Photo-control of specific types of ion channels and neurotransmitter receptors
To make specific ion channels and receptors become sensitive to light, we combine chemistry with molecular biology. In brief, we conjugate a photoswitch compound onto a protein in which we have genetically-engineered an attachment site. Once the compound is attached, it can be lengthened or shortened with different wavelengths of light to extend or retract a ligand into an important binding site on the protein. For example, the tethered photoswitch can block/unblock the pore of a voltage-gated K+ channel or block/unblock the neurotransmitter binding site of a synaptic receptor.
We are using this approach on many types of channels and receptors, including voltage-gated K+ channels (as in the example below), receptors for acetylcholine, and receptors for GABA----the main inhibitory neurotransmitter in the brain. Each of these channels and receptors exist in multiple isoforms. With optogenetic pharmacology, we exert control over one isoform at a time. We can insert the attachment site into the genome, such that endogenous channels or receptors made by the organism are “photoswitch-ready”. By adding the photoswitch, we can control their activities with high spatial, temporal and biochemical precision to learn what each type of channel and receptor normally “does for a living” in the nervous system.
Optogenetic pharmacology of GABAA receptors
We have generated a comprehensive optogenetic toolkit for all of the GABAA isoforms in the brain (Lin et al., 2015). GABAA receptors are pentameric proteins containing 2 alpha, 2 beta, and 1 gamma or delta subunit. We have designed and synthesized photoswitches and complementary cysteine-mutants for each of the 6 alpha subunits in the brain. In each case, conjugation of a photoswitch compound with a complementart mutant receptor results in receptor antagonism that can be induced or alleviated as the compound is photoisomerized between trans and cis states with different wavelengths of light. We have shown that some of these Light-regulated GABAA Receptors (LiGABARs) integrate into inhibitory synapses, allowing photo-control of inhibitory post-synaptic responses. In particular, we have found that alpha-1, alpha-2, and alpha-3 are synaptically localized, whereas alpha-4, alpha-5, and alpha-6 are entirely extrasynaptic. For two of these isoforms (alpha-1 and alpha-6) we have taken the major step of using homologous recombination to generate knock-in mice, in which the mutant form genomically replaces the wild-type receptor. In these mice, photo-control is truly exerted on the endogenous receptor, revealing its normal function in the nervous system signaling.
The photoswitch compound is attached to a genetically-engineered cysteine in the α-subunit of the receptor (red star). Access to the GABA binding site is blocked when the photoswitch is in the trans form and unblocked upon photoisomerization to cis.
Optogenetic pharmacology of acetylcholine receptors
We have used optogenetically pharmacology to confer light-sensitivity on neuronal nicotinic acetylcholine receptors (nAChRs) in the brain (Tochitsky et al., 2011). We have generated a series of photoswitchable tethered agonists and antagonists, enabling activation or inhibition of nAChRs with light. Like GABAA receptors, nAChRs are heteropentamers. We have introduced a cysteine mutation in the beta-2 and beta4 subunits, allow targeted control of specific types of nAChRs. The generation of these engineered receptors facilitates the investigation of the physiological and pathological functions of specific neuronal nAChRs in different parts of the nervous system. In collaboration with Prof. Michael Stryker, UCSF, we are exploring the role of nAChR signaling in visual attention in awake, behaving mice. We are virally expressing a light-sensitive nAChR subunit in one type of inhibitory interneuron (the VIP cell) in the visual cortex to allow dynamic photo-control of one nAChR isoform in one specific neuron type, a level of selectivity that cannot be achieved either by conventional pharmacology or optogenetics. By injecting the photoswitch into the brains of mice of different ages, we will be able to evaluate the role that this circuit connection makes both during development and in the adult brain.
Optogenetic control of voltage-gated potassium channels
Optogenetic pharmacology also allows us to exert photo-control over a range of different types of K+ channels. In this case, the photoswitchable tethered ligand contains a blocker of the pore of the K+ channel. Specifically, the photoswitch contains a cysteine-reactive maleimide (M), a photoisomerizable azobenzene (A), and a quaternary ammonium (Q), a K+ channel pore blocker. Using mutagenesis we identify the optimal extracellular cysteine attachment site where MAQ conjugation results in pore blockade only when the azobenezene moeity is in its trans configuration. With this strategy we have conferred photosensitivity on channels containing Kv1.3 subunits (which control axonal action potential repolarization), Kv3.1 subunits (which contribute to rapid firing properties of brain neurons), Kv7.2 subunits (which underlie “M-current”), and SK2 subunits (which are Ca2+-activated K+ channels that contribute to synaptic responses) (Fortin et al., 2011). These light-regulated channels can be over-expressed in genetically-targeted neurons or substituted for native channels with gene knock-in technology. We have recently generated a knock-in mouse with the mutant form of Kv3.1 genomically substituting for the wild-type gene, and we have verified that light can modulate K+ current in neurons that normally expressing the channel, but not in neurons that do not. At present, there are no specific pharmacological blockers for discriminating between the various Kv3 channels in the nervous system, but this mouse gives us the means to control KV3.1 with “built-in” specificity. With this in mind, we are evaluating the channel’s role in various cellular and system functions, from action potential propagation at nodes of Ranvier, to signaling in the motor and auditory systems.