Ion-conducting Pore Research Articles

Pore Determinants of Mechanosensitive Piezo Channels

Piezo proteins have been proposed as the long-sought-after mechanosensitive (MS) cation channels in mammals that play critical roles in various mechanotransduction processes, such as touch sensation and vascular development. However, their ion-conducting pore and ion permeation mechanisms have remained undefined. Here we identify domains and specific residues in Piezo1 that control the essential pore properties, including unitary conductance, ion selectivity and pore blockage, pinpointing the ion-conducting pathway. By uncovering the bona fide ion-conducting pore, these findings not only provide definitive proof that Piezo proteins are genuine pore-forming subunits of MS cation channels, but also shed light on elucidating the ion permeation and gating mechanisms of this prototypic class of mammalian MS cation channels.

Structural and Functional Characterizations of the Mechanosensitive Piezo Channel

Piezo proteins are evolutionarily conserved and functionally diverse mechanosensitive cation channels that play critical roles in various mechanotransduction processes. However, the overall structural architecture, ion permeation and gating mechanisms of Piezo channels have remained unknown. Here we determine the cryo-electron microscopy structure of the full-length (2,547 amino acids) mouse Piezo1 (Piezo1) at a resolution of 4.8 A. Piezo1 forms a trimeric propeller-like structure (about 900 kilodalton), with the extracellular domains resembling three distal blades and a central cap. The transmembrane region has 14 apparently resolved segments per subunit. These segments form three peripheral wings and a central pore module that encloses a potential ion-conducting pore. The rather flexible extracellular blade domains are connected to the central intracellular domain by three long beam-like structures. This trimeric architecture suggests that Piezo1 may use its peripheral regions as force sensors to gate the central ion-conducting pore. Importantly, by characterizing the structurally revealed featured domains, we provide functional evidence to support this hypothesis. Taken together, these findings significantly advance our understanding of the structure-function relationship of this novel class of mechanosensitive cation channels.

Dimeric E. Coli YidC Forms a Translocation Pore in the Membrane

The universally conserved protein YidC functions as a membrane protein insertase. It facilitates transmembrane domains’ passage out of SecYEG's lateral gate and inserts short or closely spaced membrane proteins independent of SecYEG. According to a recent cryo-EM density map of a ribosome-nascent chain complex (RNC) bound to YidC in detergent in addition to X-ray structures of YidC in a monoolein membrane, monomeric YidC assists in membrane protein insertion by offering a hydrophilic groove that surrounds a charged amino acid located at the membrane midplane. These structures raise the question: how is the membrane barrier to protons maintained? We show that purified E. coli YidC forms an aqueous pore upon reconstitution. The pore is closed to ions in the resting state and opens upon binding of RNC. This indicates that YidC is able to preserve the membrane barrier in its idle state. As monomeric YidC cannot form a gated ion channel, we exploited fluorescence correlation spectroscopy to count the number of particles upon dissolution of proteoliposomes by a mild detergent and subsequent exposure of the resulting micelles to a harsh detergent1. The number of particles doubled, indicating that YidC was reconstituted as a dimer. To confirm dimer formation and the causal connection between ribosome binding and channel activity, we monitored single-molecule FRET between labeled ribosomes and labeled YidC. Unitary channel conductivity was decreased as compared to channels activated by the RNC, suggesting that the nascent chain is located at the interface between the two pore-forming monomers and the lipid. Thus, our data suggest that YidC forms an ion-conducting pore in the membrane, which is opened by ribosome binding similar to SecYEG2.1. Horner et al. (2015). Science Advances 1, e1400083.2. Knyazev et al. (2013). J. Biol. Chem. 288, 17941-17946.

Functional Modification of Bacterial Voltage-Gated Sodium Channels by Batrachotoxin

Batrachotoxin (BTX) is an excitatory component in the skin secretions of dendrobatid frogs, which advertise their armament with the gaudy colors of their lethal skin. BTX is a hydrophobic alkaloid, which crosses cell membranes and activates voltage-gated sodium (Nav) channels of muscle, nerve and heart. BTX binding causes a shift of the activation curve toward more negative voltages, and suppresses fast and slow inactivation resulting in a massive increase in excitability of the tissue and ultimately causing paralysis. BTX is thought to act by binding in the cytoplasmic cavity of the ion-conducting pore, of pseudo-symmetric eukaryotic Nav channels. This binding site is near, or overlapping, receptor sites for amphiphylic inhibitor drugs including local anesthetics, anticonvulsants and anti-arrhythmics.The four homologous domains of eukaryotic Navs parallel the homo-tetrameric arrangement of BacNav channels, which have yielded high-resolution crystal structures. Despite the absence of a “ball-and chain” or “hinged-lid” fast inactivation mechanism, BacNavs display a “pore based” inactivation, that in some cases is quite rapid. Thus, they offer an opportunity to explore BTX actions on a molecular system of gating that is amenable to structural verification.The BacNav channels NaChBac and NavSp1 both show dramatic modification by BTX, with negative shifts of activation and inhibition of inactivation. These actions are enhanced by phenylalanine substitutions of pore domain (S6) residues, at positions which align with Phe residues that are important for BTX activation in eukaryotic Nav channels.

The Stoichiometry and Activation Mechanism of TRPV1 by Vanilloids

Vanilloids are pain evoking molecules that serve as ligands of the ‘heat and capsaicin receptor’ TRPV1. It is expressed by primary afferent sensory neurons of the pain pathway, where it functions as a sensor of noxious heat (>42°C) and various noxious chemicals. TRPV1 channel is formed by the symmetrical arrangement of four identical subunits around a central ion-conducting pore. Binding of vanilloids evokes channel opening and subsequent neuronal activation, leading to pain sensation. Despite its pivotal physiological role, the molecular basis of TRPV1 activation by vanilloids is not fully understood. Here, we combine the use of site-directed mutagenesis, concatemeric constructs harboring mutated binding sites with patch-clamp recordings and calcium imaging in order to determine the stoichiometry and activation mechanism of TRPV1 by the exo-vanilloid capsaicin, the ‘hot’ component of chili peppers. We show that a single capsaicin-bound subunit is sufficient to achieve a maximal open-channel lifetime, while the high sensitivity for this agonist requires all four subunits. Moreover, we defined the role of residues within TRPV1 vanilloid binding site in the channel activation mechanism, by acting to stabilize ligand-receptor interaction.

Class I Anti-Arrhythmics Differentially Regulate Cardiac Sodium Channel Voltage-Sensing Domains

Background: Class I anti-arrhythmics target cardiac voltage-gated Na+ channels (NaV1.5). The NaV1.5 α-subunit contains four domains (DI-DIV) with six membrane-spanning segments (S1-S6). S1-S4 form voltage-sensing domains (VSDs), and S5-S6 create the ion-conducting pore. The distinct therapeutic action of subclasses Ia, Ib, and Ic have been traditionally attributed to differences in NaV1.5 access and pore-binding rate. However, others have shown that lidocaine, a local anesthetic and Class Ib anti-arrhythmic interacts with the muscle Na+ channel, NaV1.4, DIII-VSD. Thus, we tested the hypothesis that Class I drug interaction with the NaV1.5 VSDs significantly determines the therapeutic phenotype. Methods: Previously, we created four NaV1.5 DNA constructs with a cysteine introduced to the extracellular S4 of individual VSDs. Channels encoded by these constructs are expressed in Xenopus oocytes and cysteine-labeled with the TAMRA-MTS fluorophore. Ionic current and fluorescence emission corresponding to changes in VSD conformation are then simultaneously recorded using the cut-open oocyte configuration. After control recordings, anti-arrhythmics are administered to the internal solution. When currents are 80% blocked, VSD kinetics are measured. Results: We have not observed significant interaction of Class I drugs with the DI, DII, or DIV VSDs. In contrast, quinidine, lidocaine, and ranolazine all uniquely shift DIII-VSD activation. During control recordings, we observe DIII-VSD activation has V1/2=-108.96±2.72mV. Lidocaine and ranolazine both induced a hyperpolarizing DIII-VSD activation shift (V1/2=-147.28±4.17mV, S.E.M., p=0.003, V1/2=-143.09±1.94mV, S.E.M, p=0.001, respectively) while quinidine caused a large depolarizing shift (V1/2=-80.67±4.39mV, S.E.M, p=0.007). Conclusions: Drug interaction with the DIII-VSD has been tightly linked to use-dependent block of the late Na+ current, a hallmark class Ib drugs. In contrast, class Ia drugs typically reduce peak Na+. Because the DIII-VSD is tightly coupled to NaV channel gating, we propose that the differences in DIII-VSD interaction are determining their unique therapeutic phenotypes.

Diseases caused by mutations in ORAI1 and STIM1.

Ca(2+) release-activated Ca(2+) (CRAC) channels mediate a specific form of Ca(2+) influx called store-operated Ca(2+) entry (SOCE) that contributes to the function of many cell types. CRAC channels are composed of ORAI1 proteins located in the plasma membrane, which form its ion-conducting pore. ORAI1 channels are activated by stromal interaction molecule (STIM) 1 and STIM2 located in the endoplasmic reticulum. Loss- and gain-of-function gene mutations in ORAI1 and STIM1 in human patients cause distinct disease syndromes. CRAC channelopathy is caused by loss-of-function mutations in ORAI1 and STIM1 that abolish CRAC channel function and SOCE; it is characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia, with defects in sweat gland function and dental enamel formation. The latter defect emphasizes an important role of CRAC channels in tooth development. By contrast, autosomal dominant gain-of-function mutations in ORAI1 and STIM1 result in constitutive CRAC channel activation, SOCE, and increased intracellular Ca(2+) levels that are associated with an overlapping spectrum of diseases, including nonsyndromic tubular aggregate myopathy (TAM) and York platelet and Stormorken syndromes. The latter two syndromes are defined, besides myopathy, by thrombocytopenia, thrombopathy, and bleeding diathesis. The fact that myopathy results from both loss- and gain-of-function mutations in ORAI1 and STIM1 highlights the importance of CRAC channels for Ca(2+) homeostasis in skeletal muscle function. The cellular dysfunction and clinical disease spectrum observed in mutant patients provide important information about the molecular regulation of ORAI1 and STIM1 proteins and the role of CRAC channels in human physiology.

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(-) and 1,648(+) cm(-1). These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.

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 are fascinating for the biological roles they fulfill as well as for their intriguing biophysical properties, including conductance levels ten times most other K+ channels and gating sensitivity to intracellular Na+. Here we present the structure a complete Na+-activated K+ channel, Slo2.2, in the Na+-free state, determined by cryo-electron microscopy at a nominal resolution of 4.5 Å. The channel is composed of a large cytoplasmic gating ring within 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 provides a first view of a member of the Slo K+ channel family, which reveals features explaining their high conductance and gating mechanism.

Architecture of the mammalian mechanosensitive Piezo1 channel.

Piezo proteins are evolutionarily conserved and functionally diverse mechanosensitive cation channels. However, the overall structural architecture and gating mechanisms of Piezo channels have remained unknown. Here we determine the cryo-electron microscopy structure of the full-length (2,547 amino acids) mouse Piezo1 (Piezo1) at a resolution of 4.8 Å. Piezo1 forms a trimeric propeller-like structure (about 900 kilodalton), with the extracellular domains resembling three distal blades and a central cap. The transmembrane region has 14 apparently resolved segments per subunit. These segments form three peripheral wings and a central pore module that encloses a potential ion-conducting pore. The rather flexible extracellular blade domains are connected to the central intracellular domain by three long beam-like structures. This trimeric architecture suggests that Piezo1 may use its peripheral regions as force sensors to gate the central ion-conducting pore.

Physical basis of apparent pore dilation of ATP-activated P2X receptor channels.

The selectivity of ion channels is fundamental for their roles in electrical and chemical signaling and in ion homeostasis. Although most ion channels exhibit stable ion selectivity, the prevailing view of purinergic P2X receptor channels, transient receptor potential V1 (TRPV1) channels and acid-sensing ion channels (ASICs) is that their ion conduction pores dilate upon prolonged activation. We investigated this mechanism in P2X receptors and found that the hallmark shift in equilibrium potential observed with prolonged channel activation does not result from pore dilation, but from time-dependent alterations in the concentration of intracellular ions. We derived a physical model to calculate ion concentration changes during patch-clamp recordings, which validated our experimental findings and provides a quantitative guideline for effectively controlling ion concentration. Our results have fundamental implications for understanding ion permeation and gating in P2X receptor channels, as well as more broadly for using patch-clamp techniques to study ion channels and neuronal excitability.

Resin-acid derivatives as potent electrostatic openers of voltage-gated K channels and suppressors of neuronal excitability.

Voltage-gated ion channels generate cellular excitability, cause diseases when mutated, and act as drug targets in hyperexcitability diseases, such as epilepsy, cardiac arrhythmia and pain. Unfortunately, many patients do not satisfactorily respond to the present-day drugs. We found that the naturally occurring resin acid dehydroabietic acid (DHAA) is a potent opener of a voltage-gated K channel and thereby a potential suppressor of cellular excitability. DHAA acts via a non-traditional mechanism, by electrostatically activating the voltage-sensor domain, rather than directly targeting the ion-conducting pore domain. By systematic iterative modifications of DHAA we synthesized 71 derivatives and found 32 compounds more potent than DHAA. The most potent compound, Compound 77, is 240 times more efficient than DHAA in opening a K channel. This and other potent compounds reduced excitability in dorsal root ganglion neurons, suggesting that resin-acid derivatives can become the first members of a new family of drugs with the potential for treatment of hyperexcitability diseases.

Geometric rules of channel gating inferred from computational models of the P2X receptor transmembrane domain

The P2X receptors are trimeric ATP-gated ion channels and mediate chemical communication between eukaryotic cells. Each P2X subunit contains two transmembrane helices, M1 and M2, and the M2 helix packs around an ion conduction pore. Here, I have reconstructed the three-dimensional models of the zebrafish P2X4 transmembrane domain using spatial restraints on helical packing. The models are stable in lipid bilayers during molecular dynamics simulation and adopt different conformations depending on bilayer hydrophobic thickness. Comparison of these conformations shows that the pore-lining residues L340, A344 and A347 each have multiple packing sites that define the pore configurations. Shift of L340 packing between different sites alters the side-chain orientation that occludes the pore or removes this occlusion. L340, A344 and A347 also gate the pore by expansion–contraction mechanism based on their packing patterns. Finally, pore expansions at the L340 and A344 levels are mutually exclusive, so the P2X gating may involve sequential pore opening at L340 and A344 levels to allow ion conduction. In summary, the current study shows that the computational assembly of the helical membrane protein is not only possible, but also necessary to provide insights into the mechanisms of channel gating.

Relevance of Viroporin Ion Channel Activity on Viral Replication and Pathogenesis.

Modification of host-cell ionic content is a significant issue for viruses, as several viral proteins displaying ion channel activity, named viroporins, have been identified. Viroporins interact with different cellular membranes and self-assemble forming ion conductive pores. In general, these channels display mild ion selectivity, and, eventually, membrane lipids play key structural and functional roles in the pore. Viroporins stimulate virus production through different mechanisms, and ion channel conductivity has been proved particularly relevant in several cases. Key stages of the viral cycle such as virus uncoating, transport and maturation are ion-influenced processes in many viral species. Besides boosting virus propagation, viroporins have also been associated with pathogenesis. Linking pathogenesis either to the ion conductivity or to other functions of viroporins has been elusive for a long time. This article summarizes novel pathways leading to disease stimulated by viroporin ion conduction, such as inflammasome driven immunopathology.

A Coronavirus E Protein Is Present in Two Distinct Pools with Different Effects on Assembly and the Secretory Pathway.

Coronaviruses (CoVs) assemble by budding into the lumen of the early Golgi complex prior to exocytosis. The small CoV envelope (E) protein plays roles in assembly, virion release, and pathogenesis. CoV E has a single hydrophobic domain (HD), is targeted to Golgi complex membranes, and has cation channel activity in vitro. However, the precise functions of the CoV E protein during infection are still enigmatic. Structural data for the severe acute respiratory syndrome (SARS)-CoV E protein suggest that it assembles into a homopentamer. Specific residues in the HD regulate the ion-conducting pore formed by SARS-CoV E in artificial bilayers and the pathogenicity of the virus during infection. The E protein from the avian infectious bronchitis virus (IBV) has dramatic effects on the secretory system which require residues in the HD. Here, we use the known structural data from SARS-CoV E to infer the residues important for ion channel activity and the oligomerization of IBV E. We present biochemical data for the formation of two distinct oligomeric pools of IBV E in transfected and infected cells and the residues required for their formation. A high-order oligomer of IBV E is required for the production of virus-like particles (VLPs), implicating this form of the protein in virion assembly. Additionally, disruption of the secretory pathway by IBV E correlates with a form that is likely monomeric, suggesting that the effects on the secretory pathway are independent of E ion channel activity. CoVs are important human pathogens with significant zoonotic potential, as demonstrated by the emergence of SARS-CoV and Middle East respiratory syndrome (MERS)-CoV. Progress has been made toward identifying potential vaccine candidates in mouse models of CoV infection, including the use of attenuated viruses that lack the CoV E protein or express E-protein mutants. However, no approved vaccines or antiviral therapeutics exist. We previously reported that the hydrophobic domain of the IBV E protein, a putative viroporin, causes disruption of the mammalian secretory pathway when exogenously expressed in cells. Understanding the mechanism of this disruption could lead to the identification of novel antiviral therapeutics. Here, we present biochemical evidence for two distinct oligomeric forms of IBV E, one essential for assembly and the other with a role in disruption of the secretory pathway. Discovery of two forms of CoV E protein will provide additional targets for antiviral therapeutics.

Golgi Anti-apoptotic Proteins Are Highly Conserved Ion Channels That Affect Apoptosis and Cell Migration

Golgi anti-apoptotic proteins (GAAPs) are multitransmembrane proteins that are expressed in the Golgi apparatus and are able to homo-oligomerize. They are highly conserved throughout eukaryotes and are present in some prokaryotes and orthopoxviruses. Within eukaryotes, GAAPs regulate the Ca(2+) content of intracellular stores, inhibit apoptosis, and promote cell adhesion and migration. Data presented here demonstrate that purified viral GAAPs (vGAAPs) and human Bax inhibitor 1 form ion channels and that vGAAP from camelpox virus is selective for cations. Mutagenesis of vGAAP, including some residues conserved in the recently solved structure of a related bacterial protein, BsYetJ, altered the conductance (E207Q and D219N) and ion selectivity (E207Q) of the channel. Mutation of residue Glu-207 or -178 reduced the effects of GAAP on cell migration and adhesion without affecting protection from apoptosis. In contrast, mutation of Asp-219 abrogated the anti-apoptotic activity of GAAP but not its effects on cell migration and adhesion. These results demonstrate that GAAPs are ion channels and define residues that contribute to the ion-conducting pore and affect apoptosis, cell adhesion, and migration independently.

Computational and Functional Analyses of a Small-Molecule Binding Site in ROMK

The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val168 or Asn171 in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK.

Conformational Changes that Opens TrkH Ion Channel

All living cells must accumulate a high intracellular concentration of K+ to maintain essential physiological functions. While in animals, K+ uptake is mediated by the well-studied Na+/K+ transporters, bacteria, yeast and plants accumulate K+ with a superfamily of K+ transporters known as the SKT proteins. TrkH is a member of the SKT family that assembles with a cytosolic domain, TrkA. Previously, we have shown that TrkH is an ion channel regulated by binding of ATP or ADP to the TrkA protein: ATP activates the channel while ADP inhibits it (1). We have also solved the crystal structures of TrkH and TrkH in complex with TrkA (1, 2). The structure of TrkH shows that it forms a homodimer and that each protomer has an ion conduction pore with an architecture similar to that of a K+ channel. The structure also revealed a feature not observed in K+ channels: a loop within the transmembrane region that blocks the ion permeation pathway. Further structural and functional analyses showed that the position of this loop is likely controlled by conformational changes on TrkA, which is in turn controlled by binding of ATP or ADP. We have applied single-particle cryo-electron microscopy to visualize conformational changes associated with channel gating. Preliminary data show that the TrkH-TrkA complex likely assumes a different conformation than that observed in the crystal structure.1. Cao, Y. et al. 2013. Gating of the TrkH ion channel by its associated RCK protein TrkA. Nature 496:317-322.2. Cao, Y. et al. 2011. Crystal structure of a potassium ion transporter, TrkH. Nature 471:336-340.

Conformational Changes in Voltage-Sensing Domains: Concerted Simulation and Scattering Studies

Electrical signals in excitable tissues such as nerves and muscles are generated by the synchronized opening and closing of ion channels in cell membranes, which generate transient transmembrane gradients in the concentrations of sodium and potassium ions. These channels open and close in response to local changes in transmembrane potential as the signal propagates, and it is well established that their voltage sensitivity is conferred by charged structural elements, referred to as voltage-sensing domains. The details of how conformational changes in the voltage-sensing domains lead to opening/closing of the ion-conducting pores of the channels are still being worked out. Crystal structures of the open states of voltage-gated potassium channels are available, but the structure of the closed/resting state has not been determined to high resolution experimentally. In this talk I will report our efforts to generate a model of the resting state of the archaeal KvAP potassium channel based on atomistic molecular dynamics (MD) simulations in explicit membrane environments, with restraints derived from experimental functional data. I will also present results from simulation studies of an isolated voltage-sensing domain in a hydrated membrane, which include validation by neutron diffraction measurements, and direct observations of elementary gating charge displacement events during a 30-microsecond MD simulation under applied transmembrane potential. Finally, I will discuss prospects for and present preliminary results on using x-ray and neutron interferometry measurements, carried out by our collaborators, to validate and refine simulation-based models of the resting state and the voltage-sensing mechanism.

Small Molecule Modulation of Voltage-Gated Ion Channels

Small Molecule Modulation of Voltage-Gated Ion Channels