Ion Permeation Pathway Research Articles

Ion Selectivity and Permeation in a Potassium Ion Transporter

The TrkH/TrkG/KtrB family of proteins mediate K+ uptake in bacteria and likely evolved from simple K+ channels by multiple gene duplications or fusions. We have solved the crystal structure of a TrkH from Vibrio parahaemolyticus to 3.5 A. TrkH is a homodimer, and each protomer contains an ion permeation pathway. A selectivity filter, similar in architecture to that in K+ channels but significantly shorter, is lined by backbone and side chain oxygen atoms. Functional studies showed that TrkH allows permeation of K+ and Rb+ but not smaller ions such as Na+ or Li+. Immediately intracellular to the selectivity filter is an intramembrane loop and an arginine residue, both highly conserved, that constrict an otherwise open permeation pathway. Functions of the loop and the positive charge (Arg) will be addressed.

Modeling KCNQ1 Channel and KCNE1 Interactions

KCNQ1 is the pore-forming component of the slow delayed rectifier (Iks) channel in human heart. Although KCNQ1 can function as a voltage-gated (Kv) channel on its own, it requires KCNE1 to modulate its functions. To understand the mechanism of how KCNE1 modulate KCNQ1, molecular simulations are employed to model the interactions between KCNQ1 and KCNE1. A homology model of KCNQ1 was constructed based on the crystal structure of Kv1.2-Kv2.1 paddle chimera channel (PDB entry: 2R9R) template. For KCNE1, we used a NMR structure as an initial conformation, and manually adjusted the orientations of its N- and C- terminal domains to avoid their folding into membrane. Then 100ns molecular dynamics (MD) simulations were conducted to sample possible conformations of KCNE1 in POPC lipid bilayers. The cluster analysis was conducted on KCNE1 tansmembrane domain (KCNE1-TMD) from 40ns to 100ns simulation period. Five representative structures of KCNE1-TMD were selected, and used for docking simulations to KCNQ1. We performed 2 dimensional Brownian Dynamics (2D-BD) protein-docking simulations on each representative structure of KCNE1-TMD and KCNQ1 homology model. After selecting compact complexes by using a distance-based filter to remove loose contact complexes, a series of experimental restraints were applied to select one final KCNQ1/KCNE1-TMD complex structure. The N- and C- terminal domains of KCNE1 were reconstructed to generate the whole complex of KCNQ1/KCNE1, and followed by 100ns MD simulations in explicit membrane environment. For comparison, we also conducted 100ns MD simulations on KCNQ1 alone. Based on the two simulation studies, we analyzed the dynamics of the KCNQ1 channel with and without KCNE1 present through a systematic examination of hydrogen bond network patterns and ion permeation pathway of the channel. The results could provide us helpful insights of the molecular mechanism of how KCNE1 modulating KCNQ1 channel.

The Cytoplasmic Ion Permeation Pathway of the Cardiac Na +-Ca 2+ Exchanger

The Na+-Ca2+ Exchanger (NCX) is a plasma membrane protein that exchanges three Na+ for one Ca2+. Transport function has been associated with two regions of NCX that have similar sequence: the α-repeats. Mutations here alter the transport properties of NCX. These two regions, which consist of residues spanning TMSs 2 and 3 (α-1 repeat) and a portion of TMS 7 with the following intracellular loop (α-2 repeat), are modeled to be in close proximity and may form an ion conduction pathway. The goal of this work was to understand transport function and define the pathway through which Na+ and Ca2+ diffuse from the cytoplasm to their intracellular binding sites. We mutated TMS 2 residues to cysteine and determined the affinities of these mutants for Na+ and Ca2+. Also, the reactivity of cysteines to MTSET was investigated to demonstrate intracellular accessibility. Of mutations at 10 positions, 3 behaved like the wild-type exchanger (A106C, P112A, S117C). Mutant M105C had altered affinity for Na+ but not Ca2+ and, like A106C, was blocked by cytoplasmic MTSET in the presence or absence of Na+. In contrast, Na+ blocked the sensitivity of L107C, S109C and S110C to MTSET. This suggests that Na+ binding prevents the cytoplasmic accessibility of these residues. Additionally, mutations at L107, S109 and S110 had altered affinities for intracellular Na+ and Ca2+. For S110C, the selectivity properties of NCX were also altered allowing Li+/Ca2+ exchange. We conclude that TMS 2 is involved in ion binding and transport and is accessible to cytoplasmic sulfhydryl agents in a substrate-dependent manner. The data also suggest the presence of an aqueous cavity used by Na+ and Ca2+ to reach their binding sites involving TMS 2.

Helical Rotation Associated with the Gating of the CFTR Chloride Channel

CFTR, a member of the ABC transporter superfamily, is an ATP-gated chloride channel. Its two transmembrane domains each encompassing six transmembrane segments (TMs), form the ion permeation pathway. However, which TMs contribute to the pore-lining and how conformational changes in the TMs lead to the opening and closing of the pore remain largely unknown. Our previous cysteine scanning studies reveal that TM6, a potential pore-lining candidate, is likely involved in a rotational movement during gating. Here, we presented the cysteine scanning results of TM12 (residues 1129-1150), which is the equivalent TM to TM6 based on crystallographic studies that show a pseudo two-fold symmetry in several ABC proteins. Three cysteine residues, S1141C, W1145C and N1148C, react with a negatively charged MTS reagent at a rate approaching that of free thiols with the same reagent, suggesting that they face the aqueous pore. Consistent with this idea, cysteine modification by a positively charged MTS reagent affects the single-channel current amplitude as well as the potency of the anionic, hydrophobic open-pore blocker, glibenclamide. Furthermore, blockade of the pore by glibenclamide completely protect cysteines at 1141 and 1145 from modification by MTS reagents, further supporting the idea that these residues line the pore. Interestingly, by measuring the reaction rates when the channel is gated by high (2 mM, Po ∼ 0.60) and low (30 uM, Po ∼ 0.15) concentrations of ATP, we inferred that whereas 1145C reacts faster in the open state, 1141C and 1148C react faster in the closed state. Thus, our results suggest that the cytoplasmic gate has been degenerated during evolution, and that helical rotations of TMs, which are proposed to be important for the function of ABC transporters, also play a role in the gating transitions of the CFTR channel.

Energetic Coupling at the Nucleotide-Binding Domain/Transmembrane Domain Interface of CFTR

CFTR belongs to the superfamily of ABC transporters, which generally couple vectorial transport of diverse compounds across membranes to hydrolytic cycles at conserved nucleotide-binding domains (NBDs). CFTR, alone, functions as an ion channel. As in other ABC proteins, ATP binds on the surface of the two NBDs, which then can form a head-to-tail NBD dimer, with two composite catalytic sites at the interface (site 1 and site 2). ATP hydrolysis at site 2 triggers dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains (TMDs) where they result in opening and closing, respectively, of the ion-permeation pathway.

Voltage Profile along the Permeation Pathway of an Open Channel

For ion channels, the transmembrane potential plays a critical role by acting as a driving force for permeant ions. At the microscopic level, the transmembrane potential is thought to decay nonlinearly across the ion permeation pathway because of the irregular three-dimensional shape of the channel's pore. By taking advantage of the current structural and functional understanding of cyclic nucleotide-gated channels, in this study we experimentally explore the transmembrane potential's distribution across the open pore. As a readout for the voltage drop, we engineered cysteine residues along the selectivity filter and scanned the sensitivity of their modification rates by Ag + to the transmembrane potential. The experimental data, which indicate that the majority of the electric field drops across the selectivity filter, are in good agreement with continuum electrostatic calculations using a homology model of an open CNG channel. By focusing the transmembrane potential across the selectivity filter, the electromotive driving force is coupled with the movement of permeant ions in the filter, maximizing the efficiency of this process.

Allosteric Inhibition of the Epithelial Na+ Channel through Peptide Binding at Peripheral Finger and Thumb Domains

The epithelial Na(+) channel (ENaC) mediates the rate-limiting step in transepithelial Na(+) transport in the distal segments of the nephron and in the lung. ENaC subunits are cleaved by proteases, resulting in channel activation due to the release of inhibitory tracts. Peptides derived from these tracts inhibit channel activity. The mechanism by which these intrinsic inhibitory tracts reduce channel activity is unknown, as are the sites where these tracts interact with other residues within the channel. We performed site-directed mutagenesis in large portions of the predicted periphery of the extracellular region of the α subunit and measured the effect of mutations on an 8-residue inhibitory tract-derived peptide. Our data show that the inhibitory peptide likely binds to specific residues within the finger and thumb domains of ENaC. Pairwise interactions between the peptide and the channel were identified by double mutant cycle experiments. Our data suggest that the inhibitory peptide has a specific peptide orientation within its binding site. Extended to the intrinsic inhibitory tract, our data suggest that proteases activate ENaC by removing residues that bind at the finger-thumb domain interface.

Glutamate receptor ion channels: structure, regulation, and function.

The mammalian ionotropic glutamate receptor family encodes 18 gene products that coassemble to form ligand-gated ion channels containing an agonist recognition site, a transmembrane ion permeation pathway, and gating elements that couple agonist-induced conformational changes to the opening or closing of the permeation pore. Glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system and are localized on neuronal and non-neuronal cells. These receptors regulate a broad spectrum of processes in the brain, spinal cord, retina, and peripheral nervous system. Glutamate receptors are postulated to play important roles in numerous neurological diseases and have attracted intense scrutiny. The description of glutamate receptor structure, including its transmembrane elements, reveals a complex assembly of multiple semiautonomous extracellular domains linked to a pore-forming element with striking resemblance to an inverted potassium channel. In this review we discuss International Union of Basic and Clinical Pharmacology glutamate receptor nomenclature, structure, assembly, accessory subunits, interacting proteins, gene expression and translation, post-translational modifications, agonist and antagonist pharmacology, allosteric modulation, mechanisms of gating and permeation, roles in normal physiological function, as well as the potential therapeutic use of pharmacological agents acting at glutamate receptors.

Probing Pore Constriction in a Ligand-gated Ion Channel by Trapping a Metal Ion in the Pore upon Agonist Dissociation

Eukaryotic pentameric ligand-gated ion channels (pLGICs) are receptors activated by neurotransmitters to rapidly transport ions across cell membranes, down their electrochemical gradients. Recent crystal structures of two prokaryotic pLGICs were interpreted to imply that the extracellular side of the transmembrane pore constricts to close the channel (Hilf, R. J., and Dutzler, R. (2009) Nature 457, 115-118; Bocquet, N., Nury, H., Baaden, M., Le Poupon, C., Changeux, J. P., Delarue, M., and Corringer, P. J. (2009) Nature 457, 111-114). Here, we utilized a eukaryotic acetylcholine (ACh)-serotonin chimeric pLGIC that was engineered with histidines to coordinate a metal ion within the channel pore, at its cytoplasmic side. In a previous study, the access of Zn(2+) ions to the engineered histidines had been explored when the channel was either at rest (closed) or active (open) (Paas, Y., Gibor, G., Grailhe, R., Savatier-Duclert, N., Dufresne, V., Sunesen, M., de Carvalho, L. P., Changeux, J. P., and Attali, B. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 15877-15882). In this study, the interactions of Zn(2+) with the pore were probed upon agonist (ACh) dissociation that triggers the transition of the receptor from the active conformation to the resting conformation (i.e. during deactivation). Application of Zn(2+) onto ACh-bound open receptors obstructed their pore and prevented ionic flow. Removing ACh from its extracellular binding sites to trigger deactivation while Zn(2+) is still bound led to tight trapping of Zn(2+) within the pore. Together with single-channel recordings, made to explore single pore-blocking events, we show that dissociation of ACh causes the gate to shut on a Zn(2+) ion that effectively acts as a "foot in the door." We infer that, upon deactivation, the cytoplasmic side of the pore of the ACh-serotonin receptor chimera constricts to close the channel.

Structural Analysis of the Extracellular Entrance to the Serotonin Transporter Permeation Pathway

Neurotransmitter transporters are responsible for removal of biogenic amine neurotransmitters after release into the synapse. These transporters are the targets for many clinically relevant drugs, such as antidepressants and psychostimulants. A high resolution crystal structure for the monoamine transporters has yet to be solved. We have developed a homology model for the serotonin transporter (SERT) based on the crystal structure of the leucine transporter (LeuT(Aa)) from Aquifex aeolicus. The objective of the present studies is to identify the structural determinants forming the entrance to the substrate permeation pathway based on predictions from the SERT homology model. Using the substituted cysteine accessibility method, we identified residues predicted to reside at the entrance to the substrate permeation pathway that were reactive with methanethiosulfonate (MTS) reagents. Of these residues, Gln(332) in transmembrane helix (TMH) VI was protected against MTS inactivation in the presence of serotonin. Surprisingly, the reactivity of Gln(332) to MTS reagents was enhanced in the presence of cocaine. Bifunctional MTS cross-linkers also were used to examine the distances between helices predicted to form the entrance into the substrate and ion permeation pathway. Our studies suggest that substrate and ligand binding may induce conformational shifts in TMH I and/or VI, providing new opportunities to refine existing homology models of SERT and related monoamine transporters.

The Intracellular Loop of Orai1 Plays a Central Role in Fast Inactivation of Ca2+ Release-activated Ca2+ Channels

Store-operated Ca(2+) entry (SOCE) due to activation of Ca(2+) release-activated Ca(2+) (CRAC) channels leads to sustained elevation of cytoplasmic Ca(2+) and activation of lymphocytes. CRAC channels consisting of four pore-forming Orai1 subunits are activated by STIM1, an endoplasmic reticulum Ca(2+) sensor that senses intracellular store depletion and migrates to plasma membrane proximal regions to mediate SOCE. One of the fundamental properties of CRAC channels is their Ca(2+)-dependent fast inactivation. To identify the domains of Orai1 involved in fast inactivation, we have mutated residues in the Orai1 intracellular loop linking transmembrane segment II to III. Mutation of four residues, V(151)SNV(154), at the center of the loop (MutA) abrogated fast inactivation, leading to increased SOCE as well as higher CRAC currents. Point mutation analysis identified five key amino acids, N(153)VHNL(157), that increased SOCE in Orai1 null murine embryonic fibroblasts. Expression or direct application of a peptide comprising the entire intracellular loop or the sequence N(153)VHNL(157) blocked CRAC currents from both wild type (WT) and MutA Orai1. A peptide incorporating the MutA mutations had no blocking effect. Concatenated Orai1 constructs with four MutA monomers exhibited high CRAC currents lacking fast inactivation. Reintroduction of a single WT monomer (MutA-MutA-MutA-WT) was sufficient to fully restore fast inactivation, suggesting that only a single intracellular loop can block the channel. These data suggest that the intracellular loop of Orai1 acts as an inactivation particle, which is stabilized in the ion permeation pathway by the N(153)VHNL(157) residues. These results along with recent reports support a model in which the N terminus and the selectivity filter of Orai1 as well as STIM1 act in concert to regulate the movement of the intracellular loop and evoke fast inactivation.

Does a Single Mutation in the P-Loop Open a Novel Current Pathway Beside the Central A-Pore in the Human Voltage-Gated Potassium Channel Kv1.3

Voltage-gated potassium channels are membrane proteins containing a potassium-selective ion conducting central α-pore. Recent studies showed that mutations in the voltage sensor of the Shaker channel can disclose another ion permeation pathway through the voltage sensing domain (S1-S4) beside the α-pore, the so called ω-pore described by Tombola et al. 2005 (Neuron45:379-388). We investigated a mutant voltage-gated hKv1.3 channel where the substitution of a cysteine in the pore-loop at position 388 (Shaker position 438) generated a current through the α-pore, and an inward-current at hyperpolarizing potentials carried by different cations (Li+∼Na+>NH4+>Cs+>K+∼Rb+). This observed inward current appeared similar to the ω-current and was not affected by the α-pore blocker CTX, in contrary to the currents through the α-pore of the hKv1.3_V388C mutant channel. Verapamil, which is acting from the intracellular side, could block both, the α-current with an IC50 of 3.5 μM and the observed inward-current at hyperpolarizing potentials with an IC50 of 2.3 μM in the hKv1.3_V388C mutant channel. Due to the block of inward-current by verapamil we suppose that the observed inward-current runs through the verapamil binding site in the cavity between S6-S6 of two adjacent subunits in the hKv1.3_V388C mutant channel. We hypothesize that the hKv1.3_V388C mutation generated a channel with a second ion conducting pathway distinct from the α-pore, but not identical to the ω-pathway, running through one part of the hKv1.3 verapamil binding site. The entry of the novel pathway is presumably located at the backside of Y395 (Shaker position 445), proceeds plane parallel to the α-pore, ending between S5 and S6 at the intracellular side of one α-subunit.Supported by grants from the 4SC AG (Martinsried) and the DFG (Gr848/14-1).

Molecular Determinants in Glycine and GABA-A Receptors that Sense Intracellular Chloride Concentration

GABA-A and glycine receptors are ligand-gated ion channels belonging to the Cys-loop superfamily. They are permeable to anions and mediate fast synaptic inhibition in the central nervous system. We recently showed that intracellular chloride can affect the time course of deactivation of these channels (1). The aim of the present work was to identify the residues that are involved in “sensing” the intracellular chloride concentration.We used the fast concentration jump technique to apply brief pulses (1-3 ms) of saturating glycine or GABA to outside-out patches from transiently-transfected HEK cells expressing alpha1 or alpha1 beta glycine receptors or alpha1 beta2 gamma2L GABA-A receptors. The effect of intracellular chloride could be mediated either by residues in intracellular loops, such as M1-M2 and M3-M4, or by the residues lining the channel itself. We used a combination of deletion and Ala/Cys-scanning mutagenesis approaches to address this question.In other nicotinic channels, the long M3-M4 intracellular loop can be replaced by the equivalent domain (7 amino acids) of the orthologous prokaryotic channel from Gloeobacter violaceus without losing channel function (2). We found that chimeric constructs of glycine and GABA-A receptors containing the prokaryotic M3-M4 domain retained modulation by intracellular chloride, suggesting that the major cytoplasmic domain is unlikely to mediate this effect. The results of mutagenesis of residues facing the ion permeation pathway on the contrary strongly suggests that chloride ions affect channel kinetic by binding to a site along the pore; we have identified conserved residues that reduce the differences between the deactivation rates measured at different chloride concentrations.(1) Pitt, Sivilotti and Beato (2008) J.Neurosci 28, 11454-11467(2) Jansen, Bali and Akabas (2008) J.Gen.Physiol. 131, 137-146

Identification of a Novel Organic Anion Transporter Mediating Carnitine Transport in Mouse Liver and Kidney

This study identifies a novel organic anion transporter Oat9 expressed in mouse liver and kidney. Two variants were detected by screening a mouse liver cDNA library; these varients consist of 1815 (designated Oat9S) and 2165 (Oat9L) base pairs which encode 443 and 551 amino acid proteins, respectively. Oat9S has a predicted structure containing eight transmembrane domains (TMD); whereas, Oat9L possesses twelve TMD. Oat9 mRNA expression was detected in kidney and liver. This transporter was located at the apical side of the late portion of proximal tubules and at the sinusoidal side of hepatocytes. When expressed in Xenopus oocytes, Oat9S mediated the transport of L-carnitine (Km = 2.9 µM), a representative zwitterion, as well as cimetidine (Km = 16.1 µM) and salicylic acid (Km = 175.5 µM), while Oat9L did not show any transport activity. Oat9S-mediated L-carnitine uptake was inhibited by D-carnitine, acetylcarnitine, octanoylcarnitine, betaine, and other organic compounds, suggesting that quaternary ammonium cation bulkiness and relative hydrophobicity are important factors for Oat9S-substrate interactions. Among OATs, Oat9S appears to be the first member to mediate the transport of carnitine and possesses eight TMD. Overall, these new results provide added insight into the structure-activity relationship comprising the organic ion-permeation pathway.

Temporal and Steric Analysis Of Ionic Permeation and Binding in Na+,K+-ATPase via Molecular Dynamic Simulations

Abstract: The Na+, K+-ATPase is ubiquitous in animal cells. Yet, many atomic-level details of the structure/function relationship of its electrogenic translocation process remain unanswered. This work employs computational methods to investigate the specific amino acid residues that constitute the two K+ and three Na+ ion binding sites. Putative lumenal and cytoplasmic ion permeation pathways are also determined. Homology models of the human α1 isoform of the Na+, K+-ATPase based on X-ray structures of the SERCA Ca2+-ATPase in several conformations (E1, E2, and E2P) were created using the Modeller homology modeling software. The sequence alignment incorporated an array of experimental results and consensus with similar proteins (e.g. H+, K+-ATPase). The E2P homology model presented here agrees well with the recent X-ray crystallographic structure of the Na+, K+-ATPase. The three models were simulated in a water/lipid environment with the GROMACS molecular dynamics package. Established equilibration techniques were followed by several nanoseconds of analyzable production run trajectories for each conformation. Atomic trajectories were analyzed with the steric pathway tool, CAVER, to provide putative ion permeation pathways. These pathways were consistent with regions of negative potential determined via time-averaged electrostatic calculations of the same trajectories. The electrostatic calculations provide a 3D view of the potential landscape encountered by cations. Amino acid residues (as suggested by SERCA structures and mutagenesis studies) involved in the creation of putative binding sites were investigated by placing Na+ and K+ ions at these locations and evaluating protein-ion interactions during simulation trajectories. In addition to the results of the Na+, K+-ATPase simulations, the homology models and methodology presented here provide a blueprint for the study of the larger class of P-type ATPases.

Crystal Structure Of Full-length Kcsa Trapped In Open Conformation Reveals That C-terminal Domain Fine Tunes Activation And Coupled Inactivation

Using chaperone-assisted crystallography, we have recently determined the crystal structure of full-length (FL) KcsA in its closed conformation. The FL structure reveals that the C-terminal domain (CTD) extends ~70 A towards the cytoplasm as a canonical four helix bundle stabilizing the closed state. Electrophysiological analysis of KcsA/Fab4 complex demonstrates that CTD remains as a bundle during activation gating. This motivated us to crystallize open conformation (OC) of FL KcsA using the same antibody fragments and a recently developed constitutively open mutant, which was crystallized in its truncated form. We solved the FL KcsA/Fab2 complex structure in the open state at 3.9 A resolution. FL open-structure bends at G104 and exhibits ~10 A displacement at the V115 creating four side windows large enough to accommodate hydrated K+ ions right below the gate. The CTD remains as four helix bundle, exerting strain on the bulge helices which connects the bundle and the transmembrane domain.Based on the current structure and an earlier set of truncated KcsA structures displaying different degree of openings, we suggest that the cytoplasmic domain not only stabilizes the closed state but also fine tunes the level of opening at the activation gate and thus determine the level of inactivation occurring at the selectivity filter. Brownian Dynamics, simulation, electrophysiological studies and electrostatic calculation are ongoing efforts to dissect the ion permeation pathway.

The Behavior of Ions Inside the Cytoplasmic Domain of Inward Rectifier Potassium Channels

Inward rectifiers are a subfamily of potassium channels that control the amounts of outward K+ current in a cell. These channels achieve rectification through intracellular block by strongly charged cations such as Mg2+ and polyamines like spermine (SPM4+). The large cytoplasmic domain that extends the ion permeation pathway has been shown to be an important determinant of conductance and rectification, however the organization of ions within this part of the pore is not well understood. In this study, the properties of ions inside the cytoplasmic domains of a weak (Kir1.1/ROMK) and strong (Kir2.1/IRK) rectifier are investigated via explicit solvent molecular dynamics simulations in 1M KCl. Both channels concentrate K+ ions in large amounts (local concentration > 3M), with the highest densities near the protein surface. An additional concentrating region specific to Kir2.1/IRK is observed near the cytoplasmic opening. Simulations are also carried out with Mg2+ or SPM4+ inside the domain. Mg2+ interacts directly with pore-lining residues, resulting in a depletion of K+ and increase in the local concentration of Cl−. SPM4+ shows high density throughout the central pore and selectively depletes K+ in the upper region of the pore closest to the transmembrane domain. Two long-lived states of SPM4+ are observed in Kir2.1/IRK: (i) inside the central pore in contact with residues D2591, E2241, E2242 and E2992 from two adjacent subunits, and (ii) near the cytoplasmic entrance interacting with residues D255, D259 and E224 on a single monomer. These results demonstrate a level of molecular specificity with respect to ion behavior within the cytoplasmic domains that could correspond to differences in rectification properties.

Differential Roles of Blocking Ions in KirBac1.1 Tetramer Stability

Potassium channels are tetrameric proteins that mediate K(+)-selective transmembrane diffusion. For KcsA, tetramer stability depends on interactions between permeant ions and the channel pore. We have examined the role of pore blockers on the tetramer stability of KirBac1.1. In 150 mm KCl, purified KirBac1.1 protein migrates as a monomer (approximately 40 kDa) on SDS-PAGE. Addition of Ba(2+) (K(1/2) approximately 50 microm) prior to loading results in an additional tetramer band (approximately 160 kDa). Mutation A109C, at a residue located near the expected Ba(2+)-binding site, decreased tetramer stabilization by Ba(2+) (K(1/2) approximately 300 microm), whereas I131C, located nearby, stabilized tetramers in the absence of Ba(2+). Neither mutation affected Ba(2+) block of channel activity (using (86)Rb(+) flux assay). In contrast to Ba(2+), Mg(2+) had no effect on tetramer stability (even though Mg(2+) was a potent blocker). Many studies have shown Cd(2+) block of K(+) channels as a result of cysteine substitution of cavity-lining M2 (S6) residues, with the implicit interpretation that coordination of a single ion by cysteine side chains along the central axis effectively blocks the pore. We examined blocking and tetramer-stabilizing effects of Cd(2+) on KirBac1.1 with cysteine substitutions in M2. Cd(2+) block potency followed an alpha-helical pattern consistent with the crystal structure. Significantly, Cd(2+) strongly stabilized tetramers of I138C, located in the center of the inner cavity. This stabilization was additive with the effect of Ba(2+), consistent with both ions simultaneously occupying the channel: Ba(2+) at the selectivity filter entrance and Cd(2+) coordinated by I138C side chains in the inner cavity.

An Ion Selectivity Filter in the Extracellular Domain of Cys-loop Receptors Reveals Determinants for Ion Conductance

Neurotransmitter binding to Cys-loop receptors promotes a prodigious transmembrane flux of several million ions/s, but to date, structural determinants of ion flux have been identified flanking the membrane-spanning region. Using x-ray crystallography, sequence analysis, and single-channel recording, we identified a novel determinant of ion conductance near the point of entry of permeant ions. Co-crystallization of acetylcholine-binding protein with sulfate anions revealed coordination of SO4(2-) with a ring of lysines at a position equivalent to 24 A above the lipid membrane in homologous Cys-loop receptors. Analysis of multiple sequence alignments revealed that residues equivalent to the ring of lysines are negatively charged in cation-selective receptors but are positively charged in anion-selective receptors. Charge reversal of side chains at homologous positions in the nicotinic receptor from the motor end plate decreases unitary conductance up to 80%. Selectivity filters stemming from transmembrane alpha-helices have similar pore diameters and compositions of amino acids. These findings establish that when the channel opens under a physiological electrochemical gradient, permeant ions are initially stabilized within the extracellular vestibule of Cys-loop receptors, and this stabilization is a major determinant of ion conductance.

ATP hydrolysis-driven gating in cystic fibrosis transmembrane conductance regulator

Proteins belonging to the ATP-binding cassette superfamily couple ATP binding and hydrolysis at conserved nucleotide-binding domains (NBDs) to diverse cellular functions. Most superfamily members are transporters, while cystic fibrosis transmembrane conductance regulator (CFTR), alone, is an ion channel. Despite this functional difference, recent results have suggested that CFTR shares a common molecular mechanism with other members. ATP binds to partial binding sites on the surface of the two NBDs, which then associate to form a NBD dimer, with complete composite catalytic sites now buried at the interface. ATP hydrolysis and gamma-phosphate dissociation, with the loss of molecular contacts linking the two sides of the composite site, trigger dimer dissociation. The conformational signals generated by NBD dimer formation and dissociation are transmitted to the transmembrane domains where, in transporters, they drive the cycle of conformational changes that translocate the substrate across the membrane; in CFTR, they result in opening and closing (gating) of the ion-permeation pathway.