scholarly journals Insights into the molecular mechanisms underlying the inhibition of acid-sensing ion channel 3 gating by stomatin

2020 ◽  
Vol 152 (3) ◽  
Author(s):  
Robert C. Klipp ◽  
Megan M. Cullinan ◽  
John R. Bankston
Keyword(s):  
Ion Channel ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Ph Dependence ◽  
Integral Membrane Protein ◽  
Surface Expression ◽  
C Terminus ◽  
Patch Clamp Electrophysiology ◽  
A Site ◽  
Terminal Site

Stomatin (STOM) is a monotopic integral membrane protein found in all classes of life that has been shown to regulate members of the acid-sensing ion channel (ASIC) family. However, the mechanism by which STOM alters ASIC function is not known. Using chimeric channels, we combined patch-clamp electrophysiology and FRET to search for regions of ASIC3 critical for binding to and regulation by STOM. With this approach, we found that regulation requires two distinct sites on ASIC3: the distal C-terminus and the first transmembrane domain (TM1). The C-terminal site is critical for formation of the STOM–ASIC3 complex, while TM1 is required only for the regulatory effect. We then looked at the mechanism of STOM-dependent regulation of ASIC3 and found that STOM does not alter surface expression of ASIC3 or shift the pH dependence of channel activation. However, a point mutation (Q269G) that prevents channel desensitization also prevents STOM regulation, suggesting that STOM may alter ASIC3 currents by stabilizing the desensitized state of the channel. Based on these findings, we propose a model whereby STOM is anchored to the channel via a site on the distal C-terminus and stabilizes the desensitized state of the channel via an interaction with TM1.

scholarly journals Insights into the molecular mechanisms underlying the inhibition of acid-sensing ion channel 3 gating by Stomatin

2019 ◽  
Author(s):  
Robert C. Klipp ◽  
Megan M. Cullinan ◽  
John R. Bankston
Keyword(s):  
Ion Channel ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Surface Expression ◽  
C Terminus ◽  
Functional Regulation ◽  
Patch Clamp Electrophysiology ◽  
A Site ◽  
Terminal Site

AbstractStomatin is a monotopic integral membrane protein found in all classes of life that has been shown to regulate members of the Acid-Sensing Ion Channel (ASIC) family. However, the mechanism by which Stomatin alters ASIC function is not known. Using chimeric channels, we combined patch clamp electrophysiology and FRET to search for regions of ASIC3 critical for binding to and regulation by Stomatin. With this approach, we found that regulation requires two distinct sites on ASIC3: the distal C-terminus and the first transmembrane domain. Mutation of the C-terminal site disrupts binding and regulation whereas disruption of the transmembrane site eliminates functional regulation. We then showed that Stomatin does not alter surface expression using fluorescence imaging and a surface biotinylation assay. Based on these findings, we propose a model whereby STOM is anchored to the channel via a site on the distal C-terminus but alters ASIC3 gating through action on TM1.

scholarly journals Site-specific deacylation by ABHD17a controls BK channel splice variant activity

2020 ◽  
Vol 295 (49) ◽  
pp. 16487-16496 ◽  
Author(s):  
Heather McClafferty ◽  
Hamish Runciman ◽  
Michael J. Shipston
Keyword(s):  
Ion Channels ◽  
Ion Channel ◽  
Membrane Trafficking ◽  
Transmembrane Protein ◽  
Surface Expression ◽  
Bk Channels ◽  
Bk Channel ◽  
Site Specific ◽  
And Function ◽  
A Site

S-Acylation, the reversible post-translational lipid modification of proteins, is an important mechanism to control the properties and function of ion channels and other polytopic transmembrane proteins. However, although increasing evidence reveals the role of diverse acyl protein transferases (zDHHC) in controlling ion channel S-acylation, the acyl protein thioesterases that control ion channel deacylation are very poorly defined. Here we show that ABHD17a (α/β-hydrolase domain-containing protein 17a) deacylates the stress-regulated exon domain of large conductance voltage- and calcium-activated potassium (BK) channels inhibiting channel activity independently of effects on channel surface expression. Importantly, ABHD17a deacylates BK channels in a site-specific manner because it has no effect on the S-acylated S0–S1 domain conserved in all BK channels that controls membrane trafficking and is deacylated by the acyl protein thioesterase Lypla1. Thus, distinct S-acylated domains in the same polytopic transmembrane protein can be regulated by different acyl protein thioesterases revealing mechanisms for generating both specificity and diversity for these important enzymes to control the properties and functions of ion channels.

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

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.

scholarly journals Interactions between the N- and C-termini of the mechanosensitive ion channel AtMSL10 are consistent with a three-step mechanism for activation

2020 ◽  
Vol 71 (14) ◽  
pp. 4020-4032 ◽  
Author(s):  
Debarati Basu ◽  
Jennette M Shoots ◽  
Elizabeth S Haswell
Keyword(s):  
Cell Death ◽  
Ion Channel ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Adaptive Responses ◽  
C Terminus ◽  
N Terminus ◽  
Mechanosensitive Ion Channel ◽  
Green Fluorescent

Abstract Although a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to green fluorescent protein (GFP) triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag, and could also be induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus could be transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants.

scholarly journals Biosynthesis of the dystonia-associated AAA+ ATPase torsinA at the endoplasmic reticulum

2006 ◽  
Vol 401 (2) ◽  
pp. 607-612 ◽  
Author(s):  
Anna C. Callan ◽  
Sandra Bunning ◽  
Owen T. Jones ◽  
Stephen High ◽  
Eileithyia Swanton
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Transmembrane Domain ◽  
Signal Sequence ◽  
Integral Membrane Protein ◽  
Signal Peptidase ◽  
Aaa Atpase ◽  
C Terminus ◽  
Membrane Spanning ◽  
Aaa Atpases

TorsinA is a widely expressed AAA+ (ATPases associated with various cellular activities) ATPase of unknown function. Previous studies have described torsinA as a type II protein with a cleavable signal sequence, a single membrane spanning domain, and its C-terminus located in the ER (endoplasmic reticulum) lumen. However, in the present study we show that torsinA is not in fact an integral membrane protein. Instead we find that the mature protein associates peripherally with the ER membrane, most likely through an interaction with an integral membrane protein. Consistent with this model, we provide evidence that the signal peptidase complex cleaves the signal sequence of torsinA, and we show that the region previously suggested to form a transmembrane domain is translocated into the lumen of the ER. The finding that torsinA is a peripheral, and not an integral membrane protein as previously thought, has important implications for understanding the function of this novel ATPase.

scholarly journals Interaction of the transmembrane domain of lysis protein E from bacteriophage ϕX174 with bacterial translocase MraY and peptidyl-prolyl isomerase SlyD

Microbiology ◽  
2006 ◽  
Vol 152 (10) ◽  
pp. 2959-2967 ◽  
Author(s):  
Sharon Mendel ◽  
Joanne M. Holbourn ◽  
James A. Schouten ◽  
Timothy D. H. Bugg
Keyword(s):  
Transmembrane Domain ◽  
Integral Membrane Protein ◽  
Cd Spectroscopy ◽  
Prolyl Isomerase ◽  
Peptidyl Prolyl Isomerase ◽  
E Coli ◽  
Protein Protein Interaction ◽  
Peptidoglycan Biosynthesis ◽  
Membrane Bound ◽  
A Site

The molecular target for the bacteriolytic E protein from bacteriophage ϕX174, responsible for host cell lysis, is known to be the enzyme phospho-MurNAc-pentapeptide translocase (MraY), an integral membrane protein involved in bacterial cell wall peptidoglycan biosynthesis, with an essential role being played by peptidyl-prolyl isomerase SlyD. A synthetic 37 aa peptide Epep, containing the N-terminal transmembrane α-helix of E, was found to be bacteriolytic against Bacillus licheniformis, and inhibited membrane-bound MraY. The solution conformation of Epep was found by circular dichroism (CD) spectroscopy to be 100 % α-helical. No change in the CD spectrum was observed upon addition of purified Escherichia coli SlyD, implying that SlyD does not catalyse prolyl isomerization upon E. However, Epep was found to be a potent inhibitor of SlyD-catalysed peptidyl-prolyl isomerization (IC50 0.15 μM), implying a strong interaction between E and SlyD. Epep was found to inhibit E. coli MraY activity when assayed in membranes (IC50 0.8 μM); however, no inhibition of solubilized MraY was observed, unlike nucleoside natural product inhibitor tunicamycin. These results imply that the interaction of E with MraY is not at the MraY active site, and suggest that a protein–protein interaction is formed between E and MraY at a site within the transmembrane region.

scholarly journals ASIC2b-dependent Regulation of ASIC3, an Essential Acid-sensing Ion Channel Subunit in Sensory Neurons via the Partner Protein PICK-1

2004 ◽  
Vol 279 (19) ◽  
pp. 19531-19539 ◽  
Author(s):  
Emmanuel Deval ◽  
Miguel Salinas ◽  
Anne Baron ◽  
Eric Lingueglia ◽  
Michel Lazdunski
Keyword(s):  
Ion Channel ◽  
Sensory Neurons ◽  
Ph Dependence ◽  
Pdz Domain ◽  
G Protein Coupled Receptors ◽  
Drg Neurons ◽  
Kinase C ◽  
C Terminus ◽  
First Time ◽  
G Protein Coupled

ASIC3, an acid-sensing ion channel subunit expressed essentially in sensory neurons, has been proposed to be involved in pain. We show here for the first time that native ASIC3-like currents were increased in cultured dorsal root ganglion (DRG) neurons following protein kinase C (PKC) stimulation. This increase was induced by the phorbol ester PDBu and by pain mediators, such as serotonin, which are known to activate the PKC pathway through their binding to G protein-coupled receptors. We demonstrate that this regulation involves the silent ASIC2b subunit, an ASIC subunit also expressed in sensory neurons. Indeed, heteromultimeric ASIC3/ASIC2b channels, but not homomeric ASIC3 channels, are positively regulated by PKC. The increase of ASIC3/ASIC2b current is accompanied by a shift in its pH dependence toward more physiological pH values and may lead to an increase of sensory neuron excitability. This regulation by PKC requires PICK-1 (protein interacting with C kinase), a PDZ domain-containing protein, which interacts with the ASIC2b C terminus.

scholarly journals The NHE3 Juxtamembrane Cytoplasmic Domain Directly Binds Ezrin: Dual Role in NHE3 Trafficking and Mobility in the Brush Border

2006 ◽  
Vol 17 (6) ◽  
pp. 2661-2673 ◽  
Author(s):  
Boyoung Cha ◽  
Ming Tse ◽  
Chris Yun ◽  
Olga Kovbasnjuk ◽  
Sachin Mohan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Brush Border ◽  
Half Life ◽  
Double Mutant ◽  
Pdz Domain ◽  
Point Mutations ◽  
Surface Expression ◽  
Triple Mutant ◽  
C Terminus ◽  
A Site

Based on physiological studies, the epithelial brush-border (BB) Na+/H+ antiporter3 (NHE3) seems to associate with the actin cytoskeleton both by binding to and independently of the PDZ domain containing proteins NHERF1 and NHERF2. We now show that NHE3 directly binds ezrin at a site in its C terminus between aa 475-589, which is separate from the PSD95/dlg/zonular occludens-1 (PDZ) interacting domain. This is an area predicted to be α-helical, with a positive aa cluster on one side (K516, R520, and R527). Point mutations of these positively charged aa reduced (NHE3 double mutant [R520F, R527F]) or abolished (NHE3 triple mutant [K516Q, R520F, R 527F]) ezrin binding. Functional consequences of these NHE3 point mutants included the following. 1) A marked decrease in surface amount with a greater decrease in NHE3 activity. 2) Decreased surface expression due to decreased rates of exocytosis and plasma membrane delivery of newly synthesized NHE3, with normal total expression levels and slightly reduced endocytosis rates. 3) A longer plasma membrane half-life of mutant NHE3 with normal total half-life. 4) Decreased BB mobile fraction of NHE3 double mutant. These results show that NHE3 binds ezrin directly as well as indirectly and suggest that the former is related to 1) the exocytic trafficking of and plasma membrane delivery of newly synthesized NHE3, which determines the amount of plasma membrane NHE3 and partially determines NHE3 activity, and 2) BB mobility of NHE3, which may increase its delivery from microvilli to the intervillus clefts, perhaps for NHE3-regulated endocytosis.

scholarly journals Interactions between the N- and C- termini of mechanosensitive ion channel AtMSL10 are consistent with a three-step mechanism for activation

2019 ◽  
Author(s):  
Debarati Basu ◽  
Jennette M. Shoots ◽  
Elizabeth S. Haswell
Keyword(s):  
Cell Death ◽  
Ion Channel ◽  
Conformational Changes ◽  
Molecular Mechanisms ◽  
Adaptive Responses ◽  
C Terminus ◽  
Step Model ◽  
N Terminus ◽  
Mechanosensitive Ion Channel ◽  
Cellular Phenotypes

ABSTRACTAlthough a growing number of mechanosensitive ion channels are being identified in plant systems, the molecular mechanisms by which they function are still under investigation. Overexpression of the mechanosensitive ion channel MSL (MscS-Like)10 fused to GFP triggers a number of developmental and cellular phenotypes including the induction of cell death, and this function is influenced by seven phosphorylation sites in its soluble N-terminus. Here, we show that these and other phenotypes required neither overexpression nor a tag and could be also induced by a previously identified point mutation in the soluble C-terminus (S640L). The promotion of cell death and hyperaccumulation of H2O2 in 35S:MSL10S640L-GFP overexpression lines was suppressed by N-terminal phosphomimetic substitutions, and the soluble N- and C-terminal domains of MSL10 physically interacted. We propose a three-step model by which tension-induced conformational changes in the C-terminus are transmitted to the N-terminus, leading to its dephosphorylation and the induction of adaptive responses. Taken together, this work expands our understanding of the molecular mechanisms of mechanotransduction in plants.HIGHLIGHTCell death is triggered by mutations in either the cytoplasmic N- or C-terminus of AìMSLlü. Our proposed model explains how membrane tension may activate signaling through the interaction of these two domains.

scholarly journals NMDA Receptor Opening and Closing—Transitions of a Molecular Machine Revealed by Molecular Dynamics

Biomolecules ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 546 ◽  
Author(s):  
Jiří Černý ◽  
Paulína Božíková ◽  
Aleš Balík ◽  
Sérgio M. Marques ◽  
Ladislav Vyklický
Keyword(s):  
Molecular Dynamics ◽  
Nmda Receptor ◽  
Ion Channel ◽  
Structural Transition ◽  
Molecular Mechanisms ◽  
Transmembrane Domain ◽  
Molecular Machine ◽  
Large Rearrangement ◽  
Extracellular Domains ◽  
Opening And Closing

We report the first complete description of the molecular mechanisms behind the transition of the N-methyl-d-aspartate (NMDA) receptor from the state where the transmembrane domain (TMD) and the ion channel are in the open configuration to the relaxed unliganded state where the channel is closed. Using an aggregate of nearly 1 µs of unbiased all-atom implicit membrane and solvent molecular dynamics (MD) simulations we identified distinct structural states of the NMDA receptor and revealed functionally important residues (GluN1/Glu522, GluN1/Arg695, and GluN2B/Asp786). The role of the “clamshell” motion of the ligand binding domain (LBD) lobes in the structural transition is supplemented by the observed structural similarity at the level of protein domains during the structural transition, combined with the overall large rearrangement necessary for the opening and closing of the receptor. The activated and open states of the receptor are structurally similar to the liganded crystal structure, while in the unliganded receptor the extracellular domains perform rearrangements leading to a clockwise rotation of up to 45 degrees around the longitudinal axis of the receptor, which closes the ion channel. The ligand-induced rotation of extracellular domains transferred by LBD–TMD linkers to the membrane-anchored ion channel is responsible for the opening and closing of the transmembrane ion channel, revealing the properties of NMDA receptor as a finely tuned molecular machine.

Sign in / Sign up

Export Citation Format

Share Document