Prokaryotic Ion Channel Research Articles

The insights into calcium ion selectivity provided by ancestral prokaryotic ion channels.

Prokaryotic channels play an important role in the structural biology of ion channels. At the end of the 20th century, the first structure of a prokaryotic ion channel was revealed. Subsequently, the reporting of structures of various prokaryotic ion channels have provided fundamental insights into the structure of ion channels of higher organisms. Voltage-dependent Ca2+ channels (Cavs) are indispensable for coupling action potentials with Ca2+ signaling. Similar to other proteins, Cavs were predicted to have a prokaryotic counterpart; however, it has taken more than 20 years for one to be identified. The homotetrameric channel obtained from Meiothermus ruber generates the calcium ion specific current, so it is named as CavMr. Its selectivity filter contains a smaller number of negatively charged residues than mutant Cavs generated from other prokaryotic channels. CavMr belonged to a different cluster of phylogenetic trees than canonical prokaryotic cation channels. The glycine residue of the CavMr selectivity filter is a determinant for calcium selectivity. This glycine residue is conserved among eukaryotic Cavs, suggesting that there is a universal mechanism for calcium selectivity. A family of homotetrameric channels has also been identified from eukaryotic unicellular algae, and the investigation of these channels can help to understand the mechanism for ion selection that is conserved from prokaryotes to eukaryotes.

Preparation of Giant Escherichia coli spheroplasts for Electrophysiological Recordings.

Prokaryotic ion channels have been instrumental in furthering our understanding of many fundamental aspects of ion channels' structure and function. However, characterizing the biophysical properties of a prokaryotic ion channel in a native membrane system using patch-clamp electrophysiology is technically challenging. Patch-clamp is regarded as a gold standard technique to study ion channel properties in both native and heterologous expression systems. The presence of a cell wall and the small size of bacterial cells makes it impossible to directly patch clamp using microelectrodes. Here, we describe a method for the preparation of giant E. coli spheroplasts in order to investigate the electrophysiological properties of bacterial cell membranes. Spheroplasts are formed by first inhibiting bacterial cell wall synthesis, followed by enzymatic digestion of the outer cell wall in the presence of a permeabilizing agent. This protocol can be used to characterize the function of any heterologous ion channels or ion transporters expressed in E. coli membranes.

In vitro discovery of novel prokaryotic ion channel candidates for antiarrhythmic gene therapy.

Sudden cardiac death continues to have a devastating impact on public health prompting the continued efforts to develop more effective therapies for cardiac arrhythmias. Among different approaches to normalize function of ion channels and prevent arrhythmogenic remodeling of tissue substrate, cardiac cell and gene therapies are emerging as promising strategies to restore and maintain normal heart rhythm. Specifically, the ability to genetically enhance electrical excitability of diseased hearts through voltage-gated sodium channel (VGSC) gene transfer could improve velocity of action potential conduction and act to stop reentrant circuits underlying sustained arrhythmias. For this purpose, prokaryotic VGSC genes are promising therapeutic candidates due to their small size (<1kb) and potential to be effectively packaged in adeno-associated viral (AAV) vectors and delivered to cardiomyocytes for stable, long-term expression. This article describes a versatile method to discover and characterize novel prokaryotic ion channels for use in gene and cell therapies for heart disease including cardiac arrhythmias. Detailed protocols are provided for: (1) identification of potential ion channel candidates from large genomic databases, (2) candidate screening and characterization using site-directed mutagenesis and engineered human excitable cell system and, (3) candidate validation using electrophysiological techniques and an in vitro model of impaired cardiac impulse conduction.

Resonance-driven ion transport and selectivity in prokaryotic ion channels.

Ion channels exhibit a remarkably high accuracy in selecting uniquely its associated type of ion. The mechanisms behind ion selectivity are not well understood. Current explanations build mainly on molecular biology and bioinformatics. Here we propose a simple physical model for ion selectivity based on the driven damped harmonic oscillator (DDHO). The driving force for this oscillator is provided by self-organizing harmonic turbulent structures in the dehydrating ionic flow through the ion channel, namely, oscillating pressure waves in one dimension, and toroidal vortices in two and three dimensions. Density fluctuations caused by these turbulences efficiently transmit their energy to aqua ions that resonate with the driving frequency. Consequently, these release their hydration shell and exit the ion channel as free ions. Existing modeling frameworks do not express the required complex spatiotemporal dynamics, hence we introduce a macroscopic continuum model for ionic dehydration and transport, based on the hydrodynamics of a dissipative ionic flow through an ion channel, subject to electrostatic and amphiphilic interactions. This model combines three classical physical fields: Navier-Stokes equations from hydrodynamics, Gauss's law from Maxwell theory, and the convection-diffusion equation from continuum physics. Numerical experiments with mixtures of chemical species of ions in various degrees of hydration indeed reveal the emergence of strong oscillations in the ionic flow that are instrumental in the efficient dehydration and cause a strong ionic jet into the cell. As such, they provide a powerful engine for the DDHO mechanism. Theoretical predictions of the modeling framework match significantly with empirical patch-clamp data. The DDHO standard response curve defines a unique resonance frequency that depends on the mass and charge of the ion. In this way, the driving oscillations act as a selection mechanism that filters out one specific ion. Application of the DDHO model to real ion data shows that this mechanism indeed clearly distinguishes between chemical species and between aqua and bare ions with a large Mahalanobis distance and high oscillator quality. The DDHO framework helps to understand how SNP mutations can cause severe genetic pathologies as they destroy the geometry of the channel and so alter the resonance peaks of the required ion type.

Characterizing Functional States of a Model Ligand-Gated Ion Channel by Cryo-Electron Microscopy

Pentameric ligand-gated ion channels are important mediators of electrochemical signal transduction in the brain and other systems. Recent progress in protein biochemistry and structural biology has shed light on topology and possible gating mechanisms in these channels, yet the functionally relevant endpoints of activation and modulation remain unclear. In order to obtain alternative structural insights, we optimized protein purification, reconstitution, and grid preparation conditions for structure determination of a prokaryotic ion channel using cryo-electron microscopy (EM). We prepared samples at various presumed distributions of active and inactive states, with the goal of quantifying distinct classes within individual cryo-EM grids. Modification of carbon support, glow-discharging vapor, and blotting time were critical to improving particle distribution and orientation, and molecular simulations were harnessed to validate structural models. This work promises to inform methods development in cryo-EM of membrane proteins, and to populate the gating landscape in this important ion channel family.

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis.

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine and remain largely unknown. With recent advances in single-particle cryo-electron microscopy (cryo-EM) shedding light on the structural features of agonist binding sites and the activation mechanism of several ion channels, the stage is set for an in-depth dynamic analysis of their gating mechanisms using spectroscopic approaches. Spectroscopic techniques such as electron paramagnetic resonance (EPR) and double electron-electron resonance (DEER) have been mainly restricted to the study of prokaryotic ion channels that can be purified in large quantities. The requirement for large amounts of functional and stable membrane proteins has hampered the study of mammalian ion channels using these approaches. EPR and DEER offer many advantages, including determination of the structure and dynamic changes of mobile protein regions, albeit at low resolution, that might be difficult to obtain by X-ray crystallography or cryo-EM, and monitoring reversible gating transition (i.e., closed, open, sensitized, and desensitized). Here, we provide protocols for obtaining milligrams of functional detergent-solubilized transient receptor potential cation channel subfamily V member 1 (TRPV1) that can be labeled for EPR and DEER spectroscopy.

Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

Molecular Dynamics Study of Ion Conduction and Selectivity in a Prokaryotic Ion Channel

Since the publication of crystal structure of NavAb, the first of a (prokaryotic) voltage gated sodium channel, several computational studies have been aimed at determining the mechanisms of ion selectivity and ion conduction in voltage gated sodium channels. We provide a two-part study, well-converged free energy surfaces involving one, two and three ions provide results consistent with microsecond timescale simulations of ion conduction over concentration and voltages. The position of ions in the pore and cavity correlate to coordination number and side chain orientation, showing, as suggested by Chakrabarti et al. (PNAS (2013) 110, 11331). More surprising, presence of an ion in the aqueous cavity beneath the selectivity filter was sufficient to influence glutamate conformation.Because the crystal structure of NavAb was in a closed pore conformation, we truncated the S5 and S6 helices and restrained these outer helices harmonically in a membrane represented by supporting lattice of neon. The turrets, pore helices and selectivity filter were free to move. Conductances were in the range of the experimental values; however, our studies show no preference for sodium conduction over potassium conduction at physiological conditions (−200mV, 0.15 M salt). While sodium conductance varied little with respect to concentration and presence of potassium in solution, stronger potassium selectivity is observed at 1 M and in mixed solutions. Our results are consistent with the observation of anomalous mole fraction effect in NaChBac by Finol-Urdaneta et al. (JGP (2014) 143, 157-171), though not sufficient to confirm such an effect. Additionally, we provide mechanisms for ion conduction showing a greater variety of states and transitions occupied during potassium conduction, and note that at higher concentrations of salt the mechanism of conduction is not as clearly cut.

Heteromultimerization of prokaryotic bacterial cyclic nucleotide-gated (bCNG) ion channels, members of the mechanosensitive channel of small conductance (MscS) superfamily.

Traditionally, prokaryotic channels are thought to exist as homomultimeric assemblies, while many eukaryotic ion channels form complex heteromultimers. Here we demonstrate that bacterial cyclic nucleotide-gated channels likely form heteromultimers in vivo. Heteromultimer formation is indicated through channel modeling, pull-down assays, and real-time polymerase chain reaction analysis. Our observations demonstrate that prokaryotic ion channels can display complex behavior and regulation akin to that of their eukaryotic counterparts.

Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

Recent studies highlighted the importance of loop 2 of α1 glycine receptors (GlyRs) in the propagation of ligand-binding energy to the channel gate. Mutations that changed polarity at position 52 in the β hairpin of loop 2 significantly affected sensitivity to ethanol. The present study extends the investigation to charged residues. We found that substituting alanine with the negative glutamate at position 52 (A52E) significantly left-shifted the glycine concentration response curve and increased sensitivity to ethanol, whereas the negative aspartate substitution (A52D) significantly right-shifted the glycine EC₅₀ but did not affect ethanol sensitivity. It is noteworthy that the uncharged glutamine at position 52 (A52Q) caused only a small right shift of the glycine EC₅₀ while increasing ethanol sensitivity as much as A52E. In contrast, the shorter uncharged asparagine (A52N) caused the greatest right shift of glycine EC₅₀ and reduced ethanol sensitivity to half of wild type. Collectively, these findings suggest that charge interactions determined by the specific geometry of the amino acid at position 52 (e.g., the 1-Å chain length difference between aspartate and glutamate) play differential roles in receptor sensitivity to agonist and ethanol. We interpret these results in terms of a new homology model of GlyR based on a prokaryotic ion channel and propose that these mutations form salt bridges to residues across the β hairpin (A52E-R59 and A52N-D57). We hypothesize that these electrostatic interactions distort loop 2, thereby changing agonist activation and ethanol modulation. This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs.

Conservation and divergence of Grb7 family of Ras-binding domains

Ras proteins are signal-transducing GTPases that cycle between inactive GDP-bound and active GTP-bound forms. Ras is a prolific signaling molecule interacting with a spectrum of effector molecules and acting through more than one signaling pathway. The Ras-effector proteins contain a Ras-associating (RA) domain through which these associate with Ras in a GTP-dependent manner. The RA domain is highly conserved among the members of the growth factor receptor-bound (Grb) 7 family of proteins which includes Grb7, Grb10 and Grb14. Our laboratory has reported an unusual observation that RA domain of Grb14 binds to the C-terminal nucleotide binding site of cyclic nucleotide gated channel (CTRCNGA1) and inhibits the channel activity. Molecular modeling of the CTR-CNGA1 displays 50%-70% tertiary structural similarity towards Ras proteins. We named this region as Ras-like domain (RLD). The interaction between RA-Grb14 and RLD-CNGA1 is mediated through a simple protein-protein interaction temporally and spatially regulated by light and cGMP. It is interesting to note that Grb14 binds to GTPase-mutant Rab5, a Ras-related small GTPase whereas Grb10 binds only to GTP-bound form of active Rab5 but not to GTPase-defective mutant Rab5. These results suggest that Grb14 might have been evolved later in the evolution that binds to both Ras and nucleotide binding proteins such as CNGA1. Our studies also suggest that eukaryotic CNG channels could be evolved through a gene fusion between prokaryotic ion channels and cyclic nucleotide binding proteins, both of which might have undergone several sequence variations for functional adaptation during evolution.

Engineering the hERG1 Selectivity Filter into the NaK Pore Domain

Comparison of the hERG1 selectivity filter sequence and pore helix with a variety of prokaryotic ion channels revealed an unusually high sequence identity (63%) with the non-selective cation channel NaK (∼19 amino acids). Taking advantage of this remarkable similarity, we have used NaK as a template to carry out a structural analysis of a hERG1-like NaK selectivity filter in K+. To engineer the NaK filter into that of hERG1, we substituted two critical residues, V59S in the pore helix and D66F at the selectivity filter (equivalent to positions Ser620 and Phe627 in hERG1, respectively). The final construct shares 73% sequence identity with the hERG1 selectivity filter, equivalent to the closest orthologue of hERG1, the bovine ether-a-go-go channel (bEAG1). Crystals of this engineered hERG-NaK diffracted to 2.8 Å resolution and were solved by molecular replacement using closed NaK as template. The crystal structure of hERG1-like NaK in 100 mM K+ revealed a striking similarity to the all canonical K+ channel filters in the conductive conformation (RMSD= 0.5 Å with the KcsA filter) and shows three major differences compared to WT NaK. First, hERG1-like NaK filter revealed four equivalent K+ binding sites. Second, the side chain of Phe66 residue establishes critical packing interactions with the adjacent aromatic residues from the pore helix. Third, hERG1-like NaK shows hydrogen bond interactions through a water molecule behind the selectivity filter, which is absent in WT NaK, but present in KcsA. We suggest that the hERG-NaK construct represents a unique tool to investigate the properties of the hERG1 channel selectivity filter at atomic level.

Modeling Anesthetic Binding Sites within the Glycine Alpha One Receptor Based on Prokaryotic Ion Channel Templates: The Problem with TM4

Ligand-gated ion channels (LGICs) significantly modulate anesthetic effects. Their exact molecular structure remains unknown. This has led to ambiguity regarding the proper amino acid alignment within their 3D structure and, in turn, the location of any anesthetic binding sites. Current controversies suggest that such a site could be located in either an intra- or intersubunit locale within the transmembrane domain of the protein. Here, we built a model of the glycine alpha one receptor (GlyRa1) based on the open-state structures of two new high-resolution ion channel templates from the prokaryote, Gloebacter violaceus (GLIC). Sequence scoring suggests reasonable homology between GlyRa1 and GLIC. Three of the residues notable for modulating anesthetic action are on transmembrane segments 1-3 (TM1-3): (ILE229, SER 267, and ALA 288). They line an intersubunit interface, in contrast to previous models. However, residues from the fourth transmembrane domain (TM4) that are known to modulate a variety of anesthetic effects are quite distant from this putative anesthetic binding site. While this model can account for a large proportion of the physicochemical data regarding such proteins, it cannot readily account for the alterations on anesthetic effects that are due to mutations within TM4.

Linking of Glycine Receptor Transmembrane Segments Three and Four Allows Assignment of Intrasubunit-Facing Residues

Glycine receptors (GlyRs) are pentameric ligand-gated ion channels that mediate inhibitory neurotransmission in the brain and spinal cord and are targets of alcohols and anesthetics. The transmembrane (TM) domain of GlyR subunits is composed of four α-helical segments (TM1-4), but there are conflicting data about the orientation of TM3 and TM4 and, therefore, also the proximity of residues (e.g., A288) that are important for alcohol and anesthetic effects. In the present study, we investigated the proximity of A288 in TM3 to residues in TM4 from M404 to K411. We generated eight double mutant GlyRs (A288C/M404C, A288C/F405C, A288C/Y406C, A288C/W407C, A288C/I408C, A288C/I409C, A288C/Y410C, and A288C/K411C), as well as the corresponding single mutants, and expressed them in Xenopus laevis oocytes. To measure glycine responses, we used two-electrode voltage clamp electrophysiology. We built homology models of the GlyR using structures of the nicotinic acetylcholine receptor (nAChR) and a prokaryotic ion channel (Gloeobacter violaceus, GLIC) as templates, and asked which model best fit our experimental data. Application of the cross-linking reagent HgCl(2) in the closed state produced a leftward shift in the glycine concentration-response curves of the A288C/W407C and A288C/Y410C mutants, suggesting they are able to form cross-links. In addition, when HgCl(2) was coapplied with glycine, responses were changed in the A288C/Y406C, A288C/I409C, and A288C/Y410C double mutants, suggesting that agonist-induced rotation of TM4 allows A288C/Y406C and A288C/I409C to cross-link. These results are consistent with a model of GlyR, based on nAChR, in which A288, Y406, W407, I409, and Y410 face into a four-helical bundle.

A novel potassium channel in photosynthetic cyanobacteria.

Elucidation of the structure-function relationship of a small number of prokaryotic ion channels characterized so far greatly contributed to our knowledge on basic mechanisms of ion conduction. We identified a new potassium channel (SynK) in the genome of the cyanobacterium Synechocystis sp. PCC6803, a photosynthetic model organism. SynK, when expressed in a K+-uptake-system deficient E.coli strain, was able to recover growth of these organisms. The protein functions as a potassium selective ion channel when expressed in Chinese Hamster Ovary cells. The location of SynK in cyanobacteria in both thylakoid and plasmamembranes was revealed by immunogold electron microscopy and Western blotting of isolated membrane fractions. SynK seems to be conserved during evolution, giving rise to a TPK (two-pore K+ channel) family member which is shown here to be located in the thylakoid membrane of Arabidopsis. Our work characterizes a novel cyanobacterial potassium channel and indicates the molecular nature of the first higher plant thylakoid cation channel, opening the way to functional studies.

Patch Clamp and Phenotypic Analyses of a Prokaryotic Cyclic Nucleotide-gated K+ Channel Using Escherichia coli as a Host

Prokaryotic ion channels have been valuable in providing structural models for understanding ion filtration and channel-gating mechanisms. However, their functional examinations have remained rare and usually been carried out by incorporating purified channel protein into artificial lipid membranes. Here we demonstrate the utilization of Escherichia coli to host the functional analyses by examining a putative cyclic nucleotide-gated K+ channel cloned from Magnetospirillum magnetotacticum, MmaK. When expressed in wild-type E. coli cells, MmaK renders the host sensitive to millimolar concentrations of externally applied K+, indicating MmaK forms a functional K+ conduit in the E. coli membrane in vivo. After enlarging these cells into giant spheroplasts, macro- and microscopic MmaK currents are readily detected in excised E. coli membrane patches by a patch clamp. We show that MmaK is indeed gated by submicromolar cAMP and approximately 10-fold higher concentration of cGMP and manifests as an inwardly rectified, K+-specific current with a 10.8 pS unitary conductance at -100 mV. Additionally, MmaK is inactivated by slightly acidic pH only from the cytoplasmic side. Our in vitro biophysical characterizations of MmaK correlate with its in vivo phenotype in E. coli, implicating its critical role as an intracellular cAMP and pH sensor for modulating bacterial membrane potential. Exemplified by MmaK functional studies, we establish that E. coli and its giant spheroplast provide a convenient and versatile system to express foreign channels for biophysical analyses that can be further dovetailed with microbial genetics.

A Superfamily of Voltage-gated Sodium Channels in Bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.

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.