Pore dimensions and the role of occupancy in unitary conductance of Shaker K channels

K channels mediate the selective passage of K⁺ across the plasma membrane by means of intimate interactions with ions at the pore selectivity filter located near the external face. Despite high conservation of the selectivity filter, the K⁺ transport properties of different K channels vary widely, with the unitary conductance spanning a range of over two orders of magnitude. Mutation of Pro475, a residue located at the cytoplasmic entrance of the pore of the small-intermediate conductance K channel Shaker (Pro475Asp (P475D) or Pro475Gln (P475Q)), increases Shaker’s reported ∼20-pS conductance by approximately six- and approximately threefold, respectively, without any detectable effect on its selectivity. These findings suggest that the structural determinants underlying the diversity of K channel conductance are distinct from the selectivity filter, making P475D and P475Q excellent probes to identify key determinants of the K channel unitary conductance. By measuring diffusion-limited unitary outward currents after unilateral addition of 2 M sucrose to the internal solution to increase its viscosity, we estimated a pore internal radius of capture of ∼0.82 Å for all three Shaker variants (wild type, P475D, and P475Q). This estimate is consistent with the internal entrance of the Kv1.2/2.1 structure if the effective radius of hydrated K⁺ is set to ∼4 Å. Unilateral exposure to sucrose allowed us to estimate the internal and external access resistances together with that of the inner pore. We determined that Shaker resistance resides mainly in the inner cavity, whereas only ∼8% resides in the selectivity filter. To reduce the inner resistance, we introduced additional aspartate residues into the internal vestibule to favor ion occupancy. No aspartate addition raised the maximum unitary conductance, measured at saturating [K⁺], beyond that of P475D, suggesting an ∼200-pS conductance ceiling for Shaker. This value is approximately one third of the maximum conductance of the large conductance K (BK) channel (the K channel of highest conductance), reducing the energy gap between their K⁺ transport rates to ∼1 kT. Thus, although Shaker’s pore sustains ion translocation as the BK channel’s does, higher energetic costs of ion stabilization or higher friction with the ion’s rigid hydration cage in its narrower aqueous cavity may entail higher resistance.     

Modular design of Cys-loop ligand-gated ion channels: functional 5-HT3 and GABA rho1 receptors lacking the large cytoplasmic M3M4 loop

Cys-loop receptor neurotransmitter-gated ion channels are pentameric assemblies of subunits that contain three domains: extracellular, transmembrane, and intracellular. The extracellular domain forms the agonist binding site. The transmembrane domain forms the ion channel. The cytoplasmic domain is involved in trafficking, localization, and modulation by cytoplasmic second messenger systems but its role in channel assembly and function is poorly understood and little is known about its structure. The intracellular domain is formed by the large (>100 residues) loop between the alpha-helical M3 and M4 transmembrane segments. Putative prokaryotic Cys-loop homologues lack a large M3M4 loop. We replaced the complete M3M4 loop (115 amino acids) in the 5-hydroxytryptamine type 3A (5-HT(3A)) subunit with a heptapeptide from the prokaryotic homologue from Gloeobacter violaceus. The macroscopic electrophysiological and pharmacological characteristics of the homomeric 5-HT(3A)-glvM3M4 receptors were comparable to 5-HT(3A) wild type. The channels remained cation-selective but the 5-HT(3A)-glvM3M4 single channel conductance was 43.5 pS as compared with the subpicosiemens wild-type conductance. Coexpression of hRIC-3, a protein that modulates expression of 5-HT(3) and acetylcholine receptors, significantly attenuated 5-HT-induced currents with wild-type 5-HT(3A) but not 5-HT(3A)-glvM3M4 receptors. A similar deletion of the M3M4 loop in the anion-selective GABA-rho1 receptor yielded functional, GABA-activated, anion-selective channels. These results imply that the M3M4 loop is not essential for receptor assembly and function and suggest that the cytoplasmic domain may fold as an independent module from the transmembrane and extracellular domains.     

UTP-dependent inhibition of Na+ absorption requires activation of PKC in endometrial epithelial cells

The objective of this study was to investigate the mechanism of uridine 5′-triphosphate (UTP)-dependent inhibition of Na⁺ absorption in porcine endometrial epithelial cells. Acute stimulation with UTP (5 μM) produced inhibition of sodium absorption and stimulation of chloride secretion. Experiments using basolateral membrane–permeabilized cell monolayers demonstrated a reduction in benzamil-sensitive Na⁺ conductance in the apical membrane after UTP stimulation. The UTP-dependent inhibition of sodium transport could be mimicked by PMA (1 μM). Several PKC inhibitors, including GF109203X and Gö6983 (both nonselective PKC inhibitors) and rottlerin (a PKCδ selective inhibitor), were shown to prevent the UTP-dependent decrease in benzamil-sensitive current. The PKCα-selective inhibitors, Gö6976 and PKC inhibitor 20–28, produced a partial inhibition of the UTP effect on benzamil-sensitive Isc. Inhibition of the benzamil-sensitive Isc by UTP was observed in the presence of BAPTA-AM (50 μM), confirming that activation of PKCs, and not increases in [Ca²⁺]_i, were directly responsible for the inhibition of apical Na⁺ channels and transepithelial Na⁺ absorption.     

The Minimum M3-M4 Loop Length of Neurotransmitter-activated Pentameric Receptors Is Critical for the Structural Integrity of Cytoplasmic Portals

The 5-HT₃A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT₃A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Å gap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT₃A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.     

Low resistance, large dimension entrance to the inner cavity of BK channels determined by changing side-chain volume

Large-conductance Ca²⁺- and voltage-activated K⁺ (BK) channels have the largest conductance (250–300 pS) of all K⁺-selective channels. Yet, the contributions of the various parts of the ion conduction pathway to the conductance are not known. Here, we examine the contribution of the entrance to the inner cavity to the large conductance. Residues at E321/E324 on each of the four α subunits encircle the entrance to the inner cavity. To determine if 321/324 is accessible from the inner conduction pathway, we measured single-channel current amplitudes before and after exposure and wash of thiol reagents to the intracellular side of E321C and E324C channels. MPA⁻ increased currents and MTSET⁺ decreased currents, with no difference between positions 321 and 324, indicating that side chains at 321/324 are accessible from the inner conduction pathway and have equivalent effects on conductance. For neutral amino acids, decreasing the size of the entrance to the inner cavity by substituting large side-chain amino acids at 321/324 decreased outward single-channel conductance, whereas increasing the size of the entrance with smaller side-chain substitutions had little effect. Reductions in outward conductance were negated by high [K⁺]_i. Substitutions had little effect on inward conductance. Fitting plots of conductance versus side-chain volume with a model consisting of one variable and one fixed resistor in series indicated an effective diameter and length of the entrance to the inner cavity for wild-type channels of 17.7 and 5.6 Å, respectively, with the resistance of the entrance ∼7% of the total resistance of the conduction pathway. The estimated dimensions are consistent with the structure of MthK, an archaeal homologue to BK channels. Our observations suggest that BK channels have a low resistance, large entrance to the inner cavity, with the entrance being as large as necessary to not limit current, but not much larger.     

Structure of S. aureus HPPK and the discovery of a new substrate site inhibitor

The first structural and biophysical data on the folate biosynthesis pathway enzyme and drug target, 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (SaHPPK), from the pathogen Staphylococcus aureus is presented. HPPK is the second essential enzyme in the pathway catalysing the pyrophosphoryl transfer from cofactor (ATP) to the substrate (6-hydroxymethyl-7,8-dihydropterin, HMDP). In-silico screening identified 8-mercaptoguanine which was shown to bind with an equilibrium dissociation constant, K_d, of ∼13 µM as measured by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). An IC₅₀ of ∼41 µM was determined by means of a luminescent kinase assay. In contrast to the biological substrate, the inhibitor has no requirement for magnesium or the ATP cofactor for competitive binding to the substrate site. The 1.65 Å resolution crystal structure of the inhibited complex showed that it binds in the pterin site and shares many of the key intermolecular interactions of the substrate. Chemical shift and ¹⁵N heteronuclear NMR measurements reveal that the fast motion of the pterin-binding loop (L2) is partially dampened in the SaHPPK/HMDP/α,β-methylene adenosine 5′-triphosphate (AMPCPP) ternary complex, but the ATP loop (L3) remains mobile on the µs-ms timescale. In contrast, for the SaHPPK/8-mercaptoguanine/AMPCPP ternary complex, the loop L2 becomes rigid on the fast timescale and the L3 loop also becomes more ordered – an observation that correlates with the large entropic penalty associated with inhibitor binding as revealed by ITC. NMR data, including ¹⁵N-¹H residual dipolar coupling measurements, indicate that the sulfur atom in the inhibitor is important for stabilizing and restricting important motions of the L2 and L3 catalytic loops in the inhibited ternary complex. This work describes a comprehensive analysis of a new HPPK inhibitor, and may provide a foundation for the development of novel antimicrobials targeting the folate biosynthetic pathway.     

Ion conduction and selectivity in acid-sensing ion channel 1

The ability of acid-sensing ion channels (ASICs) to discriminate among cations was assessed based on changes in conductance and reversal potential with ion substitution. Human ASIC1a was expressed in Xenopus laevis oocytes, and acid-induced currents were measured using two-electrode voltage clamp. Replacement of extracellular Na⁺ with Li⁺, K⁺, Rb⁺, or Cs⁺ altered inward conductance and shifted the reversal potentials consistent with a selectivity sequence of Li ∼ Na > K > Rb > Cs. Permeability decreased more rapidly than conductance as a function of atomic size, with P_K/P_Na = 0.1 and G_K/G_Na = 0.7 and P_Rb/P_Na = 0.03 and G_Rb/G_Na = 0.3. Stimulation of Cl⁻ currents when Na⁺ was replaced with Ca²⁺, Sr²⁺, or Ba²⁺ indicated a finite permeability to divalent cations. Inward conductance increased with extracellular Na⁺ in a hyperbolic manner, consistent with an apparent affinity (K_m) for Na⁺ conduction of 25 mM. Nitrogen-containing cations, including NH₄⁺, NH₃OH⁺, and guanidinium, were also permeant. In addition to passing through the channels, guanidinium blocked Na⁺ currents, implying competition for a site within the pore. The role of negative charges in an external vestibule of the pore was evaluated using the point mutation D434N. The mutant channel had a decreased single-channel conductance, measured in excised outside-out patches, and a macroscopic slope conductance that increased with hyperpolarization. It had a weakened interaction with Na⁺ (K_m = 72 mM) and a selectivity that was shifted toward larger atomic sizes. We conclude that the selectivity of ASIC1 is based at least in part on interactions with binding sites both within and internal to the outer vestibule.     

Thermodynamic examination of the pyrophosphate sensor helix in the thiamine pyrophosphate riboswitch

Riboswitches are functional mRNA that control gene expression. Thiamine pyrophosphate (TPP) binds to thi-box riboswitch RNA and allosterically inhibits genes that code for proteins involved in the biosynthesis and transport of thiamine. Thiamine binding to the pyrimidine sensor helix and pyrophosphate binding to the pyrophosphate sensor helix cause changes in RNA conformation that regulate gene expression. Here we examine the thermodynamic properties of the internal loop of the pyrophosphate binding domain by comparing the wild-type construct (RNA WT) with six modified 2 × 2 bulged RNA and one 2 × 2 bulged DNA. The wild-type construct retains five conserved bases of the pyrophosphate sensor domain, two of which are in the 2 × 2 bulge (C65 and G66). The RNA WT construct was among the most stable (ΔG°₃₇ = −7.7 kcal/mol) in 1 M KCl at pH 7.5. Breaking the A•G mismatch of the bulge decreases the stability of the construct ∼0.5–1 kcal/mol, but does not affect magnesium binding to the RNA WT. Guanine at position 48 is important for RNA–Mg²⁺ interactions of the TPP-binding riboswitch at pH 7.5. In the presence of 9.5 mM magnesium at pH 5.5, the bulged RNA constructs gained an average of 1.1 kcal/mol relative to 1 M salt. Formation of a single A+•C mismatch base pair contributes about 0.5 kcal/mol at pH 5.5, whereas two tandem A+•C mismatch base pairs together contribute about 2 kcal/mol.     

Effect of saline irrigation on plant water traits,photosynthesis and ionic balance in durum wheat genotypes

In the era of climate change, decreased precipitation and increased evapo-transpiration hampers the yield of several cereal crops along with the soil salinity and poor ground water resource. Wheat being the moderately tolerant crop face many challenges in the arid and semi-arid regions under irrigated agriculture. In view of this, the study was planned to explore the potential of durum wheat genotypes under salinity on the basis of physiological traits. Experiment was designed as RBD in three replications to evaluate 15 wheat genotypes with moderate saline irrigation (EC_iw – 6 dS m⁻¹) and extreme saline irrigation (EC_iw – 10 dS m⁻¹) along with one set of control (Best available water). Different physiological traits such as water potential (ψ_p), osmotic potential (ψ_s), relative water content (RWC), Na⁺ and K⁺ content were recorded in roots as well as shoots at the reproductive stage whereas photosynthetic rate and chlorophyll content were measured in the flag leaves. A significant variability (p < 0.001) was noted among the genotypes under different stress environments and it was observed that durum genotype HI 8728 and HI 8737 showed less reduction in plant water traits (RWC, ψ_p and ψ_s) than the salinity tolerant checks of bread wheat KRL 99 and KRL 3–4. HD 4728 and HI 8708 maintained higher photosynthetic rate as well as higher chlorophyll content under the extreme salinity level of EC_iw – 10 dSm⁻¹. No significant differences were found in root Na⁺ in genotypes KRL 99 (3.17^g), KRL 3–4 (3.34^g) and HI 8737 (3.41^g) while in shoots, lowest accumulation was seen in KRL 99, MACS 3949 and KRL 3–4 at EC_iw – 10 dSm⁻¹. The mean range of K⁺ content was 7.60–9.74% in roots and 4.21–6.61% in shoots under control environment which decreased to 50.77% in roots and 46.05% in shoots under extreme salinity condition of EC_iw – 10 dSm⁻¹. At EC_iw – 10 dSm⁻¹, KRL 99 maintained highest K^+/Na⁺ in both root and shoot followed by KRL 3–4, HI 8737, MACS 3949, HD 4728 in roots and MACS 3949, KRL 3–4, MACS 4020, HD 4758, MACS 3972 and HI 8713 in shoots. The differential response of durum wheat genotypes under salinity particularly for physiological traits, confer their adaptability towards stress environments and exhibit their potential as genetic sources in breeding programs for improving salt stress tolerance.     

Enzymatic suppression of the membrane conductance associated with the glutamine transporter SNAT3 expressed in Xenopus oocytes by carbonic anhydrase II

The transport activity of the glutamine/neutral amino acid transporter SNAT3 (former SN1, SLC38A3), expressed in oocytes of the frog Xenopus laevis is associated with a non-stoichiometrical membrane conductance selective for Na⁺ and/or H⁺ (Schneider, H.P., S. Bröer, A. Bröer, and J.W. Deitmer. 2007. J. Biol. Chem. 282:3788–3798). When we expressed SNAT3 in frog oocytes, the glutamine-induced membrane conductance was suppressed, when carbonic anhydrase isoform II (CAII) had been injected into the oocytes. Transport of substrate, however, was not affected by CAII. The reduction of the membrane conductance by CAII was dependent on the presence of CO₂/HCO₃ ⁻, and could be reversed by blocking the catalytic activity of CAII by ethoxyzolamide (10 μM). Coexpression of wild-type CAII or a N-terminal CAII mutant with SNAT3 also reduced the SNAT3- associated membrane conductance. The catalytically inactive CAII mutant V143Y coexpressed in oocytes did not affect SNAT3-associated membrane conductance. Our results reveal a new type of interaction between CAII and a transporter-associated cation conductance, and support the hypothesis that the transport of substrate and the non-stoichiometrical ion conductance are independent of each other. This study also emphasizes the importance of carbonic anhydrase activity and the presence of CO₂-bicarbonate buffers for membrane transport processes.     

Contribution of residues in second transmembrane domain of ASIC1a protein to ion selectivity

Acid-sensing ion channels (ASICs) are proton-gated cation-selective channels expressed in the peripheral and central nervous systems. The ion permeation pathway of ASIC1a is defined by residues 426–450 in the second transmembrane (TM2) segment. The gate, formed by the intersection of the TM2 segments, localizes near the extracellular boundary of the plasma membrane. We explored the contribution to ion permeation and selectivity of residues in the TM2 segment of ASIC1a. Studies of accessibility with positively charged methanethiosulfonate reagents suggest that the permeation pathway in the open state constricts below the gate, restricting the passage to large ions. Substitution of residues in the intracellular vestibule at positions 437, 438, 443, or 446 significantly increased the permeability to K⁺ versus Na⁺. ASIC1a shows a selectivity sequence for alkali metals of Na⁺>Li⁺>K⁺≫Rb⁺>Cs⁺. Alanine and cysteine substitutions at position 438 increased, to different extents, the relative permeability to Li⁺, K⁺, Rb⁺, and Cs⁺. For these mutants, ion permeation was not a function of the diameter of the nonhydrated ion, suggesting that Gly-438 encompasses an ion coordination site that is essential for ion selectivity. M437C and A443C mutants showed slightly increased permeability to K⁺, Rb⁺, and Cs⁺, suggesting that substitutions at these positions influence ion discrimination by altering molecular sieving. Our results indicate that ion selectivity is accomplished by the contribution of multiple sites in the pore of ASIC1a.     

Synergistic activation of eIF4A by eIF4B and eIF4G

eIF4A is a key component in eukaryotic translation initiation; however, it has not been clear how auxiliary factors like eIF4B and eIF4G stimulate eIF4A and how this contributes to the initiation process. Based on results from isothermal titration calorimetry, we propose a two-site model for eIF4A binding to an 83.5 kDa eIF4G fragment (eIF4G-MC), with a high- and a low-affinity site, having binding constants K_D of ∼50 and ∼1000 nM, respectively. Small angle X-ray scattering analysis shows that the eIF4G-MC fragment adopts an elongated, well-defined structure with a maximum dimension of 220 Å, able to span the width of the 40S ribosomal subunit. We establish a stable eIF4A–eIF4B complex requiring RNA, nucleotide and the eIF4G-MC fragment, using an in vitro RNA pull-down assay. The eIF4G-MC fragment does not stably associate with the eIF4A–eIF4B–RNA-nucleotide complex but acts catalytically in its formation. Furthermore, we demonstrate that eIF4B and eIF4G-MC act synergistically in stimulating the ATPase activity of eIF4A.     

Cloning, Baeyer-Villiger biooxidations, and structures of the camphor pathway 2-oxo-Δ(3)-4,5,5-trimethylcyclopentenylacetyl-coenzyme A monooxygenase of Pseudomonas putida ATCC 17453

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP⁺ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP⁺. A comparison of several crystal forms of OTEMO bound to FAD and NADP⁺ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ³-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k_cat/K_m) favors 2-n-hexyl cyclopentanone (4.3 × 10⁵ M⁻¹ s⁻¹) as a substrate, although its affinity (K_m = 32 μM) was lower than that of the CoA-activated substrate (K_m = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.     

Selective inhibition of MBNL1�CCCUG interaction by small molecules toward potential therapeutic agents for myotonic dystrophy type 2 (DM2)

Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disease caused by expanded CCUG repeats that may exhibit toxicity by sequestering the splicing regulator MBNL1. A series of triaminotriazine- and triaminopyrimidine-based small molecules (ligands 1–3) were designed, synthesized and tested as inhibitors of the MBNL1–CCUG interaction. Despite the structural similarities of the triaminotriazine and triaminopyrimidine units, the triaminopyrimidine-based ligands bind with low micromolar affinity to CCUG repeats (K_d ∼ 0.1–3.6 µM) whereas the triaminotriazine ligands do not bind CCUG repeats. Importantly, these simple and small triaminopyrimidine ligands exhibit both strong inhibition (K_i ∼ 2 µM) of the MBNL1–CCUG interaction and high selectivity for CCUG repeats over other RNA targets. These experiments suggest these compounds are potential lead agents for the treatment of DM2.     

Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry

Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimer''s disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimer''s disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimer''s disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-β peptide (Aβ) deposit. Two cases were apoϵ4/apoϵ3, and one case was apoϵ3/apoϵ3. A known quantity of ¹³C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 ± 0.1 attomoles/µm³ in the senile plaques and 2.2 ± 0.49 attomoles/µm³ in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 ± 16 femtomol) is similar to the published quantity of Aβ peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimer''s disease linked to the apoϵ4 allele, suggests new pathogenetic mechanisms.     

Fast and slow gating are inherent properties of the pore module of the K+ channel Kcv

Kcv from the chlorella virus PBCV-1 is a viral protein that forms a tetrameric, functional K⁺ channel in heterologous systems. Kcv can serve as a model system to study and manipulate basic properties of the K⁺ channel pore because its minimalistic structure (94 amino acids) produces basic features of ion channels, such as selectivity, gating, and sensitivity to blockers. We present a characterization of Kcv properties at the single-channel level. In symmetric 100 mM K⁺, single-channel conductance is 114 ± 11 pS. Two different voltage-dependent mechanisms are responsible for the gating of Kcv. “Fast” gating, analyzed by β distributions, is responsible for the negative slope conductance in the single-channel current–voltage curve at extreme potentials, like in MaxiK potassium channels, and can be explained by depletion-aggravated instability of the filter region. The presence of a “slow” gating is revealed by the very low (in the order of 1–4%) mean open probability that is voltage dependent and underlies the time-dependent component of the macroscopic current.     

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Effect of peptide PV on the ionic permeability of lipid bilayer membranes

This paper reports the effects of peptide PV (primary structure: cyclo-(D-val-L-pro-L-val-D-pro)_δ) on the electrical properties of sheep red cell lipid bilayers. The membrane conductance (G_m) induced by PV in either Na⁺ or K⁺ medium is proportional to the concentration of PV in the aqueous phase. The PV concentration required to produce a comparable increase in G_m in K⁺ medium is about 10⁴ times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K⁺ ≳ Rb⁺ > Cs⁺ > NH₄ ⁺ > TI⁺ > Na⁺ > Li⁺; the ratio of GGm in K⁺ to that in Na⁺ is about 10 for PV compared to > 10³ for val. When equal concentrations of PV are added to both sides of a bilayer, the membrane current approaches a maximum value independent of voltage when the membrane potential exceeds 100 mV. When PV is added to only one side of a bilayer separating identical salt solutions of either Na⁺ or K⁺ salts, rectification occurs such that the positive current flows more easily away rather than toward the side containing the carrier. Under these conditions, a large, stable, zero-current potential (VVm) is also observed, with the side containing PV being negative. The magnitude of this VVm is about 90 mV and relatively independent of PV concentration when the latter is larger than 2 Times; 10^–5 M. From a model which assumes that V_m equals the equilibrium potential for the PV-cation complexes (MS ⁺) and that the reaction between PV and cations is at equilibrium on the two membrane surfaces, we compute the permeability of the membrane to free PV to be about 10^–5 cm s^–1, which is about 10^–7 times the permeability of similar membranes to free val. This interpretation is supported by the fact that the observed values of V_m are in agreement with the calculated equilibrium potential for MS⁺ over a wide range of ratios of concentrations of total PV in the two bathing solutions, if the unstirred layers are taken into account in computing the MS⁺ concentrations at the membrane surfaces.     

Proteins in aggregates functionally impact multiple neurodegenerative disease models by forming proteasome‐blocking complexes

Age-dependent neurodegenerative diseases progressively form aggregates containing both shared components (e.g., TDP-43, phosphorylated tau) and proteins specific to each disease. We investigated whether diverse neuropathies might have additional aggregation-prone proteins in common, discoverable by proteomics. Caenorhabditis elegans expressing unc-54p/Q40::YFP, a model of polyglutamine array diseases such as Huntington''s, accrues aggregates in muscle 2–6 days posthatch. These foci, isolated on antibody-coupled magnetic beads, were characterized by high-resolution mass spectrometry. Three Q40::YFP-associated proteins were inferred to promote aggregation and cytotoxicity, traits reduced or delayed by their RNA interference knockdown. These RNAi treatments also retarded aggregation/cytotoxicity in Alzheimer''s disease models, nematodes with muscle or pan-neuronal Aβ_1–42 expression and behavioral phenotypes. The most abundant aggregated proteins are glutamine/asparagine-rich, favoring hydrophobic interactions with other random-coil domains. A particularly potent modulator of aggregation, CRAM-1/HYPK, contributed < 1% of protein aggregate peptides, yet its knockdown reduced Q40::YFP aggregates 72–86% (P < 10⁻⁶). In worms expressing Aβ_1–42, knockdown of cram-1 reduced β-amyloid 60% (P < 0.002) and slowed age-dependent paralysis > 30% (P < 10⁻⁶). In wild-type worms, cram-1 knockdown reduced aggregation and extended lifespan, but impaired early reproduction. Protection against seeded aggregates requires proteasome function, implying that normal CRAM-1 levels promote aggregation by interfering with proteasomal degradation of misfolded proteins. Molecular dynamic modeling predicts spontaneous and stable interactions of CRAM-1 (or human orthologs) with ubiquitin, and we verified that CRAM-1 reduces degradation of a tagged-ubiquitin reporter. We propose that CRAM-1 exemplifies a class of primitive chaperones that are initially protective and highly beneficial for early reproduction, but ultimately impair aggregate clearance and limit longevity.     

The concentration of soluble extracellular amyloid-β protein in acute brain slices from CRND8 mice

Background

Many recent studies of the effects of amyloid-β protein (Aβ) on brain tissue from amyloid precursor protein (APP) overexpressing mice have concluded that Aβ oligomers in the extracellular space can profoundly affect synaptic structure and function. As soluble proteins, oliomers of Aβ can diffuse through brain tissue and can presumably exit acute slices, but the rate of loss of Aβ species by diffusion from brain slices and the resulting reduced concentrations of Aβ species in brain slices are unknown.

Methodology/Principal Findings

Here I combine measurements of Aβ_1–42 diffusion and release from acute slices and simple numerical models to measure the concentration of Aβ_1–42 in intact mice (in vivo) and in acute slices from CRND8 mice. The in vivo concentration of diffusible Aβ_1–42 in CRND8 mice was 250 pM at 6 months of age and 425 pM at 12 months of age. The concentration of Aβ_1–42 declined rapidly after slice preparation, reaching a steady-state concentration within one hour. 50 µm from the surface of an acute slice the steady-state concentration of Aβ was 15–30% of the concentration in intact mice. In more superficial regions of the slice, where synaptic physiology is generally studied, the remaining Aβ is less than 15%. Hence the concentration of Aβ_1–42 in acute slices from CRND8 mice is less than 150 pM.

Conclusions/Significance

Aβ affects synaptic plasticity in the picomolar concentration range. Some of the effects of Aβ may therefore be lost or altered after slice preparation, as the extracellular Aβ concentration declines from the high picomolar to the low picomolar range. Hence loss of Aβ by diffusion may complicate interpretation of the effects of Aβ in experiments on acute slices from APP overexpressing mice.