Optogenetic photochemical control of designer K+ channels in mammalian neurons.
ABSTRACT Currently available optogenetic tools, including microbial light-activated ion channels and transporters, are transforming systems neuroscience by enabling precise remote control of neuronal firing, but they tell us little about the role of indigenous ion channels in controlling neuronal function. Here, we employ a chemical-genetic strategy to engineer light sensitivity into several mammalian K(+) channels that have different gating and modulation properties. These channels provide the means for photoregulating diverse electrophysiological functions. Photosensitivity is conferred on a channel by a tethered ligand photoswitch that 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 when the azobenzene moiety is in the trans but not cis 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 Ca(2+)-activated K(+) channels that contribute to synaptic responses). These light-regulated channels may be overexpressed in genetically targeted neurons or substituted for native channels with gene knockin technology to enable precise optopharmacological manipulation of channel function.
SourceAvailable from: Alexandre Mourot[Show abstract] [Hide abstract]
ABSTRACT: The optical neuroscience revolution is transforming how we study neural circuits. By providing a precise way to manipulate endogenous neuronal signaling proteins, it also has the potential to transform our understanding of molecular neuroscience. Recent advances in chemical biology have produced light-sensitive compounds that photoregulate a wide variety of proteins underlying signaling between and within neurons. Chemical tools for optopharmacology include caged agonists and antagonists and reversibly photoswitchable ligands. These reagents act on voltage-gated ion channels and neurotransmitter receptors, enabling control of neuronal signaling with a high degree of spatial and temporal precision. By covalently attaching photoswitch molecules to genetically tagged proteins, the newly emerging methodology of optogenetic pharmacology allows biochemically precise control in targeted subsets of neurons. Now that the tools for manipulating endogenous neuronal signaling proteins are available, they can be implemented in vivo to enhance our understanding of the molecular bases of brain function and dysfunctions.Nature Neuroscience 06/2013; 16(7):816-823. DOI:10.1038/nn.3424 · 14.98 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Patterns of resting potential in non-excitable cells of living tissue are now known to be instructive signals for pattern formation during embryogenesis, regeneration and cancer suppression. The development of molecular-level techniques for tracking ion flows and functionally manipulating the activity of ion channels and pumps has begun to reveal the mechanisms by which voltage gradients regulate cell behaviors and the assembly of complex large-scale structures. A recent paper demonstrated that a specific voltage range is necessary for demarcation of eye fields in the frog embryo. Remarkably, artificially setting other somatic cells to the eye-specific voltage range resulted in formation of eyes in aberrant locations, including tissues that are not in the normal anterior ectoderm lineage: eyes could be formed in the gut, on the tail, or in the lateral plate mesoderm. These data challenge the existing models of eye fate restriction and tissue competence maps, and suggest the presence of a bioelectric code-a mapping of physiological properties to anatomical outcomes. This Addendum summarizes the current state of knowledge in developmental bioelectricity, proposes three possible interpretations of the bioelectric code that functionally maps physiological states to anatomical outcomes, and highlights the biggest open questions in this field. We also suggest a speculative hypothesis at the intersection of cognitive science and developmental biology: that bioelectrical signaling among non-excitable cells coupled by gap junctions simulates neural network-like dynamics, and underlies the information processing functions required by complex pattern formation in vivo. Understanding and learning to control the information stored in physiological networks will have transformative implications for developmental biology, regenerative medicine and synthetic bioengineering.Communicative & integrative biology 01/2013; 6(1):e22595. DOI:10.4161/cib.22595
[Show abstract] [Hide abstract]
ABSTRACT: A force field to induce isomerisation of photoswitchable azobenzene groups embedded in molecular materials has been developed in the framework of force field molecular dynamics simulations. A molecular mechanics switching potential has been tuned so as to reproduce both the correct photoisomerisation timescale and mechanism that has been generated by reference nonadiabatic ab initio molecular dynamics. As a first application, we present a force field molecular dynamics study of a prototype photoswitchable foldamer in acetonitrile as solvent. Our analyses reveal that the photoisomerisation of the azobenzene unit embedded in the foldamer occurs via the so-called NN-twist mechanism, and that there exist several distinct unfolding channels for the helix that could be exploited in novel applications of photoresponsive materials.The Journal of Chemical Physics 08/2013; 139(8):084108. DOI:10.1063/1.4818489 · 3.12 Impact Factor