Photochemical tools for remote control of ion channels in excitable cells

Department of Molecular and Cell Biology, 142 Life Sciences Addition, University of California, Berkeley, Berkeley, California 94720, USA.
Nature Chemical Biology (Impact Factor: 13). 01/2006; 1(7):360-5. DOI: 10.1038/nchembio750
Source: PubMed


Various strategies have been developed recently for imparting light sensitivity onto normally insensitive cells. These include expression of natural photosensitive proteins, photolysis of caged agonists of native cell surface receptors and photoswitching of isomerizable tethered ligands that act on specially engineered ion channels and receptor targets. The development of chemical tools for optically stimulating or inhibiting signaling proteins has particular relevance for the nervous system, where precise, noninvasive control is an experimental and medical necessity.

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    • "For example, phosphorylation of Tyr in tyrosine kinases activates proteins for signal transduction [2]; 4-hydroxylation of Pro in collagen is essential for the formation of collagen triple helix [3]; and palmitoylation of Cys localizes proteins on the plasma membrane [4]. Therefore, the post-translational addition of new functionalities to proteins can help to investigate cellular events and regulate cell functions [5] [6] [7]. Various synthesized molecules that are selectively conjugated with proteins of interest have been developed [8], in addition to classical reagents that are non-specifically conjugated with functional moieties (-NH2, -COOH, -SH) of proteins. "
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    ABSTRACT: A Staphylococcus aureus transpeptidase, sortase A (SrtA), which catalyzes a peptide ligation with high substrate specificity, is a useful tool to site-specifically attach proteinaceous/peptidic functional molecules to target proteins. However, its strong Ca(2+) dependency makes SrtA difficult for use under low Ca2+ concentrations and in the presence of Ca(2+) -binding substances. To overcome this problem, we designed a SrtA mutant that Ca(2+) -independently demonstrates a high catalytic activity. The heptamutant (P94R/E105K/E108A/D160N/D165A/K190E/K196T), which resulted from a combination of known mutations at the Ca(2+) -binding site and around the substrate-binding site, successfully catalyzed a selective protein-protein ligation in the cytoplasm of Escherichia coli. Selective protein modification in living cells is a promising approach for investigating cellular events and regulating cell functions. This SrtA mutant may prove to be a versatile tool for adding new functionalities to proteins of interest by incorporating functional proteins and chemically modified peptides in living cells, which usually retain low Ca(2+) concentrations. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    Full-text · Article · Apr 2015 · Biotechnology Journal
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    • "In parallel to the generation of optogenetic tools derived by intrinsically light sensitive proteins, an alternative approach to achieve optical control of neuronal activity has been developed. This approach lies in genetically engineering existing target proteins, channels or receptors, and binding them in vivo to an exogenous chemical photoswitch (Kramer et al., 2005; Gorostiza and Isacoff, 2007; Fortin et al., 2008; Gorostiza and Isacoff, 2008a; Isacoff and Smith, 2009; Kramer et al., 2009). The core of the used phostoswitch consists of an azobenzene functional group that isomerizes when illuminated with UV and green light. "
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    Full-text · Article · Mar 2012 · Developmental Neurobiology
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    • "First, the caged neurotransmitter must be continually applied, a task difficult to do outside of a specimen chamber. A recent alternative, single neurons expressing genetically encoded light-sensitive ion channels—for example, channelrhodopsin—might extend the ability of AOD-based multisite scanning to investigate this facet of neurophysiology in vivo [87] [88] [89] [90] [91]. Somewhat implicit in our discussion, we have focused on applying photolytic stimulation techniques to studying the physiology of a single neuron. "
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    ABSTRACT: To study the complex synaptic interactions underpinning dendritic information processing in single neurons, experimenters require methods to mimic presynaptic neurotransmitter release at multiple sites with no physiological damage. We show that laser scanning systems built around large-aperture acousto-optic deflectors and high numerical aperture objective lenses provide the sub-millisecond, sub-micron precision necessary to achieve physiological, exogenous synaptic stimulation. Our laser scanning systems can produce the sophisticated spatio-temporal patterns of synaptic input that are necessary to investigate single-neuron dendritic physiology.
    Preview · Article · Aug 2010 · Journal of Neural Engineering
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