Action of nicotine and analogs on acetylcholine receptors having mutations of transmitter-binding site residue αG153

A primary target for nicotine is the acetylcholine receptor channel (AChR). Some of the ability of nicotine to activate differentially AChR subtypes has been traced to a transmitter-binding site amino acid that is glycine in lower affinity and lysine in higher affinity AChRs. We studied the effects of mutations of this residue (αG153) in neuromuscular AChRs activated by nicotine and eight other agonists including nornicotine and anabasine. All of the mutations increased the unliganded gating equilibrium constant. The affinity of the resting receptor (K_d) and the net binding energy from the agonist for gating (ΔG_B) were estimated by cross-concentration fitting of single-channel currents. In all but one of the agonist/mutant combinations there was a moderate decrease in K_d and essentially no change in ΔG_B. The exceptional case was nicotine plus lysine, which showed a large, >8,000-fold decrease in K_d but no change in ΔG_B. The extraordinary specificity of this combination leads us to speculate that AChRs with a lysine at position αG153 may be exposed to a nicotine-like compound in vivo.     

Function of Interfacial Prolines at the Transmitter-binding Sites of the Neuromuscular Acetylcholine Receptor

The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (α-ϵ and α-δ). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (ΔG₀) and the affinity change for ACh that increases the open channel probability (ΔG_B). The effects of mutations of ProD2 (ϵPro-121/δPro-123) were greater than those of its neighbor (ϵPro-120/δPro-122) and were greater at α-ϵ versus α-δ. The main consequence of the congenital myasthenic syndrome mutation ϵProD2-L was to impair the establishment of a high affinity for ACh and thus make ΔG_B less favorable. At both binding sites, most ProD2 mutations decreased constitutive activity (increased ΔG₀). LRYHQG and RL substitutions reduced substantially the net binding energy (made ΔG_B^ACh less favorable) by ≥2 kcal/mol at α-ϵ and α-δ, respectively. Mutant cycle analyses were used to estimate energy coupling between the two ProD2 residues and between each ProD2 and glycine residues (αGly-147 and αGly-153) on the primary (α subunit) side of each binding pocket. The distant binding site prolines interact weakly. ProD2 interacts strongly with αGly-147 but only at α-ϵ and only when ACh is present. The results suggest that in the low to-high affinity change there is a concerted inter-subunit strain in the backbones at ϵProD2 and αGly-147. It is possible to engineer receptors having a single functional binding site by using a α-ϵ or α-δ ProD2-R knock-out mutation. In adult-type ACh receptors, the energy from the affinity change for ACh is approximately the same at the two binding sites (approximately −5 kcal/mol).     

Acetylcholine Receptor Channels Activated by a Single Agonist Molecule

The neuromuscular acetylcholine receptor (AChR) is an allosteric protein that alternatively adopts inactive versus active conformations (R↔R^∗). The R^∗ shape has a higher agonist affinity and ionic conductance than R. To understand how agonists trigger this gating isomerization, we examined single-channel currents from adult mouse muscle AChRs that isomerize normally without agonists but have only a single site able to use agonist binding energy to motivate gating. We estimated the monoliganded gating equilibrium constant E₁ and the energy change associated with the R versus R^∗ change in affinity for agonists. AChRs with only one operational binding site gave rise to a single population of currents, indicating that the two transmitter binding sites have approximately the same affinity for the transmitter ACh. The results indicated that E₁ ≈ 4.3 × 10⁻³ with ACh, and ≈1.7 × 10⁻⁴ with the partial-agonist choline. From these values and the diliganded gating equilibrium constants, we estimate that the unliganded AChR gating constant is E₀ ≈ 6.5 × 10⁻⁷. Gating changes the stability of the ligand-protein complex by ∼5.2 kcal/mol for ACh and ∼3.3 kcal/mol for choline.     

The Role of Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH^o) and entropy (ΔS^o) upon channel gating. The magnitude, sign, and temperature dependence of ΔH^o and ΔS^o, the last given by an associated change in heat capacity (ΔC_p), can determine a channel’s temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔC_p makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH^o and ΔS^o, including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH^o/ΔS^o ratio associated with them, can also determine a channel’s temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔC_p-independent manner.     

Cooperative Unfolding of Residual Structure in Heat Denatured Proteins by Urea and Guanidinium Chloride

The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, α-chymotrypsinogen A and α-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (ΔG ⁰ _X→D) associated with the process heat denatured (X) state ↔ GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ΔG ⁰ _N→X (native (N) state ↔ X state), ΔG ⁰ _X→D and ΔG ⁰ _N→D (N state ↔ D state), and (c) there is not a good enthalpy difference between X and D states, which is the reason for the absence of endothermic peak in DSC scan for the transition, X state ↔ D state.     

Predicting Thermodynamic Properties of PBXTHs with New Quantum Topological Indexes

Novel group quantitative structure-property relationship (QSPR) models on the thermodynamic properties of PBXTHs were presented, by the multiple linear regression (MLR) analysis method. Four thermodynamic properties were studied: the entropy (S^θ), the standard enthalpy of formation (Δ_fH^θ), the standard Gibbs energy of formation (Δ_fG^θ), and the relative standard Gibbs energy of formation (Δ_RG^θ). The results by the formula indicate that the calculated and predicted data in this study are in good agreement with those in literature and the deviation is within the experimental errors. To validate the estimation reliability for internal samples and the predictive ability for other samples, leave-one-out (LOO) cross validation (CV) and external validation were performed, and the results show that the models are satisfactory.     

Pyranose Dehydrogenase Ligand Promiscuity: A Generalized Approach to Simulate Monosaccharide Solvation,Binding, and Product Formation

The flavoenzyme pyranose dehydrogenase (PDH) from the litter decomposing fungus Agaricus meleagris oxidizes many different carbohydrates occurring during lignin degradation. This promiscuous substrate specificity makes PDH a promising catalyst for bioelectrochemical applications. A generalized approach to simulate all 32 possible aldohexopyranoses in the course of one or a few molecular dynamics (MD) simulations is reported. Free energy calculations according to the one-step perturbation (OSP) method revealed the solvation free energies (ΔG_solv) of all 32 aldohexopyranoses in water, which have not yet been reported in the literature. The free energy difference between β- and α-anomers (ΔG_β-α) of all d-stereoisomers in water were compared to experimental values with a good agreement. Moreover, the free-energy differences (ΔG) of the 32 stereoisomers bound to PDH in two different poses were calculated from MD simulations. The relative binding free energies (ΔΔG_bind) were calculated and, where available, compared to experimental values, approximated from K _m values. The agreement was very good for one of the poses, in which the sugars are positioned in the active site for oxidation at C1 or C2. Distance analysis between hydrogens of the monosaccharide and the reactive N5-atom of the flavin adenine dinucleotide (FAD) revealed that oxidation is possible at HC1 or HC2 for pose A, and at HC3 or HC4 for pose B. Experimentally detected oxidation products could be rationalized for the majority of monosaccharides by combining ΔΔG_bind and a reweighted distance analysis. Furthermore, several oxidation products were predicted for sugars that have not yet been tested experimentally, directing further analyses. This study rationalizes the relationship between binding free energies and substrate promiscuity in PDH, providing novel insights for its applicability in bioelectrochemistry. The results suggest that a similar approach could be applied to study promiscuity of other enzymes.     

Thermodynamic Analyses of Nucleotide Binding to an Isolated Monomeric β Subunit and the α3β3γ Subcomplex of F1-ATPase

Rotation of the γ subunit of the F₁-ATPase plays an essential role in energy transduction by F₁-ATPase. Hydrolysis of an ATP molecule induces a 120° step rotation that consists of an 80° substep and 40° substep. ATP binding together with ADP release causes the first 80° step rotation. Thus, nucleotide binding is very important for rotation and energy transduction by F₁-ATPase. In this study, we introduced a βY341W mutation as an optical probe for nucleotide binding to catalytic sites, and a βE190Q mutation that suppresses the hydrolysis of nucleoside triphosphate (NTP). Using a mutant monomeric βY341W subunit and a mutant α₃β₃γ subcomplex containing the βY341W mutation with or without an additional βE190Q mutation, we examined the binding of various NTPs (i.e., ATP, GTP, and ITP) and nucleoside diphosphates (NDPs, i.e., ADP, GDP, and IDP). The affinity (1/K_d) of the nucleotides for the isolated β subunit and third catalytic site in the subcomplex was in the order ATP/ADP > GTP/GDP > ITP/IDP. We performed van’t Hoff analyses to obtain the thermodynamic parameters of nucleotide binding. For the isolated β subunit, NDPs and NTPs with the same base moiety exhibited similar ΔH⁰ and ΔG⁰ values at 25°C. The binding of nucleotides with different bases to the isolated β subunit resulted in different entropy changes. Interestingly, NDP binding to the α₃β(Y341W)₃γ subcomplex had similar K_d and ΔG⁰ values as binding to the isolated β(Y341W) subunit, but the contributions of the enthalpy term and the entropy term were very different. We discuss these results in terms of the change in the tightness of the subunit packing, which reduces the excluded volume between subunits and increases water entropy.     

Mutation in the M1 Domain of the Acetylcholine Receptor α Subunit Decreases the Rate of Agonist Dissociation

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) α subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing αN217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC₅₀ to lower agonist concentrations for αN217K. The apparent affinity of ACh binding, measured by competition against the rate of ¹²⁵I-α-bungarotoxin binding, is also enhanced 20-fold by αN217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the β, ε, or δ subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.     

Influence of Hydroxyl Group Position and Temperature on Thermophysical Properties of Tetraalkylammonium Hydroxide Ionic Liquids with Alcohols

In this work, we have explored the thermophysical properties of tetraalkylammonium hydroxide ionic liquids (ILs) such as tetrapropylammonium hydroxide (TPAH) and tetrabutylammonium hydroxide (TBAH) with isomers of butanol (1-butanol, 2-butanol and 2-methyl-2-propanol) within the temperature range 293.15–313.15 K, with interval of 5 K and over the varied concentration range of ILs. The molecular interactions between ILs and butanol isomers are essential for understanding the function of ILs in related measures and excess functions are sensitive probe for the molecular interactions. Therefore, we calculated the excess molar volume (V^E) and the deviation in isentropic compressibility (Δκ_s) using the experimental values such as densities (ρ) and ultrasonic sound velocities (u) that are measured over the whole compositions range at five different temperatures (293.15, 298.15, 303.15, 308.15 and 313.15 K) and atmospheric pressure. These excess functions were adequately correlated by using the Redlich–Kister polynomial equation. It was observed that for all studied systems, the V^E and Δκ_s values are negative for the whole composition range at 293.15 K. And, the excess function follows the sequence: 2-butanol>1-butanol>2-methyl-2-propanol, which reveals that (primary or secondary or tertiary) position of hydroxyl group influence the magnitude of interactions with ILs. The negative values of excess functions are contributions from the ion-dipole interaction, hydrogen bonding and packing efficiency between the ILs and butanol isomers. Hence, the position of hydroxyl group plays an important role in the interactions with ILs. The hydrogen bonding features between ILs and alcohols were analysed using molecular modelling program by using HyperChem 7.     

Expression of non-neuronal cholinergic system in maxilla of rat in vivo

Background

Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, which is also produced in a variety of non-neuronal tissues and cell. The existence of ACh in maxilla in vivo and potential regulation role for osteogenesis need further study.

Results

Components of the cholinergic system (ACh, esterase, choline acetyltransferase, high-affinity choline uptake, n- and mAChRs) were determined in maxilla of rat in vivo, by means of Real-Time PCR and immunohistochemistry. Results showed RNA for CarAT, carnitine/acylcarnitine translocase member 20 (Slc25a20), VAChT, OCTN2, OCT1, OCT3, organic cation transporter member 4 (Slc22a4), AChE, BChE, nAChR subunits α1, α2, α3, α5, α7, α10, β1, β2, β4, γ and mAChR subunits M1, M2, M3, M4, M5 were detected in rat’s maxilla. RNA of VAChT, AChE, nAChR subunits α2, β1, β4 and mAChR subunits M4 had abundant expression (2^-ΔCt > 0.03). Immunohistochemical staining was conducted for ACh, VAChT, nAChRα7 and AChE. ACh was expressed in mesenchymal cells, chondroblast, bone and cartilage matrix and bone marrow cells, The VAChT expression was very extensively while ACh receptor α7 was strongly expressed in newly formed bone matrix of endochondral and bone marrow ossification, AchE was found only in mesenchymal stem cells, cartilage and bone marrow cells.

Conclusions

ACh might exert its effect on the endochondral and bone marrow ossification, and bone matrix mineralization in maxilla.

Electronic supplementary material

The online version of this article (doi:10.1186/0717-6287-47-72) contains supplementary material, which is available to authorized users.     

Single-channel analysis of the anion channel-forming protein from the plant pathogenic bacterium Clavibacter michiganense ssp. nebraskense

The anion channel protein from Clavibacter michiganense ssp. nebraskense (Schürholz, Th. et al. 1991, J. Membrane Biol. 123: 1-8) was analyzed at different concentrations of KCl and KF. At 0.8 M KCl the conductance G(V_m) increases exponentially from 21 pS at 50 mV up to 53 pS at V_m = 200 mV, 20°C. The concentration dependence of G(V_m) corresponds to a Michaelis-Menten type saturation function at all membrane voltage values applied (0-200 mV). The anion concentration K_0.5, where G(V_m) has its half-maximum value, increases from 0.12 M at 50 mV to 0.24 M at 175 mV for channels in a soybean phospholipid bilayer. The voltage dependence of the single channel conductance, which is different for charged and neutral lipid bilayers, can be described either by a two-state flicker (2SF) model and the Nernst-Planck continuum theory, or by a two barrier, one-site (2B1S) model with asymmetric barriers. The increase in the number of open channels after a voltage jump from 50 mV to 150 mV has a time constant of 0.8 s. The changes of the single-channel conductance are much faster (<1 ms). The electric part of the gating process is characterized by the (reversible) molar electrical work ΔG^θ_el = ρZ_gFV_m ≈ -1.3 RT, which corresponds to the movement of one charge of the gating charge number |Z_g| = 1 across the fraction ρ = ΔV_m/V_m = 0.15 of the membrane voltage V_m = 200 mV. Unlike with chloride, the single channel conductance of fluoride has a maximum at about 150 mV in the presence of the buffer PIPES (≥5 mM, pH 6.8) with K_0.5 ≈ 1 M. It is shown that the decrease in conductance is due to a blocking of the channel by the PIPES anion. In summary, the results indicate that the anion transport by the Clavibacter anion channel (CAC) does not require a voltage dependent conformation change of the CAC.     

Determination of the Equilibrium Constants of Associating Protein Systems: VII. Thermodynamic Analysis of Linear and Helical Association

The expression of free energy change for linear and helical association based on Casper's definition as applied to Oosawa and Kasai's model is described. Redefinition of the thermodynamic quantity ζ indicates that it is dependent on Δf_B, the free energy change in the formation of B-bonds, rather than mean distortion energy as previously believed.     

Nicotinic Acetylcholine Receptors Containing the α7-Like Subunit Mediate Contractions of Muscles Responsible for Space Positioning of the Snail,Helix pomatia L. Tentacle

Three recently discovered tentacle muscles are crucial to perform patterned movements of upper tentacles of the terrestrial snail, Helix pomatia. The muscles receive central and peripheral excitatory cholinergic innervation lacking inhibitory innervation. Here, we investigate the pharmacology of acetylcholine (ACh) responses in muscles to determine the properties of the ACh receptor (AChR), the functional availability of which was assessed using isotonic contraction measurement. Using broad spectrum of nicotinic and muscarinic ligands, we provide the evidence that contractions in the muscles are attributable to the activation of nAChRs that contain the α7-like subunit. Contractions could be evoked by nicotine, carbachol, succinylchloride, TMA, the selective α7-nAChR agonist choline chloride, 3-Bromocytisine and PNU-282987, and blocked by nAChR selective antagonists such as mytolon, hexamethonium, succinylchloride, d-tubocurarine, hemicholinium, DMDA (decamethonium), methyllycaconitine, α-Bungarotoxin (αBgTx) and α-Conotoxin IMI. The specific muscarinic agonist oxotremorine and arecoline failed to elicit contractions. Based on these pharmacological properties we conclude that the Na⁺ and Ca²⁺ permeable AChRs of the flexor muscle are nicotinic receptors that contain the α7-like subunit. Immunodetection experiments confirmed the presence of α7- or α7-like AChRs in muscle cells, and α4-AChRs in nerves innervating the muscle. These results support the conclusion that the slowly desensitizing αBgTx-sensitive responses obtained from flexor muscles are produced by activation of α7- like AChRs. This is the first demonstration of postsynaptic expression and an obligatory role for a functional α7-like nAChR in the molluscan periphery.     

Tryptophan 207 is crucial to the unique properties of the human voltage-gated proton channel,hHV1

Part of the “signature sequence” that defines the voltage-gated proton channel (H_V1) is a tryptophan residue adjacent to the second Arg in the S4 transmembrane helix: RxWRxxR, which is perfectly conserved in all high confidence H_V1 genes. Replacing Trp²⁰⁷ in human H_V1 (hH_V1) with Ala, Ser, or Phe facilitated gating, accelerating channel opening by 100-fold, and closing by 30-fold. Mutant channels opened at more negative voltages than wild-type (WT) channels, indicating that in WT channels, Trp favors a closed state. The Arrhenius activation energy, E_a, for channel opening decreased to 22 kcal/mol from 30–38 kcal/mol for WT, confirming that Trp²⁰⁷ establishes the major energy barrier between closed and open hH_V1. Cation–π interaction between Trp²⁰⁷ and Arg²¹¹ evidently latches the channel closed. Trp²⁰⁷ mutants lost proton selectivity at pH_o >8.0. Finally, gating that depends on the transmembrane pH gradient (ΔpH-dependent gating), a universal feature of H_V1 that is essential to its biological functions, was compromised. In the WT hH_V1, ΔpH-dependent gating is shown to saturate above pH_i or pH_o 8, consistent with a single pH sensor with alternating access to internal and external solutions. However, saturation occurred independently of ΔpH, indicating the existence of distinct internal and external pH sensors. In Trp²⁰⁷ mutants, ΔpH-dependent gating saturated at lower pH_o but not at lower pH_i. That Trp²⁰⁷ mutation selectively alters pH_o sensing further supports the existence of distinct internal and external pH sensors. Analogous mutations in H_V1 from the unicellular species Karlodinium veneficum and Emiliania huxleyi produced generally similar consequences. Saturation of ΔpH-dependent gating occurred at the same pH_o and pH_i in H_V1 of all three species, suggesting that the same or similar group(s) is involved in pH sensing. Therefore, Trp enables four characteristic properties: slow channel opening, highly temperature-dependent gating kinetics, proton selectivity, and ΔpH-dependent gating.     

Thermodynamic and hydration effects for the incorporation of a cationic 3-aminopropyl chain into DNA

The introduction of cationic 5-(ω-aminoalkyl)-2′-deoxypyrimidines into duplex DNA has been shown to induce DNA bending. In order to understand the energetic and hydration contributions for the incorporation of a cationic side chain in DNA a combination of spectroscopy, calorimetry and density techniques were used. Specifically, the temperature unfolding and isothermal formation was studied for a pair of duplexes with sequence d(CGTAGUCG TGC)/d(GCACGACTACG), where U represents 2′-deoxyuridine (‘control’) or 5-(3-aminopropyl)-2′-deoxyuridine (‘modified’). Continuous variation experiments confirmed 1:1 stoichiometries for each duplex and the circular dichroism spectra show that both duplexes adopted the B conformation. UV and differential scanning calorimetry melting experiments reveal that each duplex unfolds in two-state transitions. In low salt buffer, the ‘modified’ duplex is more stable and unfolds with a lower endothermic heat and lower release of counterion and water. This electrostatic stabilization is entropy driven and disappears at higher salt concentrations. Complete thermodynamic profiles at 15°C show that the favorable formation of each duplex results from the compensation of a favorable exothermic heat with an unfavorable entropy contribution. However, the isothermal profiles yielded a differential enthalpy of 8.8 kcal/mol, which is 4.3 kcal/mol higher than the differential enthalpy observed in the unfolding profiles. This indicates that the presence of the aminopropyl chain induces an increase in base stacking interactions in the modified single strand and a decrease in base stacking interactions in the modified duplex. Furthermore, the formation of the ‘control’ duplex releases water while the ‘modified’ duplex takes up water. Relative to the control duplex, formation of the modified duplex at 15°C yielded a marginal differential ΔG° term, positive ΔΔH_ITC–Δ(TΔS) compensation, negative ΔΔV and a net release of counterions. The opposite signs of the differential enthalpy–entropy compensation and differential volume change terms show a net uptake of structural water around polar and non-polar groups. This indicates that incorporation of the aminopropyl chain induces a higher exposure of aromatic bases to the solvent, which may be consistent with a small and local bend in the ‘modified’ duplex.     

Functional Dissection of the Nascent Polypeptide-Associated Complex in Saccharomyces cerevisiae

Both the yeast nascent polypeptide-associated complex (NAC) and the Hsp40/70-based chaperone system RAC-Ssb are systems tethered to the ribosome to assist cotranslational processes such as folding of nascent polypeptides. While loss of NAC does not cause phenotypic changes in yeast, the simultaneous deletion of genes coding for NAC and the chaperone Ssb (nacΔssbΔ) leads to strongly aggravated defects compared to cells lacking only Ssb, including impaired growth on plates containing L-canavanine or hygromycin B, aggregation of newly synthesized proteins and a reduced translational activity due to ribosome biogenesis defects. In this study, we dissected the functional properties of the individual NAC-subunits (α-NAC, β-NAC and β’-NAC) and of different NAC heterodimers found in yeast (αβ-NAC and αβ’-NAC) by analyzing their capability to complement the pleiotropic phenotype of nacΔssbΔ cells. We show that the abundant heterodimer αβ-NAC but not its paralogue αβ’-NAC is able to suppress all phenotypic defects of nacΔssbΔ cells including global protein aggregation as well as translation and growth deficiencies. This suggests that αβ-NAC and αβ’-NAC are functionally distinct from each other. The function of αβ-NAC strictly depends on its ribosome association and on its high level of expression. Expression of individual β-NAC, β’-NAC or α-NAC subunits as well as αβ’-NAC ameliorated protein aggregation in nacΔssbΔ cells to different extents while only β-NAC was able to restore growth defects suggesting chaperoning activities for β-NAC sufficient to decrease the sensitivity of nacΔssbΔ cells against L-canavanine or hygromycin B. Interestingly, deletion of the ubiquitin-associated (UBA)-domain of the α-NAC subunit strongly enhanced the aggregation preventing activity of αβ-NAC pointing to a negative regulatory role of this domain for the NAC chaperone activity in vivo.     

Contributions of Ca2+-Independent Thin Filament Activation to Cardiac Muscle Function

Although Ca²⁺ is the principal regulator of contraction in striated muscle, in vitro evidence suggests that some actin-myosin interaction is still possible even in its absence. Whether this Ca²⁺-independent activation (CIA) occurs under physiological conditions remains unclear, as does its potential impact on the function of intact cardiac muscle. The purpose of this study was to investigate CIA using computational analysis. We added a structurally motivated representation of this phenomenon to an existing myofilament model, which allowed predictions of CIA-dependent muscle behavior. We found that a certain amount of CIA was essential for the model to reproduce reported effects of nonfunctional troponin C on myofilament force generation. Consequently, those data enabled estimation of ΔG_CIA, the energy barrier for activating a thin filament regulatory unit in the absence of Ca²⁺. Using this estimate of ΔG_CIA as a point of reference (∼7 kJ mol⁻¹), we examined its impact on various aspects of muscle function through additional simulations. CIA decreased the Hill coefficient of steady-state force while increasing myofilament Ca²⁺ sensitivity. At the same time, CIA had minimal effect on the rate of force redevelopment after slack/restretch. Simulations of twitch tension show that the presence of CIA increases peak tension while profoundly delaying relaxation. We tested the model’s ability to represent perturbations to the Ca²⁺ regulatory mechanism by analyzing twitch records measured in transgenic mice expressing a cardiac troponin I mutation (R145G). The effects of the mutation on twitch dynamics were fully reproduced by a single parameter change, namely lowering ΔG_CIA by 2.3 kJ mol⁻¹ relative to its wild-type value. Our analyses suggest that CIA is present in cardiac muscle under normal conditions and that its modulation by gene mutations or other factors can alter both systolic and diastolic function.     

Thermodynamic criteria for high hit rate antisense oligonucleotide design

Antisense oligonucleotides are used for therapeutic applications and in functional genomic studies. In practice, however, many of the oligonucleotides complementary to an mRNA have little or no antisense activity. Theoretical strategies to improve the ‘hit rate’ in antisense screens will reduce the cost of discovery and may lead to identification of antisense oligonucleotides with increased potency. Statistical analysis performed on data collected from more than 1000 experiments with phosphorothioate-modified oligonucleotides revealed that the oligo-probes, which form stable duplexes with RNA (ΔG^o₃₇ ≤ –30 kcal/mol) and have small self-interaction potential, are more frequently efficient than molecules that form less stable oligonucleotide–RNA hybrids or more stable self-structures. To achieve optimal statistical preference, the values for self-interaction should be (ΔG^o₃₇) ≥ –8 kcal/mol for inter-oligonucleotide pairing and (ΔG^o₃₇) ≥ –1.1 kcal/mol for intra-molecular pairing. Selection of oligonucleotides with these thermodynamic values in the analyzed experiments would have increased the ‘hit rate’ by as much as 6-fold.     

Electrogenic transport of protons driven by the plasma membrane ATPase in membrane vesicles from radish : biochemical characterization

Mg:ATP-dependent H⁺ pumping has been studied in microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO₃⁻. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H⁺-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO₄²⁻, Cl⁻, Br⁻, NO₃⁻. ATP-dependent H⁺ pumping strictly requires Mg²⁺. It is very specific for ATP (apparent K_m 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO₄ and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K⁺ (+ 80%) and to a lesser extent by Na⁺ and choline (+28% and +14%, respectively). The characteristics of H⁺ pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials.