the dark either in their cis or trans isomer following brief illuminations. These data establish a stable photocontrol of channels.…”

Section: Resultsmentioning

confidence: 99%

Optical control of an ion channel gate

Lemoine

Habermacher

Martz

et al. 2013

“…One promising alternative to microbial opsins is an optopharmacological strategy that uses synthetic azobenzene-based photoswitches to endow light sensitivity either to native ion channels of neurons (17,18) or to engineered mammalian receptors and channels that, like the microbial opsins, allow for genetic targeting to specific cells (19)(20)(21)(22). We previously showed that an engineered light-gated ionotropic glutamate receptor (LiGluR) restores light responses to blind retina degeneration (rd1) mice (23).…”

Section: Significancementioning

confidence: 99%

Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells

Gaub

Berry²,

Holt³

et al. 2014

108

137

Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second-and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0 460 ). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform lightevoked firing when LiGluR-MAG0 460 was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0 460 was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0 460 in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rodcone dystrophy dog model of blindness, LiGluR-MAG0 460 in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0 460 was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.retinal gene therapy | visual prosthetics | retinitis pigmentosa | optogenetic pharmacology | azobenzene photoswitches

“…The increase in our knowledge of molecular and atomic structure of ligand-gated channels over the past 10 years has allowed one such approach (photoswitchable tethered ligands) to become much more sophisticated, because cysteines can be introduced into the channel protein exactly where required to form an attachment point. The method has been applied to pentameric nicotinic receptors (6) and tetrameric glutamate receptors (7,8). Although attaching ligands through photoswitchable tethers is proving extremely valuable, an intimate structural knowledge of a closed and open state of a channel also allows for optical control of conformation at parts of the protein that are remote from the agonist binding site (9)(10)(11) 16).…”

mentioning

confidence: 99%

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

Browne¹,

Nunes

Sim

et al. 2013

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. Lightactivated 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. P 2X receptors and acid-sensing ion channels (ASICs) are trimeric membrane ion channels gated by binding extracellular ligands. P2X receptors are gated by extracellular ATP, and their physiological roles include neuroeffector transmission, primary afferent transmission (e.g., taste, hearing, chemoreception), central control of respiration, and neuroinflammation (1-3). ASICs are gated by protons and are involved in pain sensation (4, 5). The experimental study of ligand-gated channels in intact tissues is often hampered by difficulties in application of the appropriate ligand while recording ion channel activity in the millisecond time domain, and there are advantages to controlling channel activation by surrogate optical methods. The increase in our knowledge of molecular and atomic structure of ligand-gated channels over the past 10 years has allowed one such approach (photoswitchable tethered ligands) to become much more sophisticated, because cysteines can be introduced into the channel protein exactly where required to form an attachment point. The method has been applied to pentameric nicotinic receptors (6) and tetrameric glutamate receptors (7,8). Although attaching ligands through photoswitchable tethers is proving extremely valuable, an intimate structural knowledge of a closed and open state of a channel also allows for optical control of conformation at parts of the protein that are remote from the agonist binding site (9-11).High-resolution structures are available for P2X receptors (closed: ref. 12; open: ref. 13) and ASICs (closed: refs. 14 and 15; open: ref. 16). In both these trimeric channels the second of the...