Specific residues in the cytoplasmic domain modulate photocurrent kinetics of channelrhodopsin from Klebsormidium nitens

AbstractChannelrhodopsins (ChRs) are light-gated ion channels extensively applied as optogenetics tools for manipulating neuronal activity. All currently known ChRs comprise a large cytoplasmic domain, whose function is elusive. Here, we report the cation channel properties of KnChR, one of the photoreceptors from a filamentous terrestrial alga Klebsormidium nitens, and demonstrate that the cytoplasmic domain of KnChR modulates the ion channel properties. KnChR is constituted of a 7-transmembrane domain forming a channel pore, followed by a C-terminus moiety encoding a peptidoglycan binding domain (FimV). Notably, the channel closure rate was affected by the C-terminus moiety. Truncation of the moiety to various lengths prolonged the channel open lifetime by more than 10-fold. Two Arginine residues (R287 and R291) are crucial for altering the photocurrent kinetics. We propose that electrostatic interaction between the rhodopsin domain and the C-terminus domain accelerates the channel kinetics. Additionally, maximal sensitivity was exhibited at 430 and 460 nm, the former making KnChR one of the most blue-shifted ChRs characterized thus far, serving as a novel prototype for studying the molecular mechanism of color tuning of the ChRs. Furthermore, KnChR would expand the optogenetics tool kit, especially for dual light applications when short-wavelength excitation is required.

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Unique Light-gated Ion Channel Properties of a Novel Modular Cation Channelrhodopsin from an Evolutionary Important Terrestrial Green Alga

10.21203/rs.3.rs-68371/v1 ◽

2020 ◽

Author(s):

Rintaro Tashiro ◽

Kumari Sushmita ◽

Shoko Hososhima ◽

Sunita Sharma ◽

Suneel Kateriya ◽

...

Keyword(s):

Ion Channel ◽

Mammalian Cells ◽

Transmembrane Domain ◽

Action Spectrum ◽

Closure Rate ◽

C Terminus ◽

Ion Channel Properties ◽

Arginine Residues ◽

Tm Domain ◽

Channel Currents

Abstract Channelrhodopsins are a family of microbial rhodopsins that function as a light-gated ion channel. We report the molecular properties of a novel channelrhodopsin KnRh3 from an evolutionary important filamentous terrestrial alga Klebsormidium nitens. KnRh3 is constituted of a 7-transmembrane domain, followed by a long C-terminus moiety that encodes a peptidoglycan binding domain (FimV). When functionally expressed in mammalian cells, KnRh3 showed light-induced cation channel currents. The maximum action spectrum exhibited was at 430 nm and 460 nm, the former making KnRh3 one of the most blue-shifted channelrhodopsins characterized thus far. The channel closure rate was relatively fast (τ0ff = 10 ms). Surprisingly, photocurrent kinetics were affected by the C-terminus moiety of KnRh3. When this moiety was truncated to various lengths, this prolonged the channel open lifetime by more than 10-fold. We identified two arginine residues, R287 and R291, those are crucial for altering the kinetics. We propose that electrostatic interaction between the 7-TM domain and the C-terminus domain accelerates the photocycle. The most blue-shifted action spectrum of KnRh3 serves as a novel prototype of channelrhodopsin for studying the molecular mechanism of color tuning. In addition, KnRh3 would expand the optogenetics tool kit, especially for when short wavelength excitation is required.

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Identification of a di-leucine motif within the C terminus domain of the Menkes disease protein that mediates endocytosis from the plasma membrane

Journal of Cell Science ◽

10.1242/jcs.112.11.1721 ◽

1999 ◽

Vol 112 (11) ◽

pp. 1721-1732 ◽

Cited By ~ 2

Author(s):

M.J. Francis ◽

E.E. Jones ◽

E.R. Levy ◽

R.L. Martin ◽

S. Ponnambalam ◽

...

Keyword(s):

Plasma Membrane ◽

Golgi Apparatus ◽

Transmembrane Domain ◽

Menkes Disease ◽

Cytoplasmic Domain ◽

Endocytic Pathway ◽

Site Directed Mutagenesis ◽

Trans Golgi Network ◽

C Terminus ◽

Golgi Network

The protein encoded by the Menkes disease gene (MNK) is localised to the Golgi apparatus and cycles between the trans-Golgi network and the plasma membrane in cultured cells on addition and removal of copper to the growth medium. This suggests that MNK protein contains active signals that are involved in the retention of the protein to the trans-Golgi network and retrieval of the protein from the plasma membrane. Previous studies have identified a signal involved in Golgi retention within transmembrane domain 3 of MNK. To identify a motif sufficient for retrieval of MNK from the plasma membrane, we analysed the cytoplasmic domain, downstream of transmembrane domain 7 and 8. Chimeric constructs containing this cytoplasmic domain fused to the reporter molecule CD8 localised the retrieval signal(s) to 62 amino acids at the C terminus. Further studies were performed on putative internalisation motifs, using site-directed mutagenesis, protein expression, chemical treatment and immunofluorescence. We observed that a di-leucine motif (L1487L1488) was essential for rapid internalisation of chimeric CD8 proteins and the full-length Menkes cDNA from the plasma membrane. We suggest that this motif mediates the retrieval of MNK from the plasma membrane into the endocytic pathway, via the recycling endosomes, but is not sufficient on its own to return the protein to the Golgi apparatus. These studies provide a basis with which to identify other motifs important in the sorting and delivery of MNK from the plasma membrane to the Golgi apparatus.

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Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

Molecular and Cellular Biology ◽

10.1128/mcb.15.9.5043 ◽

1995 ◽

Vol 15 (9) ◽

pp. 5043-5053 ◽

Cited By ~ 81

Author(s):

A S Ho ◽

S H Wei ◽

A L Mui ◽

A Miyajima ◽

K W Moore

Keyword(s):

Transmembrane Domain ◽

Cytoplasmic Domain ◽

Interleukin 10 ◽

Proliferation Assay ◽

Wild Type ◽

Cell Surface Antigens ◽

Functional Regions ◽

Heat Stable ◽

C Terminus ◽

Heat Stable Antigen

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.

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Pervanadate activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1

Biochemical Journal ◽

10.1042/bj3190039 ◽

1996 ◽

Vol 319 (1) ◽

pp. 39-47 ◽

Cited By ~ 70

Author(s):

Jane REILAND ◽

Vanessa L. OTT ◽

Connie S. LEBAKKEN ◽

Charles YEAMAN ◽

James McCARTHY ◽

...

Keyword(s):

Tyrosine Phosphorylation ◽

Tyrosine Kinases ◽

Transmembrane Domain ◽

Consensus Sequence ◽

Signal Transduction Pathway ◽

Initial Step ◽

Cytoplasmic Domain ◽

Sodium Orthovanadate ◽

Intact Cells ◽

C Terminus

Syndecan-1 is a transmembrane haparan sulphate proteoglycan that binds extracellular matrices and growth factors, making it a candidate to act between these regulatory molecules and intracellular signalling pathways. It has a highly conserved transmembrane/cytoplasmic domain that contains four conserved tyrosines. One of these is in a consensus sequence for tyrosine kinase phosphorylation. As an initial step to investigating whether or not phosphorylation of these tyrosines is part of a signal-transduction pathway, we have monitored the tyrosine phosphorylation of syndecan-1 by cytoplasmic tyrosine kinases in intact cells. Tyrosine phosphorylation of syndecan-1 is observed when NMuMG cells are treated with sodium orthovanadate or pervanadate, which have been shown to activate intracellular tyrosine kinases. Initial studies with sodium orthovanadate demonstrate a slow accumulation of phosphotyrosine on syndecan-1 over the course of several hours. Pervanadate, a more effective inhibitor of phosphatases, allows detection of phosphotyrosine on syndecan-1 within 5 min, with peak phosphorylation seen by 15 min. Concurrently, in a second process activated by pervanadate, syndecan-1 ectodomain is cleaved and released into the culture medium. Two phosphorylated fragments of syndecan-1 of apparent sizes 6 and 8 kDa remain with the cell after shedding of the ectodomain. The 8 kDa size class appears to be a highly phosphorylated form of the 6 kDa product, as it disappears if samples are dephosphorylated. These fragments contain the C-terminus of syndecan-1 and also retain at least a portion of the transmembrane domain, suggesting that they are produced by a cell surface cleavage event. Thus pervanadate treatment of cells results in two effects of syndecan-1: (i) phosphorylation of one or more of its tyrosines via the action of a cytoplasmic kinase(s) and (ii) cleavage and release of the ectodomain into the medium, producing a C-terminal fragment containing the transmembrane/cytoplasmic domain.

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Secondary Structure of a KCNE Cytoplasmic Domain

The Journal of General Physiology ◽

10.1085/jgp.200609657 ◽

2006 ◽

Vol 128 (6) ◽

pp. 721-729 ◽

Cited By ~ 18

Author(s):

Jessica M. Rocheleau ◽

Steven D. Gage ◽

William R. Kobertz

Keyword(s):

Secondary Structure ◽

Protein Interactions ◽

Cytoplasmic Domain ◽

Type I ◽

Protein Protein Interactions ◽

Terminal Domain ◽

Periodicity Analysis ◽

C Terminus ◽

Kinetics Of ◽

Voltage Activation

Type I transmembrane KCNE peptides contain a conserved C-terminal cytoplasmic domain that abuts the transmembrane segment. In KCNE1, this region is required for modulation of KCNQ1 K+ channels to afford the slowly activating cardiac IKs current. We utilized alanine/leucine scanning to determine whether this region possesses any secondary structure and to identify the KCNE1 residues that face the KCNQ1 channel complex. Helical periodicity analysis of the mutation-induced perturbations in voltage activation and deactivation kinetics of KCNQ1-KCNE1 complexes defined that the KCNE1 C terminus is α-helical when split in half at a conserved proline residue. This helical rendering assigns all known long QT mutations in the KCNE1 C-terminal domain as protein facing. The identification of a secondary structure within the KCNE1 C-terminal domain provides a structural scaffold to map protein–protein interactions with the pore-forming KCNQ1 subunit as well as the cytoplasmic regulatory proteins anchored to KCNQ1–KCNE complexes.

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Faculty Opinions recommendation of Ion channel properties underlying axonal action potential initiation in pyramidal neurons.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1007335.92105 ◽

2002 ◽

Author(s):

Alain Destexhe

Keyword(s):

Action Potential ◽

Ion Channel ◽

Pyramidal Neurons ◽

Ion Channel Properties ◽

Action Potential Initiation ◽

Potential Initiation ◽

Channel Properties

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The Second Transmembrane Domain 7′ Position Influences Channel Kinetics in the Glycine α1 Receptor

Biological Bulletin ◽

10.1086/bblv207n2p156 ◽

2004 ◽

Vol 207 (2) ◽

pp. 156-156

Author(s):

Eric B. Gonzales ◽

Glenn Dillon

Keyword(s):

Transmembrane Domain ◽

Channel Kinetics

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Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain

International Journal of Molecular Sciences ◽

10.3390/ijms22157918 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7918

Author(s):

Jisun Hwang ◽

Bohee Jang ◽

Ayoung Kim ◽

Yejin Lee ◽

Joonha Lee ◽

...

Keyword(s):

Transmembrane Domain ◽

Growth Factor Receptor ◽

Cytoplasmic Domain ◽

Nih3t3 Cells ◽

C Elegans ◽

Downstream Signaling ◽

Downstream Effectors ◽

Signaling Events

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

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