scholarly journals Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

2019 ◽  
Author(s):  
Xiaodong Duan ◽  
Georg Nagel ◽  
Shiqiang Gao
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Ion Permeability ◽  
Platymonas Subcordiformis ◽  
Calcium Permeability

(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca2+ conductance or more specific ion permeability are still in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos (Stigeoclonium helveticum channelrhodopsin = ShChR) and PsChR (Platymonas subcordiformis channelrhodopsin) and compared them with published ChR2 (C. reinhardtii channelrhodopsin-2) aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na+ and Ca2+ conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na+ and Ca2+ conductance only slightly. Furthermore, the D173H mutant of PsChR showed a much improved photocurrent, compared to wildtype, and even higher Na+ selectivity to H+ than XXM. PsChR D173H also showed a strongly enhanced Ca2+ conductance, more than 2-fold that of the calcium translocating L132C of ChR2 (CatCh). (4) Conclusions: We found that mutating the aspartate of the TM4 to histidine influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na+ selectivity and Ca2+ conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca2+ manipulation.

scholarly journals Mutated Channelrhodopsins with Increased Sodium and Calcium Permeability

2019 ◽  
Vol 9 (4) ◽  
pp. 664 ◽  
Author(s):  
Xiaodong Duan ◽  
Georg Nagel ◽  
Shiqiang Gao
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Light Sensitivity ◽  
Ion Permeability ◽  
Wild Type ◽  
Calcium Permeability

(1) Background: After the discovery and application of Chlamydomonas reinhardtii channelrhodopsins, the optogenetic toolbox has been greatly expanded with engineered and newly discovered natural channelrhodopsins. However, channelrhodopsins of higher Ca2+ conductance or more specific ion permeability are in demand. (2) Methods: In this study, we mutated the conserved aspartate of the transmembrane helix 4 (TM4) within Chronos and PsChR and compared them with published ChR2 aspartate mutants. (3) Results: We found that the ChR2 D156H mutant (XXM) showed enhanced Na+ and Ca2+ conductance, which was not noticed before, while the D156C mutation (XXL) influenced the Na+ and Ca2+ conductance only slightly. The aspartate to histidine and cysteine mutations of Chronos and PsChR also influenced their photocurrent, ion permeability, kinetics, and light sensitivity. Most interestingly, PsChR D139H showed a much-improved photocurrent, compared to wild type, and even higher Na+ selectivity to H+ than XXM. PsChR D139H also showed a strongly enhanced Ca2+ conductance, more than two-fold that of the CatCh. (4) Conclusions: We found that mutating the aspartate of the TM4 influences the ion selectivity of channelrhodopsins. With the large photocurrent and enhanced Na+ selectivity and Ca2+ conductance, XXM and PsChR D139H are promising powerful optogenetic tools, especially for Ca2+ manipulation.

An ultra-high ion selective hybrid proton exchange membrane incorporated with zwitterion-decorated graphene oxide for vanadium redox flow batteries

2019 ◽  
Vol 7 (20) ◽  
pp. 12669-12680 ◽  
Author(s):  
Yuxia Zhang ◽  
Haixia Wang ◽  
Bo Liu ◽  
Jingli Shi ◽  
Jun Zhang ◽  
...  
Keyword(s):  
Proton Exchange Membrane ◽  
Ion Selectivity ◽  
Proton Exchange ◽  
Hybrid Membranes ◽  
Ion Permeability ◽  
Redox Flow Batteries ◽  
Good Trade ◽  
Exchange Membrane ◽  
Vanadium Redox ◽  
Vanadium Ion

A good trade-off effect between proton conductivity and vanadium ion permeability contributing ultra-high ion selectivity is demonstrated for SPEEK/ZC-GO hybrid membranes influenced by zwitterionic ZC-GO nanofillers.

Pore Formation of Thermostable Direct Hemolysin Secreted from Vibrio parahaemolyticus in Lipid Bilayers

2006 ◽  
Vol 25 (5) ◽  
pp. 409-418 ◽  
Author(s):  
Akira Takahashi ◽  
Chiyo Yamamoto ◽  
Toshio Kodama ◽  
Kanami Yamashita ◽  
Nagakatsu Harada ◽  
...  
Keyword(s):  
Lipid Bilayer ◽  
Lipid Bilayers ◽  
Lipid Membranes ◽  
Vibrio Parahaemolyticus ◽  
Cultured Cells ◽  
Ion Selectivity ◽  
Ion Permeability ◽  
Lower Extent ◽  
Thermostable Direct Hemolysin ◽  
Mutant Toxin

Vibrio parahaemolyticus secretes thermostable direct hemolysin (TDH), a major virulence factor. Earlier studies report that TDH is a pore-forming toxin. However, the characteristics of pores formed by TDH in the lipid bilayer, which is permeable to small ions, remain to be elucidated. Ion channel-like activities were observed in lipid bilayers containing TDH. Three types of conductance were identified. All the channels displayed relatively low ion selectivity, and similar ion permeability. The Cl− channel inhibitors, DIDS, glybenclamide, and NPPB, did not affect the channel activity of pores formed by TDH. R7, a mutant toxin of TDH, also forms pores with channel-like activity in lipid bilayers. The ion permeability of these channels is similar to that of TDH. R7 binds cultured cells and liposomes to a lower extent, compared to TDH. R7 does not display significant hemolytic activity and cell cytotoxicity, possibly owing to the difficulty of insertion into lipid membranes. Once R7 is assembled within lipid membranes, it may assume the same structure as TDH. The authors propose that the single glycine at position 62, substituted with serine in the R7 mutant toxin, plays an important role in TDH insertion into the lipid bilayer.

scholarly journals Regulation of Kir Channels by Intracellular pH and Extracellular K+

2004 ◽  
Vol 123 (4) ◽  
pp. 441-454 ◽  
Author(s):  
Anke Dahlmann ◽  
Min Li ◽  
ZhongHua Gao ◽  
Deirdre McGarrigle ◽  
Henry Sackin ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Ph Sensor ◽  
Outer Part ◽  
Selectivity Filter ◽  
Inward Rectification ◽  
Ion Binding ◽  
Enhanced Sensitivity ◽  
Internal Ph ◽  
Additional Channel

ROMK channels are regulated by internal pH (pHi) and extracellular K+ (K+o). The mechanisms underlying this regulation were studied in these channels after expression in Xenopus oocytes. Replacement of the COOH-terminal portion of ROMK2 (Kir1.1b) with the corresponding region of the pH-insensitive channel IRK1 (Kir 2.1) produced a chimeric channel (termed C13) with enhanced sensitivity to inhibition by intracellular H+, increasing the apparent pKa for inhibition by ∼0.9 pH units. Three amino acid substitutions at the COOH-terminal end of the second transmembrane helix (I159V, L160M, and I163M) accounted for these effects. These substitutions also made the channels more sensitive to reduction in K+o, consistent with coupling between the responses to pHi and K+o. The ion selectivity sequence of the activation of the channel by cations was K+ ≅ Rb+ > NH4+ >> Na+, similar to that for ion permeability, suggesting an interaction with the selectivity filter. We tested a model of coupling in which a pH-sensitive gate can close the pore from the inside, preventing access of K+ from the cytoplasm and increasing sensitivity of the selectivity filter to removal of K+o. We mimicked closure of this gate using positive membrane potentials to elicit block by intracellular cations. With K+o between 10 and 110 mM, this resulted in a slow, reversible decrease in conductance. However, additional channel constructs, in which inward rectification was maintained but the pH sensor was abolished, failed to respond to voltage under the same conditions. This indicates that blocking access of intracellular K+ to the selectivity filter cannot account for coupling. The C13 chimera was 10 times more sensitive to extracellular Ba2+ block than was ROMK2, indicating that changes in the COOH terminus affect ion binding to the outer part of the pore. This effect correlated with the sensitivity to inactivation by H+. We conclude that decreasing pHI increases the sensitivity of ROMK2 channels to K+o by altering the properties of the selectivity filter.

scholarly journals K2P channel C-type gating involves asymmetric selectivity filter order-disorder transitions

2020 ◽  
Author(s):  
Marco Lolicato ◽  
Andrew M. Natale ◽  
Fayal Abderemane-Ali ◽  
David Crottès ◽  
Sara Capponi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Selectivity Filter ◽  
Ion Binding ◽  
Anomalous Scattering ◽  
X Ray Crystallography ◽  
Functional Studies ◽  
Ion Binding Sites ◽  
Physical And Chemical

K2P channels regulate nervous, cardiovascular, and immune system functions1,2 through the action of their selectivity filter (C-type) gate3-6. Although structural studies show K2P conformations that impact activity7-13, no selectivity filter conformational changes have been observed. Here, combining K2P2.1 (TREK-1) X-ray crystallography in different potassium concentrations, potassium anomalous scattering, molecular dynamics, and functional studies, we uncover the unprecedented, asymmetric, potassium-dependent conformational changes underlying K2P C-type gating. Low potassium concentrations evoke conformational changes in selectivity filter strand 1 (SF1), selectivity filter strand 2 (SF2), and the SF2-transmembrane helix 4 loop (SF2-M4 loop) that destroy the S1 and S2 ion binding sites and are suppressed by C-type gate activator ML335. Shortening the uniquely long SF2-M4 loop to match the canonical length found in other potassium channels or disrupting the conserved Glu234 hydrogen bond network supporting this loop blunts C-type gate response to various physical and chemical stimuli. Glu234 network destabilization also compromises ion selectivity, but can be reversed by channel activation, indicating that the ion binding site loss reduces selectivity similar to other channels14. Together, our data establish that C-type gating occurs through potassium-dependent order-disorder transitions in the selectivity filter and adjacent loops that respond to gating cues relayed through the SF2-M4 loop. These findings underscore the potential for targeting the SF2-M4 loop for the development of new, selective K2P channel modulators.

scholarly journals Cation and anion channelrhodopsins: Sequence motifs and taxonomic distribution

2021 ◽  
Author(s):  
Elena G. Govorunova ◽  
Oleg A. Sineshchekov ◽  
Hai Li ◽  
Yumei Wang ◽  
Leonid S. Brown ◽  
...  
Keyword(s):  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Control Cell ◽  
Algal Species ◽  
Binding Pocket ◽  
Metagenomic Sequencing ◽  
Sequence Motifs ◽  
Spectral Tuning ◽  
Patch Clamp Recording ◽  
Ionic Selectivity

ABSTRACTCation and anion channelrhodopsins (CCRs and ACRs, respectively) primarily from two algal species, Chlamydomonas reinhardtii and Guillardia theta, have become widely used as optogenetic tools to control cell membrane potential with light. We mined algal and other protist polynucleotide sequencing projects and metagenomic samples to identify 75 channelrhodopsin homologs from three channelrhodopsin families, including one revealed in dinoflagellates in this study. We carried out electrophysiological analysis of 33 natural channelrhodopsin variants from different phylogenetic lineages and 10 metagenomic homologs in search of sequence determinants of ion selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins. Our results show that association of a reduced number of glutamates near the conductance path with anion selectivity depends on a wider protein context, because prasinophyte homologs with the identical glutamate pattern as in cryptophyte ACRs are cation-selective. Desensitization is also broadly context-dependent, as in one branch of stramenopile ACRs and their metagenomic homologs its extent roughly correlates with phylogenetic relationship of their sequences. Regarding spectral tuning, two prasinophyte CCRs exhibit red-shifted spectra to 585 nm, although their retinal-binding pockets do not match those of previously known similarly red-shifted channelrhodopsins. In cryptophyte ACRs we identified three specific residue positions in the retinal-binding pocket that define the wavelength of their spectral maxima. Lastly, we found that dinoflagellate rhodopsins with a TCP motif in the third transmembrane helix and a metagenomic homolog exhibit channel activity.IMPORTANCEChannelrhodopsins are widely used in neuroscience and cardiology as research tools and are considered as prospective therapeutics, but their natural diversity and mechanisms remain poorly characterized. Genomic and metagenomic sequencing projects are producing an ever-increasing wealth of data, whereas biophysical characterization of the encoded proteins lags behind. In this study we used manual and automated patch clamp recording of representative members of four channelrhodopsin families including a family that we report in this study in dinoflagellates. Our results contribute to a better understanding of molecular determinants of ionic selectivity, photocurrent desensitization, and spectral tuning in channelrhodopsins.

scholarly journals Pore-forming transmembrane domains control ion selectivity and selectivity filter conformation in the KirBac1.1 potassium channel

2021 ◽  
Vol 153 (5) ◽  
Author(s):  
Marcos Matamoros ◽  
Colin G. Nichols
Keyword(s):  
Single Molecule ◽  
Ion Selectivity ◽  
Transmembrane Helix ◽  
Selectivity Filter ◽  
K Channel ◽  
Physical Interaction ◽  
Conformationally Constrained ◽  
Single Molecule Fret ◽  
High K ◽  
Low K

Potassium (K+) channels are membrane proteins with the remarkable ability to very selectively conduct K+ ions across the membrane. High-resolution structures have revealed that dehydrated K+ ions permeate through the narrowest region of the pore, formed by the backbone carbonyls of the signature selectivity filter (SF) sequence TxGYG. However, the existence of nonselective channels with similar SF sequences, as well as effects of mutations in other regions on selectivity, suggest that the SF is not the sole determinant of selectivity. We changed the selectivity of the KirBac1.1 channel by introducing mutations at residue I131 in transmembrane helix 2 (TM2). These mutations increase Na+ flux in the absence of K+ and introduce significant proton conductance. Consistent with K+ channel crystal structures, single-molecule FRET experiments show that the SF is conformationally constrained and stable in high-K+ conditions but undergoes transitions to dilated low-FRET states in high-Na+/low-K+ conditions. Relative to wild-type channels, I131M mutants exhibit marked shifts in the K+ and Na+ dependence of SF dynamics to higher K+ and lower Na+ concentrations. These results illuminate the role of I131, and potentially other structural elements outside the SF, in controlling ion selectivity, by suggesting that the physical interaction of these elements with the SF contributes to the relative stability of the constrained K+-induced SF configuration versus nonselective dilated conformations.

scholarly journals Characterising the Ion Selectivity and Calcium Permeability of the Erwinia Ligand-Gated Ion Channel

2013 ◽  
Vol 104 (2) ◽  
pp. 636a
Author(s):  
David A. Weston ◽  
Andrew J. Thompson ◽  
Sarah C.R. Lummis
Keyword(s):  
Ion Channel ◽  
Ion Selectivity ◽  
Calcium Permeability

scholarly journals Structural implications of weak Ca2+ block in Drosophila cyclic nucleotide–gated channels

2015 ◽  
Vol 146 (3) ◽  
pp. 255-263 ◽  
Author(s):  
Yee Ling Lam ◽  
Weizhong Zeng ◽  
Mehabaw Getahun Derebe ◽  
Youxing Jiang
Keyword(s):  
Cyclic Nucleotide ◽  
Ion Selectivity ◽  
Structural Difference ◽  
Selectivity Filter ◽  
Structural Basis ◽  
Functional Studies ◽  
Cyclic Nucleotide Gated Channels ◽  
Cng Channel ◽  
High Resolution Structure ◽  
Calcium Permeability

Calcium permeability and the concomitant calcium block of monovalent ion current (“Ca2+ block”) are properties of cyclic nucleotide–gated (CNG) channel fundamental to visual and olfactory signal transduction. Although most CNG channels bear a conserved glutamate residue crucial for Ca2+ block, the degree of block displayed by different CNG channels varies greatly. For instance, the Drosophila melanogaster CNG channel shows only weak Ca2+ block despite the presence of this glutamate. We previously constructed a series of chimeric channels in which we replaced the selectivity filter of the bacterial nonselective cation channel NaK with a set of CNG channel filter sequences and determined that the resulting NaK2CNG chimeras displayed the ion selectivity and Ca2+ block properties of the parent CNG channels. Here, we used the same strategy to determine the structural basis of the weak Ca2+ block observed in the Drosophila CNG channel. The selectivity filter of the Drosophila CNG channel is similar to that of most other CNG channels except that it has a threonine at residue 318 instead of a proline. We constructed a NaK chimera, which we called NaK2CNG-Dm, which contained the Drosophila selectivity filter sequence. The high resolution structure of NaK2CNG-Dm revealed a filter structure different from those of NaK and all other previously investigated NaK2CNG chimeric channels. Consistent with this structural difference, functional studies of the NaK2CNG-Dm chimeric channel demonstrated a loss of Ca2+ block compared with other NaK2CNG chimeras. Moreover, mutating the corresponding threonine (T318) to proline in Drosophila CNG channels increased Ca2+ block by 16 times. These results imply that a simple replacement of a threonine for a proline in Drosophila CNG channels has likely given rise to a distinct selectivity filter conformation that results in weak Ca2+ block.

scholarly journals Dynamic change of electrostatic field in TMEM16F permeation pathway shifts its ion selectivity

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Wenlei Ye ◽  
Tina W Han ◽  
Mu He ◽  
Yuh Nung Jan ◽  
Lily Yeh Jan
Keyword(s):  
Ion Channel ◽  
Electrostatic Field ◽  
Chloride Channels ◽  
Ion Selectivity ◽  
Dynamic Change ◽  
Ion Permeability ◽  
Permeability Ratio ◽  
Phospholipid Scramblase ◽  
A Charge ◽  
Small Conductance

TMEM16F is activated by elevated intracellular Ca2+, and functions as a small-conductance ion channel and as a phospholipid scramblase. In contrast to its paralogs, the TMEM16A/B calcium-activated chloride channels, mouse TMEM16F has been reported as a cation-, anion-, or non-selective ion channel, without a definite conclusion. Starting with the Q559K mutant that shows no current rundown and less outward rectification in excised patch, we found that the channel shifted its ion selectivity in response to the change of intracellular Ca2+ concentration, with an increased permeability ratio of Cl- to Na+ (PCl-/PNa+) at a higher Ca2+ level. The gradual shift of relative ion permeability did not correlate with the channel activation state. Instead, it was indicative of an alteration of electrostatic field in the permeation pathway. The dynamic change of ion selectivity suggests a charge-screening mechanism for TMEM16F ion conduction, and it provides hints to further studies of TMEM16F physiological functions.

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