Dataset Information

Inverse-response Ca²⁺ indicators for optogenetic visualization of neuronal inhibition.

ABSTRACT: We have developed a series of yellow genetically encoded Ca²⁺ indicators for optical imaging (Y-GECOs) with inverted responses to Ca²⁺ and apparent dissociation constants (K_d') ranging from 25 to 2400?nM. To demonstrate the utility of this affinity series of Ca²⁺ indicators, we expressed the four highest affinity variants (K_d's?=?25, 63, 121, and 190?nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic stimulation of the laminar monopolar neuron L1 produced a decrease in intracellular Ca²⁺ in layers 8-10, and a corresponding increase in Y-GECO fluorescence. These experiments revealed that lower K_d' was associated with greater increases in fluorescence, but longer delays to reach the maximum signal change due to slower off-rate kinetics.

SUBMITTER: Zhao Y

PROVIDER: S-EPMC6079023 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Publications

Inverse-response Ca2+ indicators for optogenetic visualization of neuronal inhibition.

Zhao Yufeng Y Bushey Daniel D Zhao Yongxin Y Schreiter Eric R ER Harrison D Jed DJ Wong Allan M AM Campbell Robert E RE

Scientific reports 20180806 1

We have developed a series of yellow genetically encoded Ca2+ indicators for optical imaging (Y-GECOs) with inverted responses to Ca2+ and apparent dissociation constants (Kd') ranging from 25 to 2400 nM. To demonstrate the utility of this affinity series of Ca2+ indicators, we expressed the four highest affinity variants (Kd's = 25, 63, 121, and 190 nM) in the Drosophila medulla intrinsic neuron Mi1. Hyperpolarization of Mi1 by optogenetic ...[more]

PMID: 30082904

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Project description:Stac protein (named for its SH3- and cysteine-rich domains) was first identified in brain 20 years ago and is currently known to have three isoforms. Stac2, Stac1, and Stac3 transcripts are found at high, modest, and very low levels, respectively, in the cerebellum and forebrain, but their neuronal functions have been little investigated. Here, we tested the effects of Stac proteins on neuronal, high-voltage-activated Ca2+ channels. Overexpression of the three Stac isoforms eliminated Ca2+-dependent inactivation (CDI) of l-type current in rat neonatal hippocampal neurons (sex unknown), but not CDI of non-l-type current. Using heterologous expression in tsA201 cells (together with β and α2-δ1 auxiliary subunits), we found that CDI for CaV1.2 and CaV1.3 (the predominant, neuronal l-type Ca2+ channels) was suppressed by all three Stac isoforms, whereas CDI for the P/Q channel, CaV2.1, was not. For CaV1.2, the inhibition of CDI by the Stac proteins appeared to involve their direct interaction with the channel's C terminus. Within the Stac proteins, a weakly conserved segment containing ∼100 residues and linking the structurally conserved PKC C1 and SH3_1 domains was sufficient to fully suppress CDI. The presence of CDI for l-type current in control neonatal neurons raised the possibility that endogenous Stac levels are low in these neurons and Western blotting indicated that the expression of Stac2 was substantially increased in adult forebrain and cerebellum compared with neonate. Together, our results indicate that one likely function of neuronal Stac proteins is to tune Ca2+ entry via neuronal l-type channels.SIGNIFICANCE STATEMENT Stac protein, first identified 20 years ago in brain, has recently been found to be essential for proper trafficking and function of the skeletal muscle l-type Ca2+ channel and is the site of mutations causing a severe, inherited human myopathy. In neurons, however, functions for Stac protein have remained unexplored. Here, we report that one likely function of neuronal Stac proteins is tuning Ca2+ entry via l-type, but not that via non-l-type, Ca2+ channels. Moreover, there is a large postnatal increase in protein levels of the major neuronal isoform (Stac2) in forebrain and cerebellum, which could provide developmental regulation of l-type channel Ca2+ signaling in these brain regions.

Dataset Information

Inverse-response Ca²⁺ indicators for optogenetic visualization of neuronal inhibition.

Publications

Inverse-response Ca<sup>2+</sup> indicators for optogenetic visualization of neuronal inhibition.

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