User:Nevaldiaa/sandbox

Light-gated ion channel
Light-gated ion channels are a family of ion channels regulated by electromagnetic radiation. Other gating mechanisms for ion channels include voltage-gated ion channels, ligand-gated ion channels, mechanosensitive ion channels, and temperature-gated ion channels. Most light-gated ion channels have been synthesized in the laboratory for study, although two naturally occurring examples, Channelrhodopsin and Anion-conducting channelrhodopsin, are currently known. Photoreceptor proteins, which act in a similar manner to light-gated ion channels are generally G protein-coupled receptors and do not constitute an ion channel.

Mechanism
Light-gated ion channels function in a similar manner to other gated ion channels. These transmembrane proteins form pores through lipid bilayers to facilitate the passage of ions, whose movement is influenced by an electrochemical gradient, from one side of the membrane to another. When exposed to a certain stimulus, a conformational change occurs in the transmembrane region of the protein to open or close the ion channel. In the case of light-gated ion channels, the transmembrane proteins are usually coupled with a molecule that acts as a photoswitch, whereby photons serve to alter the conformation of these proteins, so that the pore changes from a closed state to an open state, and vice versa, thereby increasing or decreasing ion conductance. Retinal is a good example of a molecular photoswitch and is found in the naturally occurring Channelrhodopsins.

Synthetic isoforms
Once channelrhosopsin had been identified and characterized, researchers appreciated the value of modifying the channel's selectivity to increase anions permeability so as to maintain polarization of membrane potentials through optogenetic control. These mutated proteins successfully changed the charge lining the pore to exclude cations in favor of anions. Other ion channels have been modified by binding a photoswitch to confer photoregulation to the ion channel. This is done through careful selection of a tether which can lengthen or shorten through photoisomerization, binding one side of the tether to the ion channel and the other end to a blocking group with binding affinity for an exposed portion of the pore. When the tether is lengthened, it allows the blocking section to bind to the pore and preclude ion current. When the tether is shortened, it disrupts this binding and opens the pore without obstruction. Kinetic studies have demonstrated that fine temporal and spatial control can be achieved in this manner.

Azobenzene is a common choice for the functional portion of a tether for synthetically-developed light-gated ion channels because of its well documented length as either cis or trans isomers, as well as the excitation wavelength needed to induce photoisomerization. Azobenzene converts to its longer trans-isomer at λ=500 nm and its cis-isomer at λ=380 nm. [Banghart]

The expression of light-gated ion channels in a specific cell type through promoter control allows for regulation of cell potential by depolarizing the membrane to 0 mV for cation-permeant channelrhodopsin or by clamping the voltage to -67 mV for anion-conducting channelrhodopsin. [Berndt] Depolarization can occur on the timescale of action potentials and neurotransmitter exocytosis conducting a current in the range of 5 fA per channel. [Ishizuka] [Nagel] They have advantage over other types of regulation for ion channels in that they induce non-invasive, reversible membrane potential changes with fine temporal and spatial control granted by induction through laser stimuli. [Nagel] [Banghart] They reliably stimulate single action potentials with rapid depolarization and can be utilized in vivo because they do not require high intensity illumination to maintain function, unlike other techniques like photocaging. [Wietek] [Ishizuka]

Notes

Nagel - Native channelrhodopsin can be mutated to increase conductance and current flow. They can be expressed in live organisms and dissected tissue with protein functionality maintained. Physical (depolarization) and behavioral responses can be triggered through light stimulus if expressed in the appropriate location via promoters. It is permeable to sodium, potassium, protons and calcium. Advantages exist above photo-caged compounds. Resembles light-driven proton pump with seven transmembrane helices and intrinsic channel. All-trans retinal as chromophore.

Banghart - Azobenzene can act as a photoswitch to extend in trans to block a pore and contract in cis to activate the channel. This synthetic addition can be further regulated by various ligands to modify wavelength absorption. Non-invasive, reversible excitation with fine temporal and spatial control can be promoted by SPARK channels. Up to a 5 angstrom variance in block placement can change the channel from conducting to blocking. Figure 1B as good illustration of synthetic light-gated ion channel.

Ishizuka - Advantages of light-gated ion channels over contemporary techniques. Channelrhodopsin works in conjunction with a rhodopsin chromophore. Turning on takes less time than turning off and follow classical-type physics equations, at a speed comparable to exocytotic currents at fast CNS synapses. Non-selective to monovalent cations, depolarization can be caused in vivo. Criticism of artificial (SPARK) channels.

Jog - Beta-cyclodextrin together with azobenzene because of rapid isomerization coupled with large, geometric shift. Azobenzene bound to blocking site can adjust permeability through pi interactions with the azobenzene itself.

Wietek - Field of optogenetics. Reliably stimulate single AP with rapid depolarization. Light-driven proton pumps can maintain polarization, but require high intensity light. Retinal Schiff base as chromophore. Minor substitution of pore residues changed selectivity to chloride ion through higher anion affinity, increased pore flexibility and size due to H bonding disruption. Mimic GABAa receptors in inhibiting APs in vivo. Fully reversible fashion.

Berndt - 0 mV reversal potential due to non-selective cation permeability. Inability to engineer K+ selective channel. Seven Glu in channel. -61 mV reversal potential achieved with multiple substitutions, though effect remains dependent on pH. Multiple wavelengths of excitation are available for optogenetic experimentation.

Nagel - Native channelrhodopsin variants found in at least one bacteria. 7 transmembrane rhodopsin bound to retinal as chromophore. Channelrhodopsin = channelopsin + retinal. Two variants to mediate high and low intensity responses. Constant illumination can lead to desensitization of the channels. pH effects on permeability of various cations. Pore channel larger than Na+ channels, hence most ions travel through in a mostly dehydrated state. Ca+ is permeable, but Mg+ isn't, likely due to hydration. Desensitization occurs at a greater rate with positive membrane potential and high pH. Channelrhodopsin contains a channel, rather than activating one, as supported by the speed of opening. The protein is naturally expressed at a higher rate when grown in dark medium, and broken down under well-lit conditions (probably to protect the cell). It can depolarize mammalian cells. It has low, non-zero selectivity. Possible current of 5 fA per channel. Shifted absorbance can be observed through expression in different tissue. Alternative use to trigger Ca+ second messenger systems.