scholarly journals The structure of MgtE in the absence of magnesium provides new insights into channel gating

PLoS Biology ◽  
2021 ◽  
Vol 19 (4) ◽  
pp. e3001231
Author(s):  
Fei Jin ◽  
Minxuan Sun ◽  
Takashi Fujii ◽  
Yurika Yamada ◽  
Jin Wang ◽  
...  
Keyword(s):  
Channel Gating ◽  
Channel Activity ◽  
State Structure ◽  
Closed State ◽  
Channel Inactivation ◽  
Cryo Electron Microscopy ◽  
Pore Opening ◽  
Tm Domain ◽  
Functional Analyses ◽  
Cytoplasmic Side

MgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed-state, and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. There are no structures of the transmembrane (TM) domain for MgtE in Mg2+-free conditions, and the pore-opening mechanism has thus remained unclear. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE TM domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

scholarly journals Cryo-EM structure of the MgtE Mg2+ channel pore domain in Mg2+-free conditions reveals cytoplasmic pore opening

2020 ◽  
Author(s):  
Fei Jin ◽  
Minxuan Sun ◽  
Takashi Fujii ◽  
Yurika Yamada ◽  
Jin Wang ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Channel Activity ◽  
State Structure ◽  
Channel Pore ◽  
Closed State ◽  
Channel Inactivation ◽  
Pore Opening ◽  
Functional Analyses ◽  
Pore Domain ◽  
Cytoplasmic Side

ABSTRACTMgtE is a Mg2+ channel conserved in organisms ranging from prokaryotes to eukaryotes, including humans, and plays an important role in Mg2+ homeostasis. The previously determined MgtE structures in the Mg2+-bound, closed state and structure-based functional analyses of MgtE revealed that the binding of Mg2+ ions to the MgtE cytoplasmic domain induces channel inactivation to maintain Mg2+ homeostasis. However, due to the lack of a structure of the MgtE channel, including its transmembrane domain in Mg2+-free conditions, the pore-opening mechanism of MgtE has remained unclear.Here, we determined the cryoelectron microscopy (cryo-EM) structure of the MgtE-Fab complex in the absence of Mg2+ ions. The Mg2+-free MgtE transmembrane domain structure and its comparison with the Mg2+-bound, closed-state structure, together with functional analyses, showed the Mg2+-dependent pore opening of MgtE on the cytoplasmic side and revealed the kink motions of the TM2 and TM5 helices at the glycine residues, which are important for channel activity. Overall, our work provides structure-based mechanistic insights into the channel gating of MgtE.

scholarly journals Structure and assembly of double-headed Sendai virus nucleocapsids

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Na Zhang ◽  
Hong Shan ◽  
Mingdong Liu ◽  
Tianhao Li ◽  
Rui Luo ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Measles Virus ◽  
Sendai Virus ◽  
Nipah Virus ◽  
Mumps Virus ◽  
Genome Replication ◽  
Direct Visualization ◽  
Closed State ◽  
Cryo Electron Microscopy ◽  
Negative Sense

AbstractParamyxoviruses, including the mumps virus, measles virus, Nipah virus and Sendai virus (SeV), have non-segmented single-stranded negative-sense RNA genomes which are encapsidated by nucleoproteins into helical nucleocapsids. Here, we reported a double-headed SeV nucleocapsid assembled in a tail-to-tail manner, and resolved its helical stems and clam-shaped joint at the respective resolutions of 2.9 and 3.9 Å, via cryo-electron microscopy. Our structures offer important insights into the mechanism of the helical polymerization, in particular via an unnoticed exchange of a N-terminal hole formed by three loops of nucleoproteins, and unveil the clam-shaped joint in a hyper-closed state for nucleocapsid dimerization. Direct visualization of the loop from the disordered C-terminal tail provides structural evidence that C-terminal tail is correlated to the curvature of nucleocapsid and links nucleocapsid condensation and genome replication and transcription with different assembly forms.

scholarly journals Modulation of Kv2.1 channel gating and TEA sensitivity by distinct domains of SNAP-25

2006 ◽  
Vol 396 (2) ◽  
pp. 363-369 ◽  
Author(s):  
Yan He ◽  
Youhou Kang ◽  
Yuk-Man Leung ◽  
Fuzhen Xia ◽  
Xiaodong Gao ◽  
...  
Keyword(s):  
Hormone Secretion ◽  
Channel Gating ◽  
Receptor Proteins ◽  
Cell Excitability ◽  
Voltage Gated ◽  
Channel Inactivation ◽  
Protein Receptor ◽  
Outer Pore ◽  
Pore Domain ◽  
Protein Attachment

Distinct domains within the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins, STX1A (syntaxin 1A) and SNAP-25 (synaptosome-associated protein-25 kDa), regulate hormone secretion by their actions on the cell's exocytotic machinery, as well as voltage-gated Ca2+ and K+ channels. We examined the action of distinct domains within SNAP-25 on Kv2.1 (voltage gated K+ 2.1) channel gating. Dialysis of N-terminal SNAP-25 domains, S197 (SNAP-251–197) and S180 (SNAP-251–180), but not S206 (full-length SNAP-251–206) increased the rate of Kv2.1 channel activation and slowed channel inactivation. Remarkably, these N-terminal SNAP-25 domains, acting on the Kv2.1 cytoplasmic N-terminus, potentiated the external TEA (tetraethylammonium)-mediated block of Kv2.1. To further examine whether these are effects of the channel pore domain, internal K+ was replaced with Na+ and external K+ was decreased from 4 to 1 mM, which decreased the IC50 of the TEA block from 6.8±0.9 mM to >100 mM. Under these conditions S180 completely restored TEA sensitivity (7.9±1.5 mM). SNAP-25 C-terminal domains, SNAP-25198–206 and SNAP-25181–197, had no effect on Kv2.1 gating kinetics. We conclude that different domains within SNAP-25 can form distinct complexes with Kv2.1 to execute a fine allosteric regulation of channel gating and the architecture of the outer pore structure in order to modulate cell excitability.

scholarly journals Properties of L-type calcium channel gating current in isolated guinea pig ventricular myocytes.

1991 ◽  
Vol 98 (2) ◽  
pp. 265-285 ◽  
Author(s):  
R W Hadley ◽  
W J Lederer
Keyword(s):  
Guinea Pig ◽  
Ventricular Myocytes ◽  
Channel Gating ◽  
Charge Movement ◽  
Ca Channel ◽  
Dependent Manner ◽  
Gating Current ◽  
Ca Current ◽  
Channel Inactivation ◽  
Time Dependencies

Nonlinear capacitative current (charge movement) was compared to the Ca current (ICa) in single guinea pig ventricular myocytes. It was concluded that the charge movement seen with depolarizing test steps from -50 mV is dominated by L-type Ca channel gating current, because of the following observations. (a) Ca channel inactivation and the immobilization of the gating current had similar voltage and time dependencies. The degree of channel inactivation was directly proportional to the amount of charge immobilization, unlike what has been reported for Na channels. (b) The degree of Ca channel activation was closely correlated with the amount of charge moved at all test potentials between -40 and +60 mV. (c) D600 was found to reduce the gating current in a voltage- and use-dependent manner. D600 was also found to induce "extra" charge movement at negative potentials. (d) Nitrendipine reduced the gating current in a voltage-dependent manner (KD = 200 nM at -40 mV). However, nitrendipine did not increase charge movement at negative test potentials. Although contamination of the Ca channel gating current from other sources cannot be fully excluded, it was not evident in the data and would appear to be small. However, it was noted that the amount of Ca channel gating charge was quite large compared with the magnitude of the Ca current. Indeed, the gating current was found to be a significant contaminant (19 +/- 7%) of the Ca tail currents in these cells. In addition, it was found that Ca channel rundown did not diminish the gating current. These results suggest that Ca channels can be "inactivated" by means that do not affect the voltage sensor.

DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4

2006 ◽  
Vol 291 (5) ◽  
pp. C966-C976 ◽  
Author(s):  
Hong-Ling Li ◽  
Yu-Jie Qu ◽  
Yi Chun Lu ◽  
Vladimir E. Bondarenko ◽  
Shimin Wang ◽  
...  
Keyword(s):  
Steady State ◽  
Interacting Protein ◽  
Voltage Gated ◽  
Time To Peak ◽  
Kv Channel ◽  
Closed State ◽  
Channel Inactivation ◽  
Recovery From Inactivation

Voltage-gated K+ channels exist in vivo as multiprotein complexes made up of pore-forming and ancillary subunits. To further our understanding of the role of a dipeptidyl peptidase-related ancillary subunit, DPP10, we expressed it with Kv4.3 and Kv1.4, two channels responsible for fast-inactivating K+ currents. Previously, DPP10 has been shown to effect Kv4 channels. However, Kv1.4, when expressed with DPP10, showed many of the same effects as Kv4.3, such as faster time to peak current and negative shifts in the half-inactivation potential of steady-state activation and inactivation. The exception was recovery from inactivation, which is slowed by DPP10. DPP10 expressed with Kv4.3 caused negative shifts in both steady-state activation and inactivation of Kv4.3, but no significant shifts were detected when DPP10 was expressed with Kv4.3 + KChIP2b (Kv channel interacting protein). DPP10 and KChIP2b had different effects on closed-state inactivation. At −60 mV, KChIP2b nearly abolishes closed-state inactivation in Kv4.3, whereas it developed to a much greater extent in the presence of DPP10. Finally, expression of a DPP10 mutant consisting of its transmembrane and cytoplasmic 58 amino acids resulted in effects on Kv4.3 gating that were nearly identical to those of wild-type DPP10. These data show that DPP10 and KChIP2b both modulate Kv4.3 inactivation but that their primary effects are on different inactivation states. Thus DPP10 may be a general modulator of voltage-gated K+ channel inactivation; understanding its mechanism of action may lead to deeper understanding of the inactivation of a broad range of K+ channels.

scholarly journals Permeant calcium ion feed-through regulation of single inositol 1,4,5-trisphosphate receptor channel gating

2012 ◽  
Vol 140 (6) ◽  
pp. 697-716 ◽  
Author(s):  
Horia Vais ◽  
J. Kevin Foskett ◽  
Ghanim Ullah ◽  
John E. Pearson ◽  
Don-On Daniel Mak
Keyword(s):  
Posttranslational Modifications ◽  
Imaging Techniques ◽  
Channel Gating ◽  
Channel Activity ◽  
Release Channel ◽  
Solution Exchange ◽  
Inositol 1,4,5 Trisphosphate ◽  
Direct Binding ◽  
Trisphosphate Receptor ◽  
Type 3

The ubiquitous inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) Ca2+ release channel plays a central role in the generation and modulation of intracellular Ca2+ signals, and is intricately regulated by multiple mechanisms including cytoplasmic ligand (InsP3, free Ca2+, free ATP4−) binding, posttranslational modifications, and interactions with cytoplasmic and endoplasmic reticulum (ER) luminal proteins. However, regulation of InsP3R channel activity by free Ca2+ in the ER lumen ([Ca2+]ER) remains poorly understood because of limitations of Ca2+ flux measurements and imaging techniques. Here, we used nuclear patch-clamp experiments in excised luminal-side-out configuration with perfusion solution exchange to study the effects of [Ca2+]ER on homotetrameric rat type 3 InsP3R channel activity. In optimal [Ca2+]i and subsaturating [InsP3], jumps of [Ca2+]ER from 70 nM to 300 µM reduced channel activity significantly. This inhibition was abrogated by saturating InsP3 but restored when [Ca2+]ER was raised to 1.1 mM. In suboptimal [Ca2+]i, jumps of [Ca2+]ER (70 nM to 300 µM) enhanced channel activity. Thus, [Ca2+]ER effects on channel activity exhibited a biphasic dependence on [Ca2+]i. In addition, the effect of high [Ca2+]ER was attenuated when a voltage was applied to oppose Ca2+ flux through the channel. These observations can be accounted for by Ca2+ flux driven through the open InsP3R channel by [Ca2+]ER, raising local [Ca2+]i around the channel to regulate its activity through its cytoplasmic regulatory Ca2+-binding sites. Importantly, [Ca2+]ER regulation of InsP3R channel activity depended on cytoplasmic Ca2+-buffering conditions: it was more pronounced when [Ca2+]i was weakly buffered but completely abolished in strong Ca2+-buffering conditions. With strong cytoplasmic buffering and Ca2+ flux sufficiently reduced by applied voltage, both activation and inhibition of InsP3R channel gating by physiological levels of [Ca2+]ER were completely abolished. Collectively, these results rule out Ca2+ regulation of channel activity by direct binding to the luminal aspect of the channel.

Inhibition of voltage-gated K+ channels in dendritic cells by rapamycin

2010 ◽  
Vol 299 (6) ◽  
pp. C1379-C1385 ◽  
Author(s):  
Leonid Tyan ◽  
Mentor Sopjani ◽  
Miribane Dërmaku-Sopjani ◽  
Evi Schmid ◽  
Wenting Yang ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Xenopus Oocytes ◽  
Statistical Significance ◽  
Immunosuppressive Drug ◽  
Channel Activity ◽  
Voltage Gated ◽  
Kv Channel ◽  
Kv Channels ◽  
Channel Inactivation ◽  
Kv1.3 Channel

Rapamycin, an inhibitor of the serine/threonine kinase mammalian target of rapamycin (mTOR), is a widely used immunosuppressive drug. Rapamycin affects the function of dendritic cells (DCs), antigen-presenting cells participating in the initiation of primary immune responses and the establishment of immunological memory. Voltage-gated K+ (Kv) channels are expressed in and impact on the function of DCs. The present study explored whether rapamycin influences Kv channels in DCs. To this end, DCs were isolated from murine bone marrow and ion channel activity was determined by whole cell patch clamp. To more directly analyze an effect of mTOR on Kv channel activity, Kv1.3 and Kv1.5 were expressed in Xenopus oocytes with or without the additional expression of mTOR and voltage-gated currents were determined by dual-electrode voltage clamp. As a result, preincubation with rapamycin (0–50 nM) led to a gradual decline of Kv currents in DCs, reaching statistical significance within 6 h and 50 nM of rapamycin. Rapamycin accelerated Kv channel inactivation. Coexpression of mTOR upregulated Kv1.3 and Kv1.5 currents in Xenopus oocytes. Furthermore, mTOR accelerated Kv1.3 channel activation and slowed down Kv1.3 channel inactivation. In conclusion, mTOR stimulates Kv channels, an effect contributing to the immunomodulating properties of rapamycin in DCs.

scholarly journals Molecular basis for the R-type anion channel QUAC1 activity in guard cells

2021 ◽  
Author(s):  
Li Qin ◽  
Ling-hui Tang ◽  
Jia-shu Xu ◽  
Xian-hui Zhang ◽  
Yun Zhu ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Stomatal Closure ◽  
Anion Channel ◽  
Guard Cells ◽  
Channel Activity ◽  
Cytoplasmic Domains ◽  
Anion Channels ◽  
Channel Activation ◽  
Environmental Stimuli ◽  
Functional Analyses

SUMMARYThe rapid (R)-type anion channel plays a central role in controlling stomatal closure in plant guard cells, thus regulating the exchange of water and photosynthetic gas (CO2) in response to environmental stimuli. The activity of the R- type anion channel is regulated by malate. However, the molecular basis of the R-type anion channel activity remains elusive. Here, we describe the first cryo-EM structure of the R-type anion channel QUAC1 at 3.5 Å resolution in the presence of malate. The structure reveals that the QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore for passing anions across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted bi-layer architecture, with the associated dimeric interfaces nearly perpendicular. Our structural and functional analyses reveal that QUAC1 functions as an inward rectifying anion channel and suggests a mechanism for malate-mediated channel activation. Altogether, our study uncovers the molecular basis for a novel class of anion channels and provides insights into the gating and modulation of the R-type anion channel.

scholarly journals An ensemble of cryo-EM structures of TRiC reveal its conformational landscape and subunit specificity

2019 ◽  
Vol 116 (39) ◽  
pp. 19513-19522 ◽  
Author(s):  
Mingliang Jin ◽  
Wenyu Han ◽  
Caixuan Liu ◽  
Yunxiang Zang ◽  
Jiawei Li ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Ring Closure ◽  
Atomic Resolution ◽  
Protein Homeostasis ◽  
Structural Detail ◽  
Experimental Conditions ◽  
Closed State ◽  
Cryo Electron Microscopy ◽  
Conformational Landscape ◽  
Substrate Processing

TRiC/CCT assists the folding of ∼10% of cytosolic proteins through an ATP-driven conformational cycle and is essential in maintaining protein homeostasis. Here, we determined an ensemble of cryo-electron microscopy (cryo-EM) structures of yeast TRiC at various nucleotide concentrations, with 4 open-state maps resolved at near-atomic resolutions, and a closed-state map at atomic resolution, revealing an extra layer of an unforeseen N-terminal allosteric network. We found that, during TRiC ring closure, the CCT7 subunit moves first, responding to nucleotide binding; CCT4 is the last to bind ATP, serving as an ATP sensor; and CCT8 remains ADP-bound and is hardly involved in the ATPase-cycle in our experimental conditions; overall, yeast TRiC consumes nucleotide in a 2-ring positively coordinated manner. Our results depict a thorough picture of the TRiC conformational landscape and its allosteric transitions from the open to closed states in more structural detail and offer insights into TRiC subunit specificity in ATP consumption and ring closure, and potentially in substrate processing.

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