Light-activated ion channels for remote control of neuronal firing

Abstract

Neurons have ion channels that are directly gated by voltage, ligands and temperature but not by light. Using structure-based design, we have developed a new chemical gate that confers light sensitivity to an ion channel. The gate includes a functional group for selective conjugation to an engineered K + channel, a pore blocker and a photoisomerizable azobenzene. Long-wavelength light drives the azobenzene moiety into its extended trans configuration, allowing the blocker to reach the pore. Short-wavelength light generates the shorter cis configuration, retracting the blocker and allowing conduction. Exogenous expression of these channels in rat hippocampal neurons, followed by chemical modification with the photoswitchable gate, enables different wavelengths of light to switch action potential firing on and off. These synthetic photoisomerizable azobenzene-regulated K + (SPARK) channels allow rapid, precise and reversible control over neuronal firing, with potential applications for dissecting neural circuits and controlling activity downstream from sites of neural damage or degeneration.Natural photoreceptive proteins, such as rhodopsin, have a covalently attached chromophore that directly activates the protein on exposure to light. Several strategies have been used to enable light regulation of proteins that are not intrinsically light sensitive. Light can be used indirectly to activate receptor proteins, for example, by making a ligand available from a caged precursor 1 . Light can also be used to directly photoisomerize a synthetic molecule that is covalently attached to a protein, thereby imposing conformational changes 2,3 . We have combined these ideas by synthesizing a photoisomerizable tether that attaches a specific ligand on a protein near its normal binding site. Photoswitching of the tethered ligand rapidly regulates its ability to reach its binding site, thereby switching the protein on and off without causing unnatural conformational changes. Photoswitchable tethered ligands can be agonists (for example for nicotinic acetylcholine receptors 4 ), antagonists, or other regulators of protein function. We have applied this general design principle to an ion channel, where the chosen ligand is a blocker of the channel's pore. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript diffusion kinetics. Recently, the exogenous expression of genes encoding ion channels has been used to influence electrical activity in specific neurons 7-9 , but the onset and reversal of gene expression is slow. A modification of this technique, in which a receptor from one species is introduced into another that lacks a natural ligand, dramatically improves temporal control 10,11 , but this approach still relies on a diffusible ligand whose time course of removal limits reversibility. Finally, photic regulation has been conferred on neurons by introducing a rhodopsin-based signal transduction cascade 12 . This technique requires coordinated exogenous expression of three different genes and produ...

Optogenetic photochemical control of designer K+channels in mammalian neurons

“…To maximize photocontrol of current through the targeted K ϩ channel, MAQ must be properly positioned on the channel protein such that it can reach the pore in the trans but not the cis configuration. When the SPARK channel was engineered, the intramolecular distances between the Shaker channel pore and several extracellular residues had already been estimated (Blaustein et al 2000), making the selection of an optimal cysteine attachment site (E422C) a straightforward task (Banghart et al 2004). Although the K ϩ channels used here share considerable sequence homology with Shaker, identifying the optimal attachment position required some cysteine scanning.…”

“…Once MAQ becomes tethered, the QA moiety is constrained in space to the volume of a hemisphere with a radius of ϳ20 Å, which results in an effective concentration in the tens of millimolar range (Banghart et al 2004;Kramer and Karpen 1998). However, the affinity of some K ϩ channels for external QA is so low (K d Ͼ 100 mM) that the channels remain unblocked, even when the tethered MAQ is in the trans state.…”

“…The chemical component of the system is a synthetic photoswitch molecule called MAQ. We previously used MAQ to bestow light sensitivity onto a modified Drosophila Shaker K ϩ channel (Banghart et al 2004). This channel, which we named "SPARK," contains an engineered cysteine that serves as the MAQ attachment site, along with mutations that enhance voltage-dependent activation and remove fast inactivation, causing the channels to be constitutively active at typical neuronal resting potentials.…”

Light-Triggered Modulation of Cellular Electrical Activity by Ruthenium Diimine Nanoswitches

“…Yield: 345 mg, 65%. The desired metal complex was prepared by refluxing for 3 h a methanol solution containing bpy-C17 ligand (0.10 g, 0.25 mmol) and Ru(bpy) 2 (3H, t). All absorption and emission measurements were conducted in degassed 5% DMSO/aqueous solution.…”

Optogenetics in neuroscience: what we gain from studies in mammals

“…maleimide-azobenzene-quaternary ammonium) to a modified Shaker K + channel protein. Before treatment with MAL-AZO-QA, the Shaker K + channel was mutated to ensure that light was the principle modulator of gating [7] .…”