scholarly journals Structure, inhibition and regulation of two-pore channel TPC1 from Arabidopsis thaliana

2016 ◽  
Author(s):  
Alexander F Kintzer ◽  
Robert M Stroud
Keyword(s):  
Arabidopsis Thaliana ◽  
Ebola Virus ◽  
Cytosolic Calcium ◽  
Pore Channel ◽  
Ion Binding ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Activated State ◽  
Ion Binding Sites ◽  
Voltage Gated Ion Channels

Two-pore channels (TPCs) comprise a subfamily (TPC1-3) of eukaryotic voltage- and ligand-gated cation channels that contain two non-equivalent tandem pore-forming subunits that then dimerize to form quasi-tetramers. Found in vacuolar or endolysosomal membranes, they regulate the conductance of sodium and calcium ions, intravesicular pH, trafficking and excitability. TPCs are activated by a decrease in transmembrane potential, an increase in cytosolic calcium concentrations, and inhibited by luminal low pH, and calcium, and regulated by phosphorylation,. We report the crystal structure of TPC1 from Arabidopsis thaliana (TPC1) at 2.8x4.0x3.3 angstrom resolution as a basis for understanding ion permeation, channel activation, the location of voltage-sensing domains, and regulatory ion-binding sites. We determined sites of phosphorylation in the N-terminal and C-terminal domains that are positioned to allosterically modulate cytoplasmic Ca2+-activation. One of the two voltage sensing domains (VSDII) encodes voltage sensitivity and inhibition by lumenal Ca2+ and adopts a conformation distinct from the activated state observed in structures of other voltage-gated ion channels. The structure shows that potent pharmacophore trans-NED19 allosterically acts by clamping the pore domains to VSDII. In animals NED19 prevents infection by Ebola virus and Filoviruses presumably by altering their fusion with the endolysosome, and delivery of their contents into the cytoplasm.

scholarly journals Molecular basis for voltage sensitivity in membrane proteins

2018 ◽  
Author(s):  
Marina A. Kasimova ◽  
Erik Lindahl ◽  
Lucie Delemotte
Keyword(s):  
Membrane Potential ◽  
Ion Channels ◽  
Membrane Proteins ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Suggested Approach ◽  
Voltage Gated ◽  
Living Organisms ◽  
Voltage Gated Ion Channels ◽  
Gated Ion Channels

ABSTRACTVoltage-sensitive membrane proteins are united by the ability to transform changes in the membrane potential into mechanical work. They are responsible for a spectrum of key physiological processes in living organisms, including electric signaling and progression along the cell cycle. While the voltage-sensing mechanism has been well characterized for some membrane proteins such as voltage-gated ion channels, for others even the location of the voltage-sensing elements remains unknown. The detection of these elements using experimental techniques is complicated due to the large diversity of membrane proteins. Here, we suggest a computational approach to predict voltage-sensing elements in any membrane protein independent of structure or function. It relies on the estimation of the capacity of a protein to respond to changes in the membrane potential. We first show how this property correlates well with voltage sensitivity by applying our approach to a set of membrane proteins including voltage-sensitive and voltage-insensitive ones. We further show that it correctly identifies true voltage-sensitive residues in the voltage sensor domain of voltage-gated ion channels. Finally, we investigate six membrane proteins for which the voltage-sensing elements have not yet been characterized and identify residues and ions potentially involved in the response to voltage. The suggested approach is fast and simple and allows for characterization of voltage sensitivity that goes beyond mere identification of charges. We anticipate that its application prior to mutagenesis experiments will allow for significant reduction of the number of potential voltage-sensitive elements to be tested.

scholarly journals Determining the molecular basis of voltage sensitivity in membrane proteins

2018 ◽  
Vol 150 (10) ◽  
pp. 1444-1458 ◽  
Author(s):  
Marina A. Kasimova ◽  
Erik Lindahl ◽  
Lucie Delemotte
Keyword(s):  
Membrane Potential ◽  
Ion Channels ◽  
Membrane Proteins ◽  
Cell Cycle Progression ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Suggested Approach ◽  
Voltage Gated ◽  
Voltage Gated Ion Channels ◽  
Gated Ion Channels

Voltage-sensitive membrane proteins are united by their ability to transform changes in membrane potential into mechanical work. They are responsible for a spectrum of physiological processes in living organisms, including electrical signaling and cell-cycle progression. Although the mechanism of voltage-sensing has been well characterized for some membrane proteins, including voltage-gated ion channels, even the location of the voltage-sensing elements remains unknown for others. Moreover, the detection of these elements by using experimental techniques is challenging because of the diversity of membrane proteins. Here, we provide a computational approach to predict voltage-sensing elements in any membrane protein, independent of its structure or function. It relies on an estimation of the propensity of a protein to respond to changes in membrane potential. We first show that this property correlates well with voltage sensitivity by applying our approach to a set of voltage-sensitive and voltage-insensitive membrane proteins. We further show that it correctly identifies authentic voltage-sensitive residues in the voltage-sensor domain of voltage-gated ion channels. Finally, we investigate six membrane proteins for which the voltage-sensing elements have not yet been characterized and identify residues and ions that might be involved in the response to voltage. The suggested approach is fast and simple and enables a characterization of voltage sensitivity that goes beyond mere identification of charges. We anticipate that its application before mutagenesis experiments will significantly reduce the number of potential voltage-sensitive elements to be tested.

Quantitative analysis of herbivore-induced cytosolic calcium by using a Cameleon (YC 3.6) calcium sensor in Arabidopsis thaliana

2014 ◽  
Vol 171 (2) ◽  
pp. 136-139 ◽  
Author(s):  
Francesca Verrillo ◽  
Andrea Occhipinti ◽  
Chidananda Nagamangala Kanchiswamy ◽  
Massimo E. Maffei
Keyword(s):  
Arabidopsis Thaliana ◽  
Quantitative Analysis ◽  
Cytosolic Calcium ◽  
Calcium Sensor

scholarly journals Two distinct voltage-sensing domains control voltage sensitivity and kinetics of current activation in CaV1.1 calcium channels

2016 ◽  
Vol 147 (6) ◽  
pp. 437-449 ◽  
Author(s):  
Petronel Tuluc ◽  
Bruno Benedetti ◽  
Pierre Coste de Bagneaux ◽  
Manfred Grabner ◽  
Bernhard E. Flucher
Keyword(s):  
Calcium Channels ◽  
Molecular Mechanisms ◽  
Voltage Dependence ◽  
Specific Interaction ◽  
Site Directed Mutagenesis ◽  
Control Voltage ◽  
Voltage Sensitivity ◽  
Voltage Sensing ◽  
Activation Kinetics ◽  
Transmembrane Segments

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

scholarly journals Synthesis and Photoregulated Metal Coordination of Azobenzene Polymer Having Ion Binding Sites

1991 ◽  
Vol 23 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Masato Nanasawa ◽  
Takahiro Nishiyama ◽  
Hiroyoshi Kamogawa
Keyword(s):  
Binding Sites ◽  
Metal Coordination ◽  
Ion Binding ◽  
Azobenzene Polymer ◽  
Ion Binding Sites
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