Propofol rescues voltage-dependent gating of HCN1 channel epilepsy mutants.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels1 are essential for pacemaking activity and neural signalling2,3. Drugs inhibiting HCN1 are promising candidates for management of neuropathic pain4 and epileptic seizures5. The general anaesthetic propofol (2,6-di-iso-propylphenol) is a known HCN1 allosteric inhibitor6 with unknown structural basis. Here, using single-particle cryo-electron microscopy and electrophysiology, we show that propofol inhibits HCN1 by binding to a mechanistic hotspot in a groove between the S5 and S6 transmembrane helices. We found that propofol restored voltage-dependent closing in two HCN1 epilepsy-associated polymorphisms that act by destabilizing the channel closed state: M305L, located in the propofol-binding site in S5, and D401H in S6 (refs. 7,8). To understand the mechanism of propofol inhibition and restoration of voltage-gating, we tracked voltage-sensor movement in spHCN channels and found that propofol inhibition is independent of voltage-sensor conformational changes. Mutations at the homologous methionine in spHCN and an adjacent conserved phenylalanine in S6 similarly destabilize closing without disrupting voltage-sensor movements, indicating that voltage-dependent closure requires this interface intact. We propose a model for voltage-dependent gating in which propofol stabilizes coupling between the voltage sensor and pore at this conserved methionine-phenylalanine interface in HCN channels. These findings unlock potential exploitation of this site to design specific drugs targeting HCN channelopathies.

Rearrangement of a unique Kv1.3 selectivity filter conformation upon binding of a drug.

We report two structures of the human voltage-gated potassium channel (Kv) Kv1.3 in immune cells alone (apo-Kv1.3) and bound to an immunomodulatory drug called dalazatide (dalazatide-Kv1.3). Both the apo-Kv1.3 and dalazatide-Kv1.3 structures are in an activated state based on their depolarized voltage sensor and open inner gate. In apo-Kv1.3, the aromatic residue in the signature sequence (Y447) adopts a position that diverges 11 Å from other K+ channels. The outer pore is significantly rearranged, causing widening of the selectivity filter and perturbation of ion binding within the filter. This conformation is stabilized by a network of intrasubunit hydrogen bonds. In dalazatide-Kv1.3, binding of dalazatide to the channel's outer vestibule narrows the selectivity filter, Y447 occupies a position seen in other K+ channels, and this conformation is stabilized by a network of intersubunit hydrogen bonds. These remarkable rearrangements in the selectivity filter underlie Kv1.3's transition into the drug-blocked state.

Cryo-electron microscopy structure of the Slo2.2 Na(+)-activated K(+) channel.

Na(+)-activated K(+) channels are members of the Slo family of large conductance K(+) channels that are widely expressed in the brain, where their opening regulates neuronal excitability. These channels fulfil a number of biological roles and have intriguing biophysical properties, including conductance levels that are ten times those of most other K(+) channels and gating sensitivity to intracellular Na(+). Here we present the structure of a complete Na(+)-activated K(+) channel, chicken Slo2.2, in the Na(+)-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 ångströms. The channel is composed of a large cytoplasmic gating ring, in which resides the Na(+)-binding site and a transmembrane domain that closely resembles voltage-gated K(+) channels. In the structure, the cytoplasmic domain adopts a closed conformation and the ion conduction pore is also closed. The structure reveals features that can explain the unusually high conductance of Slo channels and how contraction of the cytoplasmic gating ring closes the pore.

Glibenclamide and HMR1098 normalize Cantú syndrome-associated gain-of-function currents.

Cantú syndrome (CS) is caused by dominant gain-of-function mutation in ATP-dependent potassium channels. Cellular ATP concentrations regulate potassium current thereby coupling energy status with membrane excitability. No specific pharmacotherapeutic options are available to treat CS but IKATP channels are pharmaceutical targets in type II diabetes or cardiac arrhythmia treatment. We have been suggested that IKATP inhibitors, glibenclamide and HMR1098, normalize CS channels. IKATP in response to Mg-ATP, glibenclamide and HMR1098 were measured by inside-out patch-clamp electrophysiology. Results were interpreted in view of cryo-EM IKATP channel structures. Mg-ATP IC50 values of outward current were increased for D207E (0.71 ± 0.14 mmol/L), S1020P (1.83 ± 0.10), S1054Y (0.95 ± 0.06) and R1154Q (0.75 ± 0.13) channels compared to H60Y (0.14 ± 0.01) and wild-type (0.15 ± 0.01). HMR1098 dose-dependently inhibited S1020P and S1054Y channels in the presence of 0.15 mmol/L Mg-ATP, reaching, at 30 µmol/L, current levels displayed by wild-type and H60Y channels in the presence of 0.15 mmol/L Mg-ATP. Glibenclamide (10 µmol/L) induced similar normalization. S1054Y sensitivity to glibenclamide increases strongly at 0.5 mmol/L Mg-ATP compared to 0.15 mmol/L, in contrast to D207E and S1020P channels. Experimental findings agree with structural considerations. We conclude that CS channel activity can be normalized by existing drugs; however, complete normalization can be achieved at supraclinical concentrations only.

Multi-ion free energy landscapes underscore the microscopic mechanism of ion selectivity in the KcsA channel.

Potassium (K(+)) channels are transmembrane proteins that passively and selectively allow K(+) ions to flow through them, after opening in response to an external stimulus. One of the most critical functional aspects of their function is their ability to remain very selective for K(+) over Na(+) while allowing high-throughput ion conduction at a rate close to the diffusion limit. Classically, it is assumed that the free energy difference between K(+) and Na(+) in the pore relative to the bulk solution is the critical quantity at the origin of selectivity. This is the thermodynamic view of ion selectivity. An alternative view assumes that kinetic factors play the dominant role. Recent results from a number of studies have also highlighted the great importance of the multi-ion single file on the selectivity of K(+) channels. The data indicate that having multiple K(+) ions bound simultaneously is required for selective K(+) conduction, and that a reduction in the number of bound K(+) ions destroys the multi-ion selectivity mechanism utilized by K(+) channels. In the present study, multi-ion potential of mean force molecular dynamics computations are carried out to clarify the mechanism of ion selectivity in the KcsA channel. The computations show that the multi-ion character of the permeation process is a critical element for establishing the selective ion conductivity through K(+)-channels. This article is part of a Special Issue entitled: Membrane Proteins edited by J.C. Gumbart and Sergei Noskov.

Efficient enzymatic cyclization of an inhibitory cystine knot-containing peptide.

Disulfide-rich peptides isolated from cone snails are of great interest as drug leads due to their high specificity and potency toward therapeutically relevant ion channels and receptors. They commonly contain the inhibitor cystine knot (ICK) motif comprising three disulfide bonds forming a knotted core. Here we report the successful enzymatic backbone cyclization of an ICK-containing peptide κ-PVIIA, a 27-amino acid conopeptide from Conus purpurascens, using a mutated version of the bacterial transpeptidase, sortase A. Although a slight loss of activity was observed compared to native κ-PVIIA, cyclic κ-PVIIA is a functional peptide that inhibits the Shaker voltage-gated potassium (Kv) channel. Molecular modeling suggests that the decrease in potency may be related to the loss of crucial, but previously unidentified electrostatic interactions between the N-terminus of the peptide and the Shaker channel. This hypothesis was confirmed by testing an N-terminally acetylated κ-PVIIA, which shows a similar decrease in activity. We also investigated the conformational dynamics and hydrogen bond network of cyc-PVIIA, both of which are important factors to be considered for successful cyclization of peptides. We found that cyc-PVIIA has the same conformational dynamics, but different hydrogen bond network compared to those of κ-PVIIA. The ability to efficiently cyclize ICK peptides using sortase A will enable future protein engineering for this class of peptides and may help in the development of novel therapeutic molecules. Biotechnol. Bioeng. 2016;113: 2202-2212. © 2016 Wiley Periodicals, Inc.

2dx_automator: implementation of a semiautomatic high-throughput high-resolution cryo-electron crystallography pipeline.

The introduction of direct electron detectors (DED) to cryo-electron microscopy has tremendously increased the signal-to-noise ratio (SNR) and quality of the recorded images. We discuss the optimal use of DEDs for cryo-electron crystallography, introduce a new automatic image processing pipeline, and demonstrate the vast improvement in the resolution achieved by the use of both together, especially for highly tilted samples. The new processing pipeline (now included in the software package 2dx) exploits the high SNR and frame readout frequency of DEDs to automatically correct for beam-induced sample movement, and reliably processes individual crystal images without human interaction as data are being acquired. A new graphical user interface (GUI) condenses all information required for quality assessment in one window, allowing the imaging conditions to be verified and adjusted during the data collection session. With this new pipeline an automatically generated unit cell projection map of each recorded 2D crystal is available less than 5 min after the image was recorded. The entire processing procedure yielded a three-dimensional reconstruction of the 2D-crystallized ion-channel membrane protein MloK1 with a much-improved resolution of 5Å in-plane and 7Å in the z-direction, within 2 days of data acquisition and simultaneous processing. The results obtained are superior to those delivered by conventional photographic film-based methodology of the same sample, and demonstrate the importance of drift-correction.

Direct manipulation of a single potassium channel gate with an atomic force microscope probe.

Ion channels are membrane proteins that regulate cell functions by controlling the ion permeability of cell membranes. An ion channel contains an ion-selective pore that permeates ions and a sensor that senses a specific stimulus such as ligand binding to regulate the permeability. The detailed molecular mechanisms of this regulation, or gating, are unknown. Gating is thought to occur from conformational changes in the sensor domain in response to the stimulus, which results in opening the gate to permit ion conduction. Using an atomic force microscope and artificial bilayer system, a mechanical stimulus is applied to a potassium channel, and its gating is monitored in real time. The channel-open probability increases greatly when pushing the cytoplasmic domain toward the membrane. This result shows that a mechanical stimulus at the cytoplasmic domain causes changes in the gating and is the first to show direct evidence of coupling between conformational changes in the cytoplasmic domain and channel gating. This novel technology has the potential to be a powerful tool for investigating the activation dynamics in channel proteins.

Gating and selectivity mechanisms for the lysosomal K+ channel TMEM175.

Transmembrane protein 175 (TMEM175) is a K+-selective ion channel expressed in lysosomal membranes, where it establishes a membrane potential essential for lysosomal function and its dysregulation is associated with the development of Parkinson's Disease. TMEM175 is evolutionarily distinct from all known channels, predicting novel ion-selectivity and gating mechanisms. Here we present cryo-EM structures of human TMEM175 in open and closed conformations, enabled by resolutions up to 2.6 Å. Human TMEM175 adopts a homodimeric architecture with a central ion-conduction pore lined by the side chains of the pore-lining helices. Conserved isoleucine residues in the center of the pore serve as the gate in the closed conformation. In the widened channel in the open conformation, these same residues establish a constriction essential for K+ selectivity. These studies reveal the mechanisms of permeation, selectivity and gating and lay the groundwork for understanding the role of TMEM175 in lysosomal function.

Assembly of Kch, a putative potassium channel from Escherichia coli.

Attempts to explore the structure and function of Kch, a putative potassium channel of Escherichia coli have yielded varying results; potassium-associated functions have been found in vivo but not in vitro. Here the kch gene is shown to produce two proteins, full-length Kch and the large C-terminal cytosolic domain (the RCK domain). Further, these two proteins are associated at the initial stages of purification. Previous structural studies of full-length Kch claim that the isolated protein forms large aggregates that are not suitable for analysis. The results presented here show that the purified protein sample, although heterogeneous, has one major population with a mass of about 400kDa, implying the presence of two Kch tetramers in a complex form. A three dimensional reconstruction at 25A based on electron microscopy data from negatively stained particles, revealed a 210A long and 95A wide complex in which the two tetrameric Kch units are linked by their RCK domains, giving rise to a large central ring of density. The formation of this dimer of tetramers on expression or during purification, may explain why attempts to reconstitute Kch into liposomes for activity measurements have failed.

Mutant potassium channels with altered binding of charybdotoxin, a pore-blocking peptide inhibitor.

The inhibition by charybdotoxin of A-type potassium channels expressed in Xenopus oocytes was studied for several splicing variants of the Drosophila Shaker gene and for several site-directed mutants of this channel. Charybdotoxin blocking affinity is lowered by a factor of 3.5 upon replacing glutamate-422 with glutamine, and by a factor of about 12 upon substituting lysine in this position. Replacement of glutamate-422 by aspartate had no effect on toxin affinity. Thus, the glutamate residue at position 422 of this potassium channel is near or in the externally facing mouth of the potassium conduction pathway, and the positively charged toxin is electrostatically focused toward its blocking site by the negative potential set up by glutamate-422.

Activation of a nucleotide-dependent RCK domain requires binding of a cation cofactor to a conserved site.

RCK domains regulate the activity of K+ channels and transporters in eukaryotic and prokaryotic organisms by responding to ions or nucleotides. The mechanisms of RCK activation by Ca2+ in the eukaryotic BK and bacterial MthK K+ channels are well understood. However, the molecular details of activation in nucleotide-dependent RCK domains are not clear. Through a functional and structural analysis of the mechanism of ATP activation in KtrA, a RCK domain from the B. subtilis KtrAB cation channel, we have found that activation by nucleotide requires binding of cations to an intra-dimer interface site in the RCK dimer. In particular, divalent cations are coordinated by the γ-phosphates of bound-ATP, tethering the two subunits and stabilizing the active state conformation. Strikingly, the binding site residues are highly conserved in many different nucleotide-dependent RCK domains, indicating that divalent cations are a general cofactor in the regulatory mechanism of many nucleotide-dependent RCK domains.

Interaction of anesthetics with open and closed conformations of a potassium channel studied via molecular dynamics and normal mode analysis.

A variety of experiments suggest that membrane proteins are important targets of anesthetic molecules, and that ion channels interact differently with anesthetics in their open and closed conformations. The availability of an open and a closed structural model for the KirBac1.1 potassium channel has made it possible to perform a comparative analysis of the interactions of anesthetics with the same channel in its open and closed states. To this end, all-atom molecular dynamics simulations supplemented by normal mode analysis have been employed to probe the interactions of the inhalational anesthetic halothane with both an open and closed conformer of KirBac1.1 embedded in a lipid bilayer. Normal mode analysis on the closed and open channel, in the presence and absence of halothane, reveals that the anesthetic modulates the global as well as the local dynamics of both conformations differently. In the case of the open channel, the observed reduction of flexibility of residues in the inner helices suggests a functional modification action of anesthetics on ion channels. In this context, preferential quenching of the aromatic residue motion and modulation of global dynamics by halothane may be seen as steps toward potentiating or favoring open state conformations. These molecular dynamics simulations provide the first insights into possible specific interactions between anesthetic molecules and ion channels in different conformations.

Subcellular segregation of two A-type K+ channel proteins in rat central neurons.

In the mammalian nervous system, K+ channels regulate diverse aspects of neuronal function and are encoded by a large set of K+ channel genes. The roles of different K+ channel proteins could be dictated by their localization to specific subcellular domains. We report that two K+ channel polypeptides, Kv1.4 and Kv4.2, which form transient (A-type) K+ channels when expressed in Xenopus oocytes, are segregated in rat central neurons. Kv1.4 protein is targeted to axons and possibly terminals, while Kv4.2 is concentrated in dendrites and somata. This differential distribution implies distinct roles for these channel proteins in vivo. Their localizations suggest that Kv1.4 and Kv4.2 may regulate synaptic transmission via presynaptic, or postsynaptic mechanisms, respectively.

Novel potassium channels encoded by the Shaker locus in Drosophila photoreceptors.

The Shaker gene, responsible for A-type potassium channels in Drosophila muscle, encodes a large family of transcripts capable of generating a variety of kinetically distinct A channels when expressed in oocytes. We describe a distinct class of A channel encoded by the Shaker gene in a novel preparation of dissociated Drosophila photoreceptors. Whole-cell recordings reveal a rapidly inactivating A current that is absent in Shaker mutants and that can be readily isolated in cell-attached patches. Although very similar to their muscle counterparts, the photoreceptor A channels show a striking 40-50 mV negative shift in their voltage-operating range. Two mutations (ShE62 and T(1;Y)W32), which exclude only certain classes of Shaker transcripts, were used to show that photoreceptor A channels are encoded by multiple transcripts distinct from those encoding muscle A channels, while PCR techniques identified four transcripts (ShA1, ShA2, ShG1, and ShG2) in mRNA from dissected retina.

Three-dimensional structure of I(to); Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution.

Regulatory KChIP2 subunits assemble with pore-forming Kv4.2 subunits in 4:4 complexes to produce native voltage-gated potassium (Kv) channels like cardiac I(to) and neuronal I(A) subtypes. Here, negative stain electron microscopy (EM) and single particle averaging reveal KChIP2 to create a novel approximately 35 x 115 x 115 Angstrom, intracellular fenestrated rotunda: four peripheral columns that extend down from the membrane-embedded portion of the channel to enclose the Kv4.2 "hanging gondola" (a platform held beneath the transmembrane conduction pore by four internal columns). To reach the pore from the cytosol, ions traverse one of four external fenestrae to enter the rotundal vestibule and then cross one of four internal windows in the gondola.

Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification.

Inwardly rectifying K+ channels bind intracellular magnesium and polyamines to generate inward rectification. We have examined the architecture of the inner pore of Kir2.1 channels by covalently attaching a constrained number (from one to four) of positively charged moieties of different sizes to the channel. Our results indicate that the inner pore is formed solely by the second transmembrane segment and is unprecedentedly wide. At a position critical for inward rectification (D172), the pore is sufficiently wide to bind three Mg2+ ions or polyamine molecules simultaneously. Single-channel recordings directly demonstrate that partially modified channels exhibit distinct subconductance levels. Such a wide inner pore may greatly facilitate ion permeation and high-affinity binding of multiple pore blockers to generate strong inward rectification.

Charybdotoxin block of Shaker K+ channels suggests that different types of K+ channels share common structural features.

Charybdotoxin (CTX), a 37 amino acid protein isolated from the venom of L. quinquestriatus, is a high-affinity blocker of various Ca2(+)-activated K+ channels. CTX also blocks Drosophila Shaker (Sh) clone H4 transient K+ currents expressed in Xenopus oocytes with similar affinity (Kd = 3.6 nM). CTX blocks both the open and the closed states of Sh channels with no apparent change in gating behavior. In addition, the block is enhanced as the ionic strength is lowered. These properties are identical to those of CTX block of Ca(+)-activated K+ channels, and these results suggest that the external pore openings of these two functionally dissimilar K+ channels may share common structural features.

Estrogen induction of a small, putative K+ channel mRNA in rat uterus.

Estrogen causes dramatic long-term changes in the activity of the uterus. Here we report the molecular cloning of a small (700 base) uterine mRNA species capable of inducing a slow K+ current in Xenopus oocytes. The 130 amino acid protein encoded by this mRNA species has a predicted structure that does not resemble that of previously described voltage-dependent channels from mammalian sources. It is, however, similar to structural motifs found in certain prokaryotic ion channels. The induction of this mRNA by estrogen is rapid; this uterine mRNA species is not detectable in uteri from estrogen-deprived rats, but is substantially induced after 3 hr of estrogen treatment. These results support a critical role for regulation of ion channel expression by estrogen in the uterus.

Images of purified Shaker potassium channels.

BACKGROUND: Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis. RESULTS: We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions. CONCLUSION: These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.