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 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 Molecular Analysis of ATP-sensitive K Channel Gating and Implications for Channel Inhibition by ATP

1998 ◽  
Vol 112 (3) ◽  
pp. 333-349 ◽  
Author(s):  
Stefan Trapp ◽  
Peter Proks ◽  
Stephen J. Tucker ◽  
Frances M. Ashcroft
Keyword(s):  
Transmembrane Domain ◽  
K Channel ◽  
Channel Activity ◽  
Inhibitory Effects ◽  
Open Probability ◽  
Regulatory Subunits ◽  
Closed State ◽  
Channel Inhibition ◽  
Apparent Decrease ◽  
Atp Inhibition

The β cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2ΔC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2ΔC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2ΔC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 μM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.

scholarly journals Local constraints in either the GluN1 or GluN2 subunit equally impair NMDA receptor pore opening

2011 ◽  
Vol 138 (2) ◽  
pp. 179-194 ◽  
Author(s):  
Iehab Talukder ◽  
Lonnie P. Wollmuth
Keyword(s):  
Nmda Receptor ◽  
Ligand Binding ◽  
Ion Channel ◽  
Nmda Receptors ◽  
Transmembrane Domain ◽  
Conformational Dynamics ◽  
Functional Feature ◽  
Channel Pore ◽  
Activation Gating ◽  
Pore Opening

The defining functional feature of N-methyl-d-aspartate (NMDA) receptors is activation gating, the energetic coupling of ligand binding into opening of the associated ion channel pore. NMDA receptors are obligate heterotetramers typically composed of glycine-binding GluN1 and glutamate-binding GluN2 subunits that gate in a concerted fashion, requiring all four ligands to bind for subsequent opening of the channel pore. In an individual subunit, the extracellular ligand-binding domain, composed of discontinuous polypeptide segments S1 and S2, and the transmembrane channel–forming domain, composed of M1–M4 segments, are connected by three linkers: S1–M1, M3–S2, and S2–M4. To study subunit-specific events during pore opening in NMDA receptors, we impaired activation gating via intrasubunit disulfide bonds connecting the M3–S2 and S2–M4 in either the GluN1 or GluN2A subunit, thereby interfering with the movement of the M3 segment, the major pore-lining and channel-gating element. NMDA receptors with gating impairments in either the GluN1 or GluN2A subunit were dramatically resistant to channel opening, but when they did open, they showed only a single-conductance level indistinguishable from wild type. Importantly, the late gating steps comprising pore opening to its main long-duration open state were equivalently affected regardless of which subunit was constrained. Thus, the NMDA receptor ion channel undergoes a pore-opening mechanism in which the intrasubunit conformational dynamics at the level of the ligand-binding/transmembrane domain (TMD) linkers are tightly coupled across the four subunits. Our results further indicate that conformational freedom of the linkers between the ligand-binding and TMDs is critical to the activation gating process.

scholarly journals Transmembrane Domain 3 (TM3) Governs Orai1 and Orai3 Pore Opening in an Isoform-Specific Manner

2021 ◽  
Vol 9 ◽  
Author(s):  
Adéla Tiffner ◽  
Lena Maltan ◽  
Marc Fahrner ◽  
Matthias Sallinger ◽  
Sarah Weiß ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Constitutive Activity ◽  
Channel Activation ◽  
Conserved Residues ◽  
Closed State ◽  
Specific Manner ◽  
Pore Opening ◽  
Domain 3 ◽  
Orai Channels

Graphical AbstractOrai1 and Orai3 channel activation depends in an isoform-specific manner on two non-conserved residues in TM3 (Orai1: V181, L185, Orai3: A156, F160). Mutation of these residues to alanine leads in the absence of STIM1 to small constitutive activity of the respective Orai1 mutants, however, to huge constitutive currents of the respective Orai3 mutants. Overall, two non-conserved residues in TM3 control the maintenance of the closed state as well as an opening permissive conformation of Orai channels in an isoform-specific manner.

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 Structural basis of control of inward rectifier Kir2 channel gating by bulk anionic phospholipids

2016 ◽  
Vol 148 (3) ◽  
pp. 227-237 ◽  
Author(s):  
Sun-Joo Lee ◽  
Feifei Ren ◽  
Eva-Maria Zangerl-Plessl ◽  
Sarah Heyman ◽  
Anna Stary-Weinzinger ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Membrane Lipids ◽  
Inward Rectifier ◽  
Primary Site ◽  
Structural Basis ◽  
Channel Activity ◽  
Anionic Phospholipid ◽  
X Ray ◽  
Anionic Phospholipids ◽  
Kir Channel

Inward rectifier potassium (Kir) channel activity is controlled by plasma membrane lipids. Phosphatidylinositol-4,5-bisphosphate (PIP2) binding to a primary site is required for opening of classic inward rectifier Kir2.1 and Kir2.2 channels, but interaction of bulk anionic phospholipid (PL−) with a distinct second site is required for high PIP2 sensitivity. Here we show that introduction of a lipid-partitioning tryptophan at the second site (K62W) generates high PIP2 sensitivity, even in the absence of PL−. Furthermore, high-resolution x-ray crystal structures of Kir2.2[K62W], with or without added PIP2 (2.8- and 2.0-Å resolution, respectively), reveal tight tethering of the C-terminal domain (CTD) to the transmembrane domain (TMD) in each condition. Our results suggest a refined model for phospholipid gating in which PL− binding at the second site pulls the CTD toward the membrane, inducing the formation of the high-affinity primary PIP2 site and explaining the positive allostery between PL− binding and PIP2 sensitivity.

scholarly journals Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

2019 ◽  
Vol 116 (27) ◽  
pp. 13358-13367 ◽  
Author(s):  
Mette H. Poulsen ◽  
Anahita Poshtiban ◽  
Viktoria Klippenstein ◽  
Valentina Ghisi ◽  
Andrew J. R. Plested
Keyword(s):  
Amino Acid ◽  
Conformational Changes ◽  
Transmembrane Domain ◽  
Uv Light ◽  
Unnatural Amino Acid ◽  
Selectivity Filter ◽  
Unnatural Amino Acid Mutagenesis ◽  
Amino Acid Mutagenesis ◽  
Mutant Receptors ◽  
Pore Domain

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the vertebrate nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type iGluRs using genetically encoded unnatural amino acid (UAA) photocross-linkers, p-benzoyl-l-phenylalanine (BzF) and p-azido-l-phenylalanine (AzF). We introduced these UAAs at sites throughout the TMD of the GluA2 receptor and characterized the mutants in patch-clamp recordings, exposing them to glutamate and ultraviolet (UV) light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence for an interaction between the Pre-M1 and the M4 TMD helix during desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter in the M2 segment, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric ion channels, selectivity filter gating.

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 Genomic and Functional Analyses ofRhodococcus equiPhages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7

2010 ◽  
Vol 77 (2) ◽  
pp. 669-683 ◽  
Author(s):  
E. J. Summer ◽  
M. Liu ◽  
J. J. Gill ◽  
M. Grant ◽  
T. N. Chan-Cortes ◽  
...  
Keyword(s):  
Protein Sequence ◽  
Genome Organization ◽  
Transmembrane Domain ◽  
Sequence Comparisons ◽  
Data Set ◽  
A Value ◽  
Genes Encoding ◽  
High Fraction ◽  
Functional Analyses ◽  
Relationship Of

ABSTRACTThe isolation and results of genomic and functional analyses ofRhodococcus equiphages ReqiPepy6, ReqiDocB7, ReqiPine5, and ReqiPoco6 (hereafter referred to as Pepy6, DocB7, Pine5, and Poco6, respectively) are reported. Two phages, Pepy6 and Poco6, more than 75% identical, exhibited genome organization and protein sequence likeness toLactococcus lactisphage 1706 and clostridial prophage elements. An unusually high fraction, 27%, of Pepy6 and Poco6 proteins were predicted to possess at least one transmembrane domain, a value much higher than the average of 8.5% transmembrane domain-containing proteins determined from a data set of 36,324 phage protein entries. Genome organization and protein sequence comparisons place phage Pine5 as the first nonmycobacteriophage member of the large Rosebush cluster. DocB7, which had the broadest host range among the four isolates, was not closely related to any phage or prophage in the database, and only 23 of 105 predicted encoded proteins could be assigned a functional annotation. Because of the relationship ofRhodococcustoMycobacterium, it was anticipated that these phages should exhibit some of the features characteristic of mycobacteriophages. Traits that were identified as shared by theRhodococcusphages and mycobacteriophages include the prevalent long-tailed morphology and the presence of genes encoding LysB-like mycolate-hydrolyzing lysis proteins. Application of DocB7 lysates to soils amended with a host strain ofR. equireduced recoverable bacterial CFU, suggesting that phage may be useful in limitingR. equiload in the environment while foals are susceptible to infection.

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