Ion Conduction Pathway Research Articles

Coupling of Charge Displacement to Na Current Activation is Impaired for DIIS4 Mutations in HypoPP

All eight missense mutations of NaV1.4 associated with hypokalemic periodic paralysis (HypoPP) occur at arginine residues in S4 voltage sensors. These R/X mutations create an anomalous ion conduction pathway, detected as a gating pore current (Igp) in the oocyte. A critical question is how large is the gating pore conductance in HypoPP muscle, since this will determine susceptibility to depolarization-induced weakness. Large discrepancies for estimates of Igp relative to peak INa have been reported; Igp / INa = ∼ 1:100 when currents are measured with TTX (Igp) and without (INa) in the same egg, whereas the conductance ratio γgp / γNa = ∼ 1:1000 based on measurement of Igp and maximal “on” gating charge displacement, Qon.To resolve this issue, we measured Qon, INa, and Igp in oocytes expressing WT or R672G channels. Robust gating pore currents were observed for R672G channels, with Igp/Qon = 65.2 nA/nC at −140 mV. The ratio of INa(-10 mv) to Qon was decreased 2.3-fold for R672G channels compared to WT. This decoupling may also account for the 2x reduction of INa in muscle fibers from R669H knock-in HypoPP mouse for which mRNA levels were not reduced. For R672G, the measured currents had a ratio of Igp(-140) / INa(-10) = 0.025 or 1:40. This current ratio can be converted to an equivalent conductance ratio by accounting for (i) decoupling 2.3, (ii) Popen_max = 0.25, (iii) driving force of 140 mV for Igp and 50 mV for INa. All together, these factors are ∼25 and yield an equivalent conductance ratio of 1: 40 x 25 = 1 : 1000; thereby resolving the discrepancy in the literature.Supported by NIAMS and MDA.

Neodymium-doped ceria nanomaterials: facile low-temperature synthesis and excellent electrical properties for IT-SOFCs

A novel and facile method is applied to successfully synthesize the 20 mol% neodymium-doped ceria (CNO) nanomaterials, including nanowires and nanoparticles, at 240 °C in ambient air. These special nanostructures are easily densified at 1300 °C and provide a three-dimensional (3D), continuous and fast ionic conduction pathway. Surprisingly, the CNO nanomaterials endow the homogeneous and compact ceramics with a competitive ionic conductivity of ∼1.5 S m−1 at 600 °C, down to a pO2 value of about 10−4 atm, and act as a potential electrolyte or composite anode for intermediate-temperature solid oxide fuel cells.

Crystal Structure of a Voltage-gated K+ Channel Pore Module in a Closed State in Lipid Membranes

Voltage-gated K(+) channels underlie the electrical excitability of cells. Each subunit of the functional tetramer consists of the tandem fusion of two modules, an N-terminal voltage-sensor and a C-terminal pore. To investigate how sensor coupling to the pore generates voltage-dependent channel opening, we solved the crystal structure and characterized the function of a voltage-gated K(+) channel pore in a lipid membrane. The structure of a functional channel in a membrane environment at 3.1 Å resolution establishes an unprecedented connection between channel structure and function. The structure is unique in delineating an ion-occupied ready to conduct selectivity filter, a confined aqueous cavity, and a closed activation gate, embodying a dynamic entity trapped in an unstable closed state.

Structural Basis of the Selective Block of Kv1.2 by Maurotoxin from Computer Simulations

The 34-residue polypeptide maurotoxin (MTx) isolated from scorpion venoms selectively inhibits the current of the voltage-gated potassium channel Kv1.2 by occluding the ion conduction pathway. Here using molecular dynamics simulation as a docking method, the binding modes of MTx to three closely related channels (Kv1.1, Kv1.2 and Kv1.3) are examined. We show that MTx forms more favorable electrostatic interactions with the outer vestibule of Kv1.2 compared to Kv1.1 and Kv1.3, consistent with the selectivity of MTx for Kv1.2 over Kv1.1 and Kv1.3 observed experimentally. One salt bridge in the bound complex of MTx-Kv1.2 forms and breaks in a simulation period of 20ns, suggesting the dynamic nature of toxin-channel interactions. The toxin selectivity likely arises from the differences in the shape of the channel outer vestibule, giving rise to distinct orientations of MTx on block. Potential of mean force calculations show that MTx blocks Kv1.1, Kv1.2 and Kv1.3 with an IC50 value of 6 µM, 0.6nM and 18 µM, respectively.

Structural insights into neuronal K + channel–calmodulin complexes

Calmodulin (CaM) is a ubiquitous intracellular calcium sensor that directly binds to and modulates a wide variety of ion channels. Despite the large repository of high-resolution structures of CaM bound to peptide fragments derived from ion channels, there is no structural information about CaM bound to a fully folded ion channel at the plasma membrane. To determine the location of CaM docked to a functioning KCNQ K(+) channel, we developed an intracellular tethered blocker approach to measure distances between CaM residues and the ion-conducting pathway. Combining these distance restraints with structural bioinformatics, we generated an archetypal quaternary structural model of an ion channel-CaM complex in the open state. These models place CaM close to the cytoplasmic gate, where it is well positioned to modulate channel function.

The Periplasmic Loop Provides Stability to the Open State of the CorA Magnesium Channel

Crystal structures of the CorA Mg(2+) channel have suggested that metal binding in the cytoplasmic domain stabilizes the pentamer in a closed conformation. The open "metal free" state of the channel is, however, still structurally uncharacterized. Here, we have attempted to map conformational states of CorA from Thermotoga maritima by determining which residues support the pentameric structure in the presence or absence of Mg(2+). We find that when Mg(2+) is present, the pentamer is stabilized by the putative gating sites (M1/M2) in the cytoplasmic domain. Strikingly however, we find that the conserved and functionally important periplasmic loop is vital for the integrity of the pentamer when Mg(2+) is absent from the M1/M2 sites. Thus, although the periplasmic loops were largely disordered in the x-ray structures of the closed channel, our data suggests a prominent role for the loops in stabilizing the open conformation of the CorA channels.

Ion channels and transporters in lymphocyte function and immunity

Lymphocyte function is regulated by a network of ion channels and transporters in the plasma membrane of B and T cells. These proteins modulate the cytoplasmic concentrations of diverse cations, such as calcium, magnesium and zinc ions, which function as second messengers to regulate crucial lymphocyte effector functions, including cytokine production, differentiation and cytotoxicity. The repertoire of ion-conducting proteins includes calcium release-activated calcium (CRAC) channels, P2X receptors, transient receptor potential (TRP) channels, potassium channels, chloride channels and magnesium and zinc transporters. This Review discusses the roles of ion conduction pathways in lymphocyte function and immunity.

Binding Modes of μ-Conotoxin to the Bacterial Sodium Channel (NaVAb)

Polypeptide toxins isolated from the venom of cone snails, known as μ-conotoxins, block voltage-gated sodium channels by physically occluding the ion-conducting pathway. Using molecular dynamics, we show that one subtype of μ-conotoxins, PIIIA, effectively blocks the bacterial voltage-gated sodium channel NaVAb, whose crystal structure has recently been elucidated. The spherically shaped toxin, carrying a net charge of +6 e with six basic residues protruding from its surface, is attracted by the negatively charged residues on the vestibular wall and the selectivity filter of the channel. The side chain of each of these six arginine and lysine residues can wedge into the selectivity filter, whereas the side chains of other basic residues form electrostatic complexes with two acidic residues on the channel. We construct the profile of potential of mean force for the unbinding of PIIIA from the channel, and predict that PIIIA blocks the bacterial sodium channel with subnanomolar affinity.

Structural Insights into Calmodulation of Neuronal KCNQ Channels

Calmodulin (CaM) is a ubiquitous intracellular calcium sensor for many potassium channels. For the voltage-gated KCNQ family of potassium channels, CaM binds to the intracellular C-terminus to mediate channel assembly, trafficking, and gating. Resolution of the isolated crystal structures of CaM associated with peptide fragments from various ion channels has provided some insight into “calmodulation.” Despite the large repository of these structures, structural information about CaM bound to a fully folded ion channel in the membrane is unknown. We determined the location and orientation of CaM binding with respect to the KCNQ2/KCNQ3 (Q2/Q3) ion-conducting pathway using distance restraints from a panel of intracellular tethered blockers. Molecular dynamics simulations of the Q2/Q3-CaM complex position CaM strikingly close to the gate of Q2/Q3. The structure of the Q2/Q3-CaM complex and its implications for calcium-induced calmodulation will be discussed.

Flexibility of the Third Extracellular Loop Affects Permeation of Orai1 Channels

A major calcium influx pathway in T-cells and mast cells is mediated by calcium-release-activated calcium (CRAC) channels that exhibit a high selectivity for Ca2+. In the event of ER store-depletion, the signal is perpetuated to the plasma-membrane by the stromal interaction molecule STIM1. At concrete sites, STIM1 interacts with Orai1, the pore-forming subunit of the CRAC channel to initiate Ca2+ influx. McNally et al. (2009) have shown that the architecture of the Orai1 ion conduction pathway is characterized by a flexible outer vestibule formed by the first extracellular loop, which leads to a narrow pore flanked by residues of the first, helical transmembrane segment. Oxidative crosslinking further reveals Orai1 tetrameric or higher oligomeric assembly with the first transmembrane segment centrally located (Zhou et al., 2010). In our present study we found that the flexibility of the third extracellular loop might affect Orai1 permeation as well. Cysteine-crosslinking studies targeting the third extracellular loop of Orai1 channels revealed reduced ion permeation. Symmetrically opposed residues within the third loop were tested for their ability to form disulfide bonds. We could identify several residues within the third extracelular loop that were capable of disulfide crosslinking enhanced by copper-phenanthroline (CuP). Their disruption by the compound BMS (bis(2-mercaptoethylsulfone)) resulted in an increase in Ca2+ currents. Hence, we assume that decreased flexibility of the third extracellular loop of Orai channels might directly shield or allosterically affect the permeation pathway. (supported by the Austrian Science Fund (FWF): T442 to I.F. and and P22747 to R.S.)

Sodium Channel Structure and Function at Atomic Resolution

Voltage-gated Na+ channels initiate action potentials in excitable cells and are important targets for drugs. Recent research gives new insight into the structural basis for permeation, drug-block, and voltage-dependent gating. Voltage-sensing depends on the S4 segments, which move outward through a gating pore via a ‘sliding-helix’ mechanism. Outward movement of S4 is coupled to pore-opening by tilting and rotation of the S6 segments. A structural model reveals conformational changes underlying activation and pore-opening, and disulfide locking of substituted cysteines demonstrates voltage-dependent formation of ion pairs during channel activation, as in the sliding-helix model. Na+ channel blocking drugs bind to a conserved receptor site in the inner pore, and binding is enhanced by repetitive opening of the pore and inactivation of the channel. A three-dimensional view of these structural components is provided by a new high-resolution structure of a voltage-gated Na+ channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage-sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage-sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore K+ channel structures suggest that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting around a hinge at the base of the pore. The NavAb selectivity filter is short, ∼4.6 Å wide, and water-filled, with four acidic glutamate side-chains surrounding the narrowest part of the ion-conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na+ selectivity through partial dehydration via direct interaction with glutamate side-chains. Fenestrations in the sides of the pore are unexpectedly penetrated by fatty-acyl chains that extend into the central cavity, and these portals are large enough for entry of small, hydrophobic pore-blocking drugs. Supported by NINDS/NIH.

Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca2+ channel α2δ-1 auxiliary subunit

Voltage-gated calcium (CaV) channels are transmembrane proteins that form Ca2+-selective pores gated by depolarization and are essential regulators of the intracellular Ca2+ concentration. By providing a pathway for rapid Ca2+ influx, CaV channels couple membrane depolarization to a wide array of cellular responses including neurotransmission, muscle contraction and gene expression. CaV channels fall into two major classes, low voltage-activated (LVA) and high voltage-activated (HVA). The ion-conducting pathway of HVA channels is the α1 subunit, which typically contains associated β and α2δ ancillary subunits that regulate the properties of the channel. Although it is widely acknowledged that α2δ-1 is post-translationally cleaved into an extracellular α2 polypeptide and a membrane-anchored δ protein that remain covalently linked by disulfide bonds, to date the contribution of different cysteine (Cys) residues to the formation of disulfide bridges between these proteins has not been investigated. In the present report, by predicting disulfide connectivity with bioinformatics, molecular modeling and protein biochemistry experiments we have identified two Cys residues involved in the formation of an intermolecular disulfide bond of critical importance for the structure and function of the α2δ-1 subunit. Site directed-mutagenesis of Cys404 (located in the von Willebrand factor-A region of α2) and Cys1047 (in the extracellular domain of δ) prevented the association of the α2 and δ peptides upon proteolysis, suggesting that the mature protein is linked by a single intermolecular disulfide bridge. Furthermore, co-expression of mutant forms of α2δ-1 Cys404Ser and Cys1047Ser with recombinant neuronal N-type (CaV2.2α1/β3) channels, showed decreased whole-cell patch-clamp currents indicating that the disulfide bond between these residues is required for α2δ-1 function.

External Ba2+ Block of the Two-pore Domain Potassium Channel TREK-1 Defines Conformational Transition in Its Selectivity Filter

TREK-1 is a member of the two-pore domain potassium channel family that is known as a leak channel and plays a key role in many physiological and pathological processes. The conformational transition of the selectivity filter is considered as an effective strategy for potassium channels to control the course of potassium efflux. It is well known that TREK-1 is regulated by a large volume of extracellular and intracellular signals. However, until now, little was known about the selectivity filter gating mechanism of the channel. In this research, it was found that Ba(2+) blocked the TREK-1 channel in a concentration- and time-dependent manner. A mutagenesis analysis showed that overlapped binding of Ba(2+) at the assumed K(+) binding site 4 (S4) within the selectivity filter was responsible for the inhibitory effects on TREK-1. Then, Ba(2+) was used as a probe to explore the conformational transition in the selectivity filter of the channel. It was confirmed that collapsed conformations were induced by extracellular K(+)-free and acidification at the selectivity filters, leading to nonconductive to permeable ions. Further detailed characterization demonstrated that the two conformations presented different properties. Additionally, the N-terminal truncated isoform (ΔN41), a product derived from alternative translation initiation, was identified as a constitutively nonconductive variant. Together, these results illustrate the important role of selectivity filter gating in the regulation of TREK-1 by the extracellular K(+) and proton.

Interactions of cations with the cytoplasmic pores of inward rectifier K(+) channels in the closed state.

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K+ (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg2+ by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg2+ when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg2+ indicates that the mode of binding of Mg2+ is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore.

Common Molecular Determinants of Tarantula Huwentoxin-IV Inhibition of Na+ Channel Voltage Sensors in Domains II and IV

The voltage sensors of domains II and IV of sodium channels are important determinants of activation and inactivation, respectively. Animal toxins that alter electrophysiological excitability of muscles and neurons often modify sodium channel activation by selectively interacting with domain II and inactivation by selectively interacting with domain IV. This suggests that there may be substantial differences between the toxin-binding sites in these two important domains. Here we explore the ability of the tarantula huwentoxin-IV (HWTX-IV) to inhibit the activity of the domain II and IV voltage sensors. HWTX-IV is specific for domain II, and we identify five residues in the S1-S2 (Glu-753) and S3-S4 (Glu-811, Leu-814, Asp-816, and Glu-818) regions of domain II that are crucial for inhibition of activation by HWTX-IV. These data indicate that a single residue in the S3-S4 linker (Glu-818 in hNav1.7) is crucial for allowing HWTX-IV to interact with the other key residues and trap the voltage sensor in the closed configuration. Mutagenesis analysis indicates that the five corresponding residues in domain IV are all critical for endowing HWTX-IV with the ability to inhibit fast inactivation. Our data suggest that the toxin-binding motif in domain II is conserved in domain IV. Increasing our understanding of the molecular determinants of toxin interactions with voltage-gated sodium channels may permit development of enhanced isoform-specific voltage-gating modifiers.

Where’s the gate? Gating in the deep pore of the BKCa channel

Four α-helical segments at the intracellular entrance to the ion conduction pathway splaying open is the conventional imagery of a K+ channel opening to allow K+ ions and co-traversing water molecules to hop through the channel’s pore ([Fig. 1][1]) ([Armstrong, 2003][2]; [Swartz, 2004][3]). This

Polymer chain organization in tensile-stretched poly(ethylene oxide)-based polymer electrolytes

Polymer chain orientation in tensile-stretched poly(ethylene oxide)–lithium trifluoromethanesulfonate polymer electrolytes are investigated with polarized infrared spectroscopy as a function of the degree of strain and salt composition (ether oxygen atom to lithium ion ratios of 20:1, 15:1, and 10:1). The 1359 and 1352 cm −1 bands are used to probe the crystalline PEO and P(EO) 3LiCF 3SO 3 domains, respectively, allowing a direct comparison of chain orientation for the two phases. Two-dimensional correlation FT-IR spectroscopy indicates that the two crystalline domains align at the same rate as the polymer electrolytes are stretched. Quantitative measurements of polymer chain orientation obtained through dichroic infrared spectroscopy show that chain orientation predominantly occurs between strain values of 150% and 250%, regardless of salt composition investigated. There are few changes in chain orientation for either phase when the films are further elongated to a strain of 300%; however, the PEO domains are slightly more oriented at the high strain values. The spectroscopic data are consistent with stretching-induced melt-recrystallization of the unoriented crystalline domains in the solution-cast polymer films. Stretching the films pulls polymer chains from the crystalline domains, which subsequently recrystallize with the polymer helices parallel to the stretch direction. If lithium ion conduction in crystalline polymer electrolytes is viewed as consisting of two major components (facile intra-chain lithium ion conduction and slow helix-to-helix inter-grain hopping), then alignment of the polymer helices will affect the ion conduction pathways for these materials by reducing the number of inter-grain hops required to migrate through the polymer electrolyte.

The crystal structure of a voltage-gated sodium channel.

Voltage-gated sodium channels initiate electrical signaling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity, and drug block is unknown. Here, we report the crystal structure of a voltage-gated Na+-channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage-sensors at 2.7 Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage-sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures suggest that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ~6.5 Å wide, and water-filled, with four acidic side-chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high field-strength anionic coordination site, which confers Na+-selectivity through partial dehydration via direct interaction with glutamate side-chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs.

A conserved residue, PomB-F22, in the transmembrane segment of the flagellar stator complex, has a critical role in conducting ions and generating torque

Bacterial flagellar motors exploit the electrochemical potential gradient of a coupling ion (H(+) or Na(+)) as their energy source, and are composed of stator and rotor proteins. Sodium-driven and proton-driven motors have the stator proteins PomA and PomB or MotA and MotB, respectively, which interact with each other in their transmembrane (TM) regions to form an ion channel. The single TM region of PomB or MotB, which forms the ion-conduction pathway together with TM3 and TM4 of PomA or MotA, respectively, has a highly conserved aspartate residue that is the ion binding site and is essential for rotation. To investigate the ion conductivity and selectivity of the Na(+)-driven PomA/PomB stator complex, we replaced conserved residues predicted to be near the conserved aspartate with H(+)-type residues, PomA-N194Y, PomB-F22Y and/or PomB-S27T. Motility analysis revealed that the ion specificity was not changed by either of the PomB mutations. PomB-F22Y required a higher concentration of Na(+) to exhibit swimming, but this effect was suppressed by additional mutations, PomA-N194Y or PomB-S27T. Moreover, the motility of the PomB-F22Y mutant was resistant to phenamil, a specific inhibitor for the Na(+) channel. When PomB-F22 was changed to other amino acids and the effects on swimming ability were investigated, replacement with a hydrophilic residue decreased the maximum swimming speed and conferred strong resistance to phenamil. From these results, we speculate that the Na(+) flux is reduced by the PomB-F22Y mutation, and that PomB-F22 is important for the effective release of Na(+) from PomB-D24.

A Novel Current Pathway Parallel to the Central Pore in a Mutant Voltage-gated Potassium Channel

Voltage-gated potassium channels are proteins composed of four subunits consisting of six membrane-spanning segments S1-S6, with S4 as the voltage sensor. The region between S5 and S6 forms the potassium-selective ion-conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel could disclose another ion permeation pathway through the voltage-sensing domain (S1-S4) of the channel, the ω-pore. In our studies we used the voltage-gated hKv1.3 channel, and the insertion of a cysteine at position V388C (Shaker position 438) generated a current through the α-pore in high potassium outside and an inward current at hyperpolarizing potentials carried by different cations like Na(+), Li(+), Cs(+), and NH(4)(+). The observed inward current looked similar to the ω-current described for the R1C/S Shaker mutant channel and was not affected by some pore blockers like charybdotoxin and tetraethylammonium but was inhibited by a phenylalkylamine blocker (verapamil) that acts from the intracellular side. Therefore, we hypothesize that the hKv1.3_V388C mutation in the P-region generated a channel with two ion-conducting pathways. One, the α-pore allowing K(+) flux in the presence of K(+), and the second pathway, the σ-pore, functionally similar but physically distinct from the ω-pathway. The entry of this new pathway (σ-pore) is presumably located at the backside of Y395 (Shaker position 445), proceeds parallel to the α-pore in the S6-S6 interface gap, ending between S5 and S6 at the intracellular side of one α-subunit, and is blocked by verapamil.