Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

Olson, Karl D.; Zhang, Xue Nong; Spudich, John L.

doi:10.1073/pnas.92.8.3185

Cited by 35 publications

(36 citation statements)

References 23 publications

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“…The channel became constitutively open in the dark, whereas illumination caused it to close. A similar inversion of protein function was previously observed in haloarchaeal sensory rhodopsin I (SRI), in which a single point mutation either of the photoreceptor itself or of its cognate transducer converted SRI from an attractant to a repellent photoreceptor (16,17). The SRI inversion is attributable to a switch in its conformation from the C (retinylidene Schiff base accessible from the cytoplasm) to E (Schiff base accessible from the extracellular space) conformer (18)(19)(20).…”

Section: Discussionmentioning

confidence: 77%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

102

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Anion channelrhodopsins (ACRs) are a class of light-gated channels recently identified in cryptophyte algae that provide unprecedented fast and powerful hyperpolarizing tools for optogenetics. Analysis of photocurrents generated by Guillardia theta ACR 1 (GtACR1) and its mutants in response to laser flashes showed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH and involving different amino acid residues. The first mechanism, characterized by slow opening and fast closing of the channel, is regulated by Glu-68. Neutralization of this residue (the E68Q mutation) specifically suppressed this first mechanism, but did not eliminate it completely at high pH. Our data indicate the involvement of another, yetunidentified pH-sensitive group X. Introducing a positive charge at the Glu-68 site (the E68R mutation) inverted the channel gating so that it was open in the dark and closed in the light, without altering its ion selectivity. The second mechanism, characterized by fast opening and slow closing of the channel, was not substantially affected by the E68Q mutation, but was controlled by Cys-102. The C102A mutation reduced the rate of channel closing by the second mechanism by ∼100-fold, whereas it had only a twofold effect on the rate of the first. The results show that anion conductance by ACRs has a fundamentally different structural basis than the relatively well studied conductance by cation channelrhodopsins (CCRs), not attributable to simply a modification of the CCR selectivity filter.ion transport | channel gating | channelrhodopsins | optogenetics R ecently we reported a class of rhodopsins from the cryptophyte alga Guillardia theta that act as anion-conducting channels when expressed in cultured animal cells and therefore can be used to suppress neuronal firing by light (1). These proteinsnamed anion channelrhodopsins (ACRs)-show distant sequence homology to cation channelrhodopsins (CCRs) from chlorophyte (green) algae (2), but completely lack permeability for protons and metal cations. This property, as well as large current amplitudes and fast kinetics, makes ACRs superior hyperpolarizing optogenetic tools, compared with proton and chloride pumps (3, 4), or engineered Cl − -conducting CCR variants (5, 6). Two G. theta (Gt) ACRs differ in their spectral sensitivity and channel kinetics (1). GtACR1, with its absorption peak at 515 nm (Fig. S1), has an advantage over a more blue-shifted GtACR2 with maximal efficiency at 470 nm, because it allows using light of longer wavelengths that is less scattered by biological tissue.Because a CCR could be altered to exhibit Cl − channel activity by mutation of a single amino acid residue (5), a fundamental question about ACRs is whether they differ from CCRs only by their selectivity filter. In our search for an answer, we analyzed photocurrents generated by GtACR1 in HEK293 cells under single-turnover conditions in which secondary photochemistry does not complicate the photocycle. We found that GtACR1 cond...

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Section: Discussionmentioning

confidence: 77%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

102

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“…Oxidation Procedure and Western Blot Analysis-Membranes were isolated from sonicated stationary phase cells as described (24) and suspended in 4 M NaCl, 25 mM Tris-HCl, pH 6.8. Membrane samples in sonication buffer (4 M NaCl 25 mM Tris, pH 6.8); low salt membrane dilution buffer (250 mM KCl, 20 mM Tris-HCl, pH 8.0), which previously has been shown to maintain HtrI and SRI in a molecular complex as assessed by spectroscopic criteria (7); and all other solutions were allowed to equilibrate to reaction temperature (10 or 25°C) prior to mixing.…”

Section: Methodsmentioning

confidence: 99%

“…Previously, it was found that certain substitutions at either of two positions in SRI, Asp-201 and His-166, result in inverted (i.e. repellent) responses to orange light (24,30). Three SRI mutants that mediate inverted responses to orange light (D201N, H166A, and H166Y) and one that eliminates phototactic responses to orange light (H166R) were expressed together with HtrI I64C in Pho81Wr…”

Section: Phototaxis Responses Of Cysteine-substituted Htri Mutants-mentioning

confidence: 99%

HtrI Is a Dimer Whose Interface Is Sensitive to Receptor Photoactivation and His-166 Replacements in Sensory Rhodopsin I

Zhang¹,

Spudich²

1998

Journal of Biological Chemistry

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Single cysteine substitutions were introduced into three positions of otherwise cysteineless HtrI, a phototaxis transducer found in Halobacterium salinarum that transmits signals from the photoreceptor sensory rhodopsin I (SRI) to a cytoplasmic pathway controlling the cell's motility. Oxidative cross-linking of the monocysteine HtrI mutants in membrane suspensions resulted in dimer forms evident in SDS-polyacrylamide gels. The rate of cross-linking of I64C on the cytoplasmic side of HtrI was accelerated by SRI binding in the dark and further increased by SRI photoactivation. Several residue replacements of His-166 in SRI accelerated the cross-linking rate of I64C in the dark and His-166 mutants that exhibit "inverted signaling" (mediating repellent instead of the normally attractant response to orange light) inverted the light effect on the cross-linking rate of I64C. Secondary structure prediction of HtrI indicates a coiled coil structure in the cytoplasmic region following TM2, a dimerization domain found in a diverse group of proteins. We conclude that 1) HtrI exists as a dimer both in the absence of SRI and in the SRI-HtrI complex, 2) binding of SRI in the dark increases reactivity of the two cysteines at position 64 in the dimer by increasing their proximity or mobility, 3) light activation of wild-type SRI further increases their reactivity, 4) His-166 replacements in the SRI receptor have conformational effects on the structure of HtrI at position 64, and 5) inverted signaling by His-166 mutants likely results from an inverted conformational change at this region induced by SRI photoactivation.HtrI is a transducer protein found in the archaeon Halobacterium salinarum (1, 2), which is homologous to eubacterial methyl-accepting chemotaxis proteins (MCPs), 1 such as the aspartate receptor (Tar) in Escherichia coli (3-5). It contains two transmembrane segments, connected by a 220-residue portion to methylation regions and a His-kinase binding domain that share high sequence identity with those of the MCP family. Together with its membrane partner, sensory rhodopsin I (SRI), HtrI mediates phototaxis. HtrI and SRI physically interact and form a tight complex in the membrane (6 -8). Signaling by the complex starts from the photoactivation of SRI, which is structurally similar to the visual pigment rhodopsin.The E. coli Tar also contains two transmembrane segments and exists as a dimer both in its ligand-free state and activated, ligand-occupied state (9). Extensive site-directed disulfide cross-linking studies of Tar and related MCPs have revealed a four-helix bundle of the transmembrane helices in the membrane and details of helix packing (10 -12). Activation of these types of receptors occurs when a new conformation is assumed either within a single subunit (13)(14)(15) or between subunits within a dimer (11, 12, 16 -18).In this study, we applied site-directed disulfide cross-linking to monocysteine HtrI mutants to examine whether HtrI exists as a dimer, to detect a conformational change in HtrI during SRI act...

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“…These mutations occur at the positions equivalent to Ala 117 and Gly 120 on TM helix 3 and Leu 266 on TM helix 6 of Rho, respectively. In addition, in the seven TM phototaxis receptor, sensory rhodopsin I, the D201N mutation converted the normally attractant signal of orange light to a repellent signal (24).…”

Section: Fig 4 Light-independent G T Activation By a Set Of Glymentioning

confidence: 99%

Partial Agonist Activity of 11-cis-Retinal in Rhodopsin Mutants

Han¹,

Lou²,

Nakanishi³

et al. 1997

Journal of Biological Chemistry

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Rhodopsin, the photoreceptor molecule of the vertebrate rod cell, is a G protein-coupled receptor. Rhodopsin consists of the opsin apoprotein and its 11-cis-retinal chromophore, which is covalently bound to a specific lysine residue by a stable protonated Schiff base linkage. Rhodopsin activation occurs when light causes photoisomerization of the 11-cis chromophore to its alltrans form. The all-trans chromophore is the receptor agonist. The 11-cis-retinylidene chromophore is analogous pharmacologically to a potent inverse agonist of the receptor. We report here that replacement of a highly conserved glycine residue (Gly 121) causes 11-cisretinal to become a pharmacologic partial agonist. Although the mutant apoproteins do not display constitutive activity, they are active in the dark when bound to an 11-cis-retinylidene chromophore, or to a "locked" chromophore analogue, Ret-7. The degree of partial agonism is directly related to the size of the amino acid replacement at position 121, and it can be reversed by a specific second-site replacement of Phe 261 . Thus, mutation of Gly 121 in rhodopsin causes 11-cis-retinal to act as a partial agonist rather than an inverse agonist, allowing the mutant pigment to activate transducin in the dark.

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Residue replacements of buried aspartyl and related residues in sensory rhodopsin I: D201N produces inverted phototaxis signals.

Cited by 35 publications

References 23 publications

Gating mechanisms of a natural anion channelrhodopsin

Gating mechanisms of a natural anion channelrhodopsin

HtrI Is a Dimer Whose Interface Is Sensitive to Receptor Photoactivation and His-166 Replacements in Sensory Rhodopsin I

Partial Agonist Activity of 11-cis-Retinal in Rhodopsin Mutants

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