Optogenetic control with a photocleavable protein, PhoCl

Zhang, Wei; Lohman, Alexander W.; Zhuravlova, Yevgeniya; Lu, Xiao; Wiens, Matthew D.; Hoi, Hiofan; Yaganoglu, Sine; Mohr, Manuel; Kitova, Elena N.; Klassen, John S.; Pantazis, Periklis; Thompson, Roger; Campbell, Robert E.

doi:10.1038/nmeth.4222

Cited by 123 publications

(172 citation statements)

References 39 publications

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“…; Zhang et al . ; Wang & Dahl, ). Curiously, oocytes expressing the C‐terminally truncated Panx1 (Panx1Δ378) displayed a robust CBX‐sensitive activation of the ion conductance at both negative and positive membrane potentials, while leaving the ethidium uptake unchanged, suggesting that caspase‐mediated activation of Panx1 is permeant‐selective.…”

Section: Discussionmentioning

confidence: 99%

Pannexin 1 activation and inhibition is permeant‐selective

Nielsen

Toft-Bertelsen

Lolansen

et al. 2020

The Journal of Physiology

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Key points The large‐pore channel pannexin 1 (Panx1) is expressed in many cell types and can open upon different, yet not fully established, stimuli. Panx1 permeability is often inferred from channel permeability to fluorescent dyes, but it is currently unknown whether dye permeability translates to permeability to other molecules. Cell shrinkage and C‐terminal cleavage led to a Panx1 open‐state with increased permeability to atomic ions (current), but did not alter ethidium uptake. Panx1 inhibitors affected Panx1‐mediated ion conduction differently from ethidium permeability, and inhibitor efficiency towards a given molecule therefore cannot be extrapolated to its effects on the permeability of another. We conclude that ethidium permeability does not reflect equal permeation of other molecules and thus is no measure of general Panx1 activity. Abstract Pannexin 1 (Panx1) is a large‐pore membrane channel connecting the extracellular milieu with the cell interior. While several activation regimes activate Panx1 in a variety of cell types, the selective permeability of an open Panx1 channel remains unresolved: does a given activation paradigm increase Panx1's permeability towards all permeants equally and does fluorescent dye flux serve as a proxy for biological permeation through an open channel? To explore permeant‐selectivity of Panx1 activation and inhibition, we employed Panx1‐expressing Xenopus laevis oocytes and HEK293T cells. We report that different mechanisms of activation of Panx1 differentially affected ethidium and atomic ion permeation. Most notably, C‐terminal truncation or cell shrinkage elevated Panx1‐mediated ion conductance, but had no effect on ethidium permeability. In contrast, extracellular pH changes predominantly affected ethidium permeability but not ionic conductance. High [K+]o did not increase the flux of either of the two permeants. Once open, Panx1 demonstrated preference for anionic permeants, such as Cl−, lactate and glutamate, while not supporting osmotic water flow. Panx1 inhibitors displayed enhanced potency towards Panx1‐mediated currents compared to that of ethidium uptake. We conclude that activation or inhibition of Panx1 display permeant‐selectivity and that permeation of ethidium does not necessarily reflect an equal permeation of smaller biological molecules and atomic ions.

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Section: Discussionmentioning

confidence: 99%

Pannexin 1 activation and inhibition is permeant‐selective

Nielsen

Toft-Bertelsen

Lolansen

et al. 2020

The Journal of Physiology

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“…[35] These efforts were complemented by the introductionofaphotoconvertible neuronal activity integrator called CaMPARI (calciummodulated photoactivatable ratiometric integrator). [37] In addition the red forms of DendFPa nd Dendra2h ave recently been proposed as ratiometric pH indicators. [37] In addition the red forms of DendFPa nd Dendra2h ave recently been proposed as ratiometric pH indicators.…”

Section: Other Photoconvertible Fpsmentioning

confidence: 99%

Primed Conversion: The New Kid on the Block for Photoconversion

Mohr

Pantazis

2018

Chemistry A European J

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In 2015, a novel way to convert photoconvertible fluorescent proteins was reported that uses the intercept of blue and far-red light instead of traditional violet or near-UV light illumination. This Minireview describes and contrasts this distinct two-step mechanism termed primed conversion with traditional photoconversion. We provide a comprehensive overview of what is known to date about primed conversion and focus on the molecular requirements for it to take place. We provide examples of its application to axially confined photoconversion in complex tissues as well as super-resolution microscopy. Further, we describe why and when it is useful, including its advantages and disadvantages, and give an insight into potential future development in the field.

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“…Recently, this protein was modified such that violet light irradiation causes irreversible cleavage of the protein as well as loss of green fluorescence 132 ( Figure 4B). This modified version of mMaple, called PhoCl (photocleavable protein), can be used to cage proteins in an inactive state until release by photocleavage.…”

Section: Emerging Optogenetic Technologiesmentioning

confidence: 99%

The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology

Chow

Vermot

2017

F1000Res

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The zebrafish ( Danio rerio) is a powerful vertebrate model to study cellular and developmental processes in vivo. The optical clarity and their amenability to genetic manipulation make zebrafish a model of choice when it comes to applying optical techniques involving genetically encoded photoresponsive protein technologies. In recent years, a number of fluorescent protein and optogenetic technologies have emerged that allow new ways to visualize, quantify, and perturb developmental dynamics. Here, we explain the principles of these new tools and describe some of their representative applications in zebrafish.

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Optogenetic control with a photocleavable protein, PhoCl

Cited by 123 publications

References 39 publications

Pannexin 1 activation and inhibition is permeant‐selective

Pannexin 1 activation and inhibition is permeant‐selective

Primed Conversion: The New Kid on the Block for Photoconversion

The rise of photoresponsive protein technologies applications in vivo: a spotlight on zebrafish developmental and cell biology

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