Uncorking MCU to let the calcium flow

New cryo-electron microscopy structures of the mitochondrial Ca²⁺ uniporter ion channel complex in various conformations reveal channel gating regulation by Ca²⁺-dependent unblock of the channel pore by MICU1.     

Gating at the selectivity filter of ion channels that conduct Na+ and K+ ions

The NaK channel is a cation selective channel with similar permeability for K⁺ and Na⁺. The available crystallographic structure of wild-type (WT) NaK is usually associated with a conductive state of the channel. Here, potential of mean force for complete conduction events of Na⁺ and K⁺ ions through NaK show that: i), large energy barriers prevent the passage of ions through the WT NaK structure, ii), the barriers are correlated to the presence of a hydrogen bond between Asp-66 and Asn-68, and iii), the structure of NaK mutated to mimic cyclic nucleotide-gated channels conducts Na⁺ and K⁺. These results support the hypothesis that the filter of cation selective channels can adopt at least two different structures: a conductive one, represented by the x-ray structures of the NaK-CNG chimeras, and a closed one, represented by the x-ray structures of the WT NaK.     

Slaying a giant: Structures of calmodulin and protein kinase a bound to the cardiac ryanodine receptor

Ryanodine Receptors are Ca²⁺ release channels expressed in the Endoplasmic and Sarcoplasmic Reticulum membranes. Gong et al [1] reported cryo-EM structures of the cardiac RyR2 complexed to Calmodulin, which can downregulate channel opening. Haji-Ghassemi et al [2] report crystal structures of an RyR2 domain with PKA, a kinase promoting channel opening.     

Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His³⁷ tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His³⁷ provide insight into the mechanism of proton transport. The channel is closed at both His³⁷ and Trp⁴¹ sites in the singly and doubly protonated states, but it opens at Trp⁴¹ upon further protonation. Anions access the charged His³⁷ and by doing so stabilize the protonated states of the channel. The narrow opening at the His³⁷ site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His³⁷ correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val²⁷ remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.     

Activation and Proton Transport Mechanism in Influenza A M2 Channel

Molecular dynamics trajectories 2 μs in length have been generated for the pH-activated, tetrameric M2 proton channel of the influenza A virus in all protonation states of the pH sensor located at the His³⁷ tetrad. All simulated structures are in very good agreement with high-resolution structures. Changes in the channel caused by progressive protonation of His³⁷ provide insight into the mechanism of proton transport. The channel is closed at both His³⁷ and Trp⁴¹ sites in the singly and doubly protonated states, but it opens at Trp⁴¹ upon further protonation. Anions access the charged His³⁷ and by doing so stabilize the protonated states of the channel. The narrow opening at the His³⁷ site, further blocked by anions, is inconsistent with the water-wire mechanism of proton transport. Instead, conformational interconversions of His³⁷ correlated with hydrogen bonding to water molecules indicate that these residues shuttle protons in high-protonation states. Hydrogen bonds between charged and uncharged histidines are rare. The valve at Val²⁷ remains on average quite narrow in all protonation states but fluctuates sufficiently to support water and proton transport. A proton transport mechanism in which the channel, depending on pH, opens at either the histidine or valine gate is only partially supported by the simulations.     

Structural insights into excitation—contraction coupling by electron cryomicroscopy

In muscle, excitation-contraction coupling is defined as the process linking depolarization of the surface membrane with Ca²⁺ release from cytoplasmic stores, which activates contraction of striated muscle. This process is primarily controlled by interplay between two Ca²⁺ channels—the voltage-gated L-type Ca²⁺ channel (dihydropyridine receptor, DHPR) localized in the t-tubule membrane and the Ca²⁺-release channel (ryanodine receptor, RyR) of the sarcoplasmic reticulum membrane. The structures of both channels have been extensively studied by several groups using electron cryomicroscopy and single particle reconstruction techniques. The structures of RyR, determined at resolutions of 22–30 Å, reveal a characteristic mushroom shape with a bulky cytoplasmic region and the membrane-spanning stem. While the cytoplasmic region exhibits a complex structure comprising a multitude of distinctive domains with numerous intervening cavities, at this resolution no definitive statement can be made about the location of the actual pore within the transmembrane region. Conformational changes associated with functional transitions of the Ca²⁺ release channel from closed to open states have been characterized. Further experiments determined localization of binding sites for various channel ligands. The structural studies of the DHPR are less developed. Although four 3D maps of the DHPR were reported recently at 24–30 Å resolution from studies of frozen-hydrated and negatively stained receptors, there are some discrepancies between reported structures with respect to the overall appearance and dimensions of the channel structure. Future structural studies at higher resolution are needed to refine the structures of both channels and to substantiate a proposed molecular model for their interaction.Translated from Biokhimiya, Vol. 69, No. 11, 2004, pp. 1506–1514.Original Russian Text Copyright © 2004 by Serysheva.     

大电导钾离子通道(BK)结构与功能的研究进展

大电导钙离子激活钾通道(BK)是细胞膜上唯一接受细胞内Ca2+和膜电位双重调控的离子通道.最新发表的关于BK通道电镜结构及其胞质功能域的晶体结构的文章,第一次展示了BK通道各亚基的组装,并证实通道各功能域在通道门控机制中存在紧密的相互作用.近年来,针对BK通道的功能调节及其门控动力学模拟的研究取得较多进展,有助于更好地理解BK通道发挥生理功能的门控机制,并揭示BK通道相关疾病的病理生理学基础.     

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

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.

MgtE is a magnesium-selective ion channel whose gating is regulated by cytoplasmic magnesium concentration; this cryo-EM study reveals how MgtE undergoes magnesium-dependent structural changes to open the pore on the cytoplasmic side.     

Structures of Gating Intermediates in a K+ channel

Regulation of ion conduction through the pore of a K⁺ channel takes place through the coordinated action of the activation gate at the bundle crossing of the inner helices and the inactivation gate located at the selectivity filter. The mechanism of allosteric coupling of these gates is of key interest. Here we report new insights into this allosteric coupling mechanism from studies on a W67F mutant of the KcsA channel. W67 is in the pore helix and is highly conserved in K⁺ channels. The KcsA W67F channel shows severely reduced inactivation and an enhanced rate of activation. We use continuous wave EPR spectroscopy to establish that the KcsA W67F channel shows an altered pH dependence of activation. Structural studies on the W67F channel provide the structures of two intermediate states: a pre- open state and a pre-inactivated state of the KcsA channel. These structures highlight key nodes in the allosteric pathway. The structure of the KcsA W67F channel with the activation gate open shows altered ion occupancy at the second ion binding site (S2) in the selectivity filter. This finding in combination with previous studies strongly support a requirement for ion occupancy at the S2 site for the channel to inactivate.     

Roles of the Hydrophobic Gate and Exit Channel in Vigna radiata Pyrophosphatase Ion Translocation

Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H⁺ and/or Na⁺) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH⁺-PPase), the VrH⁺-PPase–2P_i complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three VrH⁺-PPases complexes (P_i-, 2P_i- and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2P_i-bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among VrH⁺-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225–R562 is at the way out of the exit channel of VrH⁺-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225–R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer.     

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K_V and Ca_V channels. Like K_V channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca_V channels. We developed structural models of the NaChBac channel in closed and open conformations, using K⁺-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K⁺ crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K⁺ channels of known structure and Na_V, Ca_V, and TRP channels of unknown structure.     

Ion Channels in Southern Bean Mosaic Virus Capsid

The study of southern bean mosaic virus protein coat high resolution model revealed a structure with properties of a natural protein-ion channel. Coat protein pentamers form a 30-Å long channel and the amino acid composition of its wall bears some homology with the pentameric structure proposed for the nicotinic acetylcholine receptor channel. Ion transport properties were analyzed by computing ion-protein interaction energies on the basis of quantum chemistry methods. Energy maps show a channel attractive for cations, fully permeable to Li⁺ and a narrow barrier for other cations and water. The energy profiles found are similar to the profiles determined for the K⁺ channel of the sarcoplasmic reticulum. Comparisons with other icosahedral virus structures, including picornaviruses, suggest that ion channels would be a common feature of viral capsids. Biological roles for these channels are proposed.     

Structure and dynamics of the influenza A M2 Channel: a comparison of three structures

The M2 protein is an essential component of the Influenza virus’ infectivity cycle. It is a homo-tetrameric bundle forming a pH-gated H⁺ channel. The structure of M2 was solved by three different groups, using different techniques, protein sequences and pH environment. For example, solid-state NMR spectroscopy was used on a protein in lipid bilayers, while X-ray crystallography and solution NMR spectroscopy were applied on a protein in detergent micelles. The resulting structures from the above efforts are rather distinct. Herein, we examine the different structures under uniform conditions such as a lipid bilayer and specified protonation state. We employ extensive molecular dynamics simulations, in several protonation states, representing both closed and open forms of the channel. Exploring the properties of each of these structures has shown that the X-ray structure is more stable than the other structures according to various criteria, although its water conductance and water-wire formation do not correlate to the protonation state of the channel.     

Ion channels of glutamate receptors: structural modeling

Ionotropic glutamate receptors belong to the superfamily of P-loop channels as well as K⁺, Na⁺, and Ca²⁺ channels. However, the structural similarity between ion channels of the glutamate receptors and K⁺ channels is a matter of discussion. The aim of this study was to analyze differences between the structures of K⁺ channels and glutamate receptor channels. For this purpose, homology models of NMDA and AMPA receptor channels (M2 and M3 segments) were built using X-ray structures of K⁺ channels as templates. The models were optimized and used to reproduce specific data on the structure of glutamate receptor channels. Particular attention was paid to the data of the binding of channel blockers and to the results of scanning mutagenesis. The modeling demonstrates that properties of glutamate receptor channel can be reproduced assuming only local structural deformations of the K⁺ channel templates. The most valuable differences were found in the selectivity-filter region, whereas helical parts of M2 and M3 segments could have similar spatial organization with homologous segments in K⁺ channels. It is concluded that the current experimental data on glutamate receptor channels does not reveal global structural differences with K⁺ channels.     

Loss of cytosolic Mg2+ binding sites in the Thermotoga maritima CorA Mg2+ channel is not sufficient for channel opening

The CorA Mg²⁺ channel is a homopentamer with five-fold symmetry. Each monomer consists of a large cytoplasmic domain and two transmembrane helices connected via a short periplasmic loop. In the Thermotoga maritima CorA crystal structure, a Mg²⁺ is bound between D89 of one monomer and D253 of the adjacent monomer (M1 binding site). Release of Mg²⁺ from these sites has been hypothesized to cause opening of the channel. We generated mutants to disrupt Mg²⁺ interaction with the M1 site. Crystal structures of the D89K/D253K and D89R/D253R mutants, determined to 3.05 and 3.3?Å, respectively, showed no significant structural differences with the wild type structure despite absence of Mg²⁺ at the M1 sites. Both mutants still appear to be in the closed state. All three mutant CorA proteins exhibited transport of ⁶³Ni²⁺, indicating functionality. Thus, absence of Mg²⁺ from the M1 sites neither causes channel opening nor prevents function. We also provide evidence that the T. maritima CorA is a Mg²⁺ channel and not a Co²⁺ channel.     

An Extracellular Ion Pathway Plays a Central Role in the Cooperative Gating of a K2P K+ Channel by Extracellular pH

Proton-gated TASK-3 K⁺ channel belongs to the K_2P family of proteins that underlie the K⁺ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H⁺]. Use of recently solved K_2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K⁺ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K⁺ ions modulate the pK_a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K⁺ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH_o sensors.     

Direct NMR detection of the "invisible" alkali metal cations tightly bound to G-quadruplex structures

We report the first direct solution NMR detection of the alkali metal cations (²³Na⁺, ³⁹K⁺, and ⁸⁷Rb⁺) residing inside G-quadruplex channel structures formed by guanosine 5′-monophosphate and a DNA oligomer, d(TG₄T). In solution, these channel alkali metal cations are tightly bound to the G-quadruplex structure and have been considered to be “invisible” to NMR spectroscopy for many years. Our finding that it is possible to directly observe these alkali metal cations by NMR spectroscopy provides a new tool for studying cation binding affinity and dynamics in G-quadruplex DNA.     

Voltage-dependent cationic channel of Escherichia coli

Verapamil and dimethylcurine are Ca²⁺ entry blockers of essentially different chemical structures which presumably bind to the same arylalkylamine receptor of the L-type Ca channel. A systematic conformational analysis of methoxyverapamil (D-600) and dimethylcurine has been carried out using a molecular mechanics method. The lowest minimum-energy conformations of D-600 are predisposed to chelate Ca²⁺ by four oxygen atoms of the stacked methoxyphenyl moieties. Comparison of the lowest energy conformations of D-600-Ca²⁺ and dimethylcurine revealed a similar spatial disposition of cationic groups and methoxyphenyl moieties in the two compounds. A three-dimensional model of arylalkylamine receptor was suggested which incorporates two nucleophilic areas of the Ca channel. Dimethylcurine binds to these areas by its quaternary amine functions, whereas D-600 does so by amine function and via coordinated Ca²⁺. The results support the hypotheses on ternary complex formation between the ligands of Ca channel, their receptors, and Ca²⁺.     

NMR assignments of a 48 kDa tetramer of the T1 domain of the mammalian voltage gated potassium channel Kv1.4

The N-terminal cytosolic T1 domain of the mammalian voltage gated potassium channel Kv1.4 is strongly involved in the tetramerization of the Kv1.4 subunit that is required for forming a functional ion channel. The T1 domain forms a stable tetramer of 48 kDa in solution that cannot be dissociated into monomers. In spite of the high molecular mass it was possible to completely assign the backbone and part of the side chain resonances by multidimensional NMR spectroscopy on uniformly ²H, ¹³C, ¹⁵N enriched protein. The secondary structure analysis derived from the chemical shifts is in line with the expectations from X-ray structures of related proteins.