Structural Foundations of Potassium Selectivity in Channelrhodopsins

Govorunova, Elena G.; Sineshchekov, Oleg A.; Brown, Leonid S.; Bondar, Ana‐Nicoleta; Spudich, John L.

doi:10.1128/mbio.03039-22

Cited by 15 publications

(35 citation statements)

References 62 publications

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“…Several natural and engineered ChRs have been shown to exhibit a higher selectivity for H +3 , Na +4 , or Cl −5-7 compared to the originally discovered ChR1 and ChR2 of Chlamydomonas reinhardtii. More recently, ChRs with a high K + -selectivity were also discovered [8][9][10] . Accordingly, when expressed in neuronal cells or tissues, ChRs enable multidirectional manipulation of electrical cellular activity.…”

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confidence: 99%

“…10 particles/ml. Light-evoked currents and Ca 2+ signals were recorded at room temperature on an inverted microscope (IX73 Olympus) after 15-21 days in vitro.…”

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confidence: 99%

“…10 particles of an AAV encoding hSyn:EGFP (serotype 9) was added to each well. At DIV 21, samples were fixed for 10 min with 4% paraformaldehyde at room temperature, washed 2× with PBS and 1× with H 2 O before mounting.…”

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confidence: 99%

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Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

et al. 2022

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Channelrhodopsins are light-gated ion channels used to control excitability of designated cells in large networks with high spatiotemporal resolution. While ChRs selective for H+, Na+, K+ and anions have been discovered or engineered, Ca2+-selective ChRs have not been reported to date. Here, we analyse ChRs and mutant derivatives with regard to their Ca2+ permeability and improve their Ca2+ affinity by targeted mutagenesis at the central selectivity filter. The engineered channels, termed CapChR1 and CapChR2 for calcium-permeable channelrhodopsins, exhibit reduced sodium and proton conductance in connection with strongly improved Ca2+ permeation at negative voltage and low extracellular Ca2+ concentrations. In cultured cells and neurons, CapChR2 reliably increases intracellular Ca2+ concentrations. Moreover, CapChR2 can robustly trigger Ca2+ signalling in hippocampal neurons. When expressed together with genetically encoded Ca2+ indicators in Drosophila melanogaster mushroom body output neurons, CapChRs mediate light-evoked Ca2+ entry in brain explants.

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“…10 particles/ml. Light-evoked currents and Ca 2+ signals were recorded at room temperature on an inverted microscope (IX73 Olympus) after 15-21 days in vitro.…”

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confidence: 99%

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Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

et al. 2022

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“…Out of all known ChRs, protein sequences of HcKCRs show the highest homology to those of BCCRs (including conservation of the DTD motif in TM3, Figure 1), although their source organism is phylogenetically very distant from cryptophytes. Close homologs of HcKCRs have been found in other stramenopile and colponemid protists, and in metagenomic samples [41,42], but not in cryptophytes. Close homologs of KCRs form a distinct branch of the phylogenetic tree together with KCRs, but most of these channels are not K + selective [41,43], which provides a unique possibility to identify the residues involved in K + selection by comparative analysis of their sequences.…”

Section: Introductionmentioning

confidence: 99%

“…Close homologs of HcKCRs have been found in other stramenopile and colponemid protists, and in metagenomic samples [41,42], but not in cryptophytes. Close homologs of KCRs form a distinct branch of the phylogenetic tree together with KCRs, but most of these channels are not K + selective [41,43], which provides a unique possibility to identify the residues involved in K + selection by comparative analysis of their sequences. Only six K + selective homologs are currently known: in addition to HcKCRs, these are WiChR from Wobblia lunata [42], B1ChR2 from Bilabrum sp.…”

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confidence: 99%

Potassium-selective channelrhodopsins

2023

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Since their discovery 21 years ago, channelrhodopsins have come of age and have become indispensable tools for optogenetic control of excitable cells such as neurons and myocytes. Potential therapeutic utility of channelrhodopsins has been proven by partial vision restoration in a human patient. Previously known channelrhodopsins are either proton channels, non-selective cation channels almost equally permeable to Na + and K + besides protons, or anion channels. Two years ago, we discovered a group of channelrhodopsins that exhibit over an order of magnitude higher selectivity for K + than for Na + . These proteins, known as "kalium channelrhodopsins" or KCRs, lack the canonical tetrameric selectivity filter found in voltage-and ligand-gated K + channels, and use a unique selectivity mechanism intrinsic to their individual protomers. Mutant analysis has revealed that the key residues responsible for K + selectivity in KCRs are located at both ends of the putative cation conduction pathway, and their role has been confirmed by high-resolution KCR structures. Expression of KCRs in mouse neurons and human cardiomyocytes enabled optical inhibition of these cells' electrical activity. In this minireview we briefly discuss major results of KCR research obtained during the last two years and suggest some directions of future research.

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Combining different ion-selective channelrhodopsins to control water flux by light

Lin,

Tang,

Zhang

et al. 2023

Pflugers Arch - Eur J Physiol

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Water transport through water channels, aquaporins (AQPs), is vital for many physiological processes including epithelial fluid secretion, cell migration and adipocyte metabolism. Water flux through AQPs is driven by the osmotic gradient that results from concentration differences of solutes including ions. Here, we developed a novel optogenetic toolkit that combines the light-gated anion channel GtACR1 either with the light-gated K+ channel HcKCR1 or the new Na+ channelrhodopsin HcNCR1 with high Na+ permeability, to manipulate water transport in Xenopus oocytes non-invasively. Water efflux through AQP was achieved by light-activating K+ and Cl- efflux through HcKCR1 and GtACR1. Contrarily, when GtACR1 was co-expressed with HcNCR1, inward movement of Na+ and Cl- was light-triggered, and the resulting osmotic gradient led to water influx through AQP1. In sum, we demonstrate a novel optogenetic strategy to manipulate water movement into or out of Xenopus oocytes non-invasively. This approach provides a new avenue to interfere with water homeostasis as a means to study related biological phenomena across cell types and organisms.

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Structural Foundations of Potassium Selectivity in Channelrhodopsins

Cited by 15 publications

References 62 publications

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

Calcium-permeable channelrhodopsins for the photocontrol of calcium signalling

Potassium-selective channelrhodopsins

Combining different ion-selective channelrhodopsins to control water flux by light

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