Fluorescent toxins as ion channel activity sensors

Stewart, Robert; Cohen, Bruce E.; Sack, Jon T.

doi:10.1016/bs.mie.2021.02.014

Cited by 5 publications

(3 citation statements)

References 42 publications

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“…Conjugates of propargylglycine (Pra)-modified GxTX and the fluorophore JP-N 3 (GxTX Ser13Pra(JP) and GxTX Lys27Pra(JP)) were used to monitor the chemical environment surrounding GxTX when localized to the channel ( 46 ). All modified GxTX mutants were synthesized by solid-phase peptide synthesis as described ( 46 , 47 , 48 ). Stock solutions were stored at -80°C and thawed on ice on the day of experiment.…”

Section: Methodsmentioning

confidence: 99%

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

Sepela

Stewart

Valencia

et al. 2022

Biophysical Journal

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Section: Methodsmentioning

confidence: 99%

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

Sepela

Stewart

Valencia

et al. 2022

Biophysical Journal

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“…, 2021 ; Scepanovic et al. , 2021 ), the development of activity sensors to track the dynamics of channel activation during wound repair ( Stewart et al. , 2021 ), and novel optogenetic tools to manipulate channel activity in vivo, with ion specificity ( Beck et al.…”

Section: Do the Materials Properties Of Tissues Affect Aj Remodeling ...mentioning

confidence: 99%

Powering morphogenesis: multiscale challenges at the interface of cell adhesion and the cytoskeleton

Fernández-González

Peifer

2022

MBoC

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Among the defining features of the animal kingdom is the ability of cells to change shape and move. This underlies embryonic and postembryonic development, tissue homeostasis, regeneration, and wound healing. Cell shape change and motility require linkage of the cell’s force-generating machinery to the plasma membrane at cell–cell and cell–extracellular matrix junctions. Connections of the actomyosin cytoskeleton to cell–cell adherens junctions need to be both resilient and dynamic, preventing tissue disruption during the dramatic events of embryonic morphogenesis. In the past decade, new insights radically altered the earlier simple paradigm that suggested simple linear linkage via the cadherin–catenin complex as the molecular mechanism of junction–cytoskeleton interaction. In this Perspective we provide a brief overview of our current state of knowledge and then focus on selected examples highlighting what we view as the major unanswered questions in our field and the approaches that offer exciting new insights at multiple scales from atomic structure to tissue mechanics.

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“…To image where in tissue the voltage sensors of Kv2 proteins adopt a specific resting conformation, we exploited the conformation-selective binding of the tarantula peptide guangxitoxin (GxTX)-1E, which can be conjugated to fluorophores to report Kv2 conformational changes ( Tilley et al, 2014 ; Fletcher-Taylor et al, 2020 ; Stewart et al, 2021 ). Here, we synthesize GxTX-594, a Ser13Cys GxTX variant conjugated to Alexa Fluor 594, a fluorophore compatible with two-photon excitation imaging through light-scattering tissue.…”

Section: Introductionmentioning

confidence: 99%

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues

Thapa

Stewart

Sepela

et al. 2021

Journal of General Physiology

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A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane–endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

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Fluorescent toxins as ion channel activity sensors

Cited by 5 publications

References 42 publications

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

The AMIGO1 adhesion protein activates Kv2.1 voltage sensors

Powering morphogenesis: multiscale challenges at the interface of cell adhesion and the cytoskeleton

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues

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