Rates and equilibria at the acetylcholine receptor of electrophorus electroplaques. A study of neurally evoked postsynaptic currents and of voltage-jump relaxations

Kinetic measurements are employed to reconstruct the steady-state activation of acetylcholine [Ach] receptor channels in electrophorus electroplaques. Neurally evoked postsynaptic currents (PSCs) decay exponentially; at 15°C the rate constant, α, equals 1.2 ms^(-1) at 0 mV and decreases e-fold for every 86 mV as the membrane voltage is made more negative. Voltage-jump relaxations have been measured with bath-applied ACh, decamethonium, carbachol, or suberylcholine. We interpret the reciprocal relaxation time 1/τ as the sum of the rate constant α for channel closing and a first-order rate constant for channel opening. Where measureable, the opening rate increases linearly with [agonist] and does not vary with voltage. The voltage sensitivity of small steady-state conductances (e- fold for 86 mV) equals that of the closing rate α, confirming that the opening rate has little or no additional voltage sensitivity. Exposure to α-bungarotoxin irreversibly decreases the agonist-induced conductance but does not affect the relaxation kinetics. Tubocurarine reversibly reduces both the conductance and the opening rate. In the simultaneous presence of two agonist species, voltage-jump relaxations have at least two exponential components. The data are fit by a model in which (a) the channel opens as the receptor binds the second in a sequence of two agonist molecules, with a forward rate constant to 10^(7) to 2x10^(8) M^(-1)s^(-1); and (b) the channel then closes as either agonist molecule dissociates, with a voltage-dependent rate constant of 10^(2) to 3x10^(3)s^(-1)

Functional Stoichiometry at the Nicotinic Receptor. The Photon Cross Section for Phase 1 Corresponds to Two Bis-Q Molecules per Channel

These experiments examine changes in the agonist-induced conductance that occur when the agonist-receptor complex is perturbed. Voltage-clamped Electrophorus electroplaques are exposed to the photoisomerizable agonist trans-Bis-Q A 1-µs laser flash photoisomerizes some trans-Bis-Q molecules bound to receptors; because the cis configuration is not an agonist, receptor channels close within a few hundred microseconds. This effect is called phase 1. We compare (a) the fraction of channels that close during phase 1 with (b) the fraction of trans-Bis-Q molecules that undergo trans → cis photoisomerization. Parameter a is measured as the fractional diminution in voltage-clamp currents during phase 1. Parameter b is measured by changes in the optical spectra of Bis-Q solutions caused by flashes . At low flash intensities, a is twice b, which shows that the channel can be closed by photoisomerizing either of two bound agonist molecules. Conventional dose-response studies with trans-Bis-Q also give a Hill coefficient of two. As a partial control for changes in the photochemistry caused by binding of Bis-Q to receptors, spectral measurements are performed on the photoisomerizable agonist QBr, covalently bound to solubilized acetylcholine receptors from Torpedo. The bound and free agonist molecules have the same photoisomerization properties. These results verify the concept that the open state of the acetylcholine receptor channel is much more likely to be associated with the presence of two bound agonist molecules than with a single such molecule

Comparing and contrasting Escherichia coli and Mycobacterium tuberculosis mechanosensitive channels (MscL) - New gain of function mutations in the loop region

Sequence analysis of 35 putative MscL homologues was used to develop an optimal alignment for Escherichia coli and Mycobacterium tuberculosis MscL and to place these homologues into sequence subfamilies. By using this alignment, previously identified E. coli MscL mutants that displayed severe and very severe gain of function phenotypes were mapped onto the M. tuberculosis MscL sequence. Not all of the resulting M. tuberculosis mutants displayed a gain of function phenotype; for instance, normal phenotypes were noted for mutations at Ala20, the analogue of the highly sensitive Gly22 site in E. coli. A previously unnoticed intersubunit hydrogen bond in the extracellular loop region of the M. tuberculosis MscL crystal structure has been analyzed. Cross-linkable residues were substituted for the residues involved in the hydrogen bond, and cross-linking studies indicated that these sites are spatially close under physiological conditions. In general, mutation at these positions results in a gain of function phenotype, which provides strong evidence for the importance of the loop region in MscL channel function. No analogue to this interesting interaction could be found in E. coli MscL by sequence alignment. Taken together, these results indicate that caution should be exercised in using the M. tuberculosis MscL crystal structure to analyze previous functional studies of E. coli MscL

Conductance increases produced by bath application of cholinergic agonists to Electrophorus electroplaques

When solutions containing agonists are applied to the innervated face of an Electrophorus electroplaque, the membrane's conductance increases. The agonist-induced conductance is increased at more negative membrane potentials. The "instantaneous" current-voltage curve for agonist-induced currents is linear and shows a reversal potential near zero mV; chord conductances, calculated on the basis of this reversal potential, change epsilon-fold for every 62-mV change in potential when the conductance is small. Conductance depends non- linearly on small agonist concentrations; at all potentials, the dose-response curve has a Hill coefficient of 1.45 for decamethonium (Deca) and 1.90 for carbamylcholine (Carb). With agonist concentrations greater than 10^(-4) M Carb or 10^(-5) M Deca, the conductance rises to a peak 0.5-1.5 min after introduction of agonist, then declines with time; this effect resembles the "desensitization" reported for myoneural junctions. Elapid alpha-toxin, tubocurarine, and desensitization reduce the conductance without changing the effects of potential; the apparent dissociation constant for tubocurarine is 2 X 10^(-7) M. By contrast, procaine effects a greater fractional inhibition of the conductance at high negative potentials

Late diagenetic indicators of buried oil and gas. 2: Direct detection experiment at Cement and Garza fields, Oklahoma and Texas, using enhanced LANDSAT 1 and 2 images

The author has identified the following significant results. The Cement oil field, Oklahoma, was a test site for an experiment designed to evaluate LANDSAT's capability to detect an alteration zone in surface rocks caused by hydrocarbon microseepage. Loss of iron and impregnation of sandstone by carbonate cements and replacement of gypsum by calcite were the major alteration phenomena at Cement. The bedrock alterations were partially masked by unaltered overlying beds, thick soils, and dense natural and cultivated vegetation. Interpreters, biased by detailed ground truth, were able to map the alteration zone subjectively using a magnified, filtered, and sinusoidally stretched LANDSAT composite image; other interpreters, unbiased by ground truth data, could not duplicate that interpretation

Requirement of the FATC domain of protein kinase Tel1 for localization to DNA ends and target protein recognition

Two large phosphatidylinositol 3-kinase-related protein kinases (PIKKs), ATM and ATR, play a central role in the DNA damage response pathway. PIKKs contain a highly conserved extreme C-terminus called the FRAP-ATM-TRRAP-C-terminal (FATC) domain. In budding yeast, ATM and ATR correspond to Tel1 and Mec1, respectively. In this study, we characterized functions of the FATC domain of Tel1 by introducing substitution or truncation mutations. One substitution mutation, termed tel1-21, and a truncation mutation, called tel1- Î"C, did not significantly affect the expression level. The tel1-21 mutation impaired the cellular response to DNA damage and conferred moderate telomere maintenance defect. In contrast, the tel1-Î"C mutation behaved like a null mutation, conferring defects in both DNA damage response and telomere maintenance. Tel1-21 protein localized to DNA ends as effectively as wild-type Tel1 protein, whereas Tel1-Î"C protein failed. Introduction of a hyperactive TEL1-hy mutation suppressed the tel1-21 mutation but not the tel1-Î"C mutation. In vitro analyses revealed that both Tel1-21 and Tel1-Î"C proteins undergo efficient autophosphorylation but exhibit decreased kinase activities toward the exogenous substrate protein, Rad53. Our results show that the FATC domain of Tel1 mediates localization to DNA ends and contributes to phosphorylation of target proteins. © 2015 Ogi, Goto, Ghosh, et al

The narrative coherence of witness transcripts in children on the autism spectrum

Background and Aims. Autistic children often recall fewer details about witnessed events than typically developing children (of comparable age and ability), although the information they recall is generally no less accurate. Previous research has not examined the narrative coherence of such accounts, despite higher quality narratives potentially being perceived more favourably by criminal justice professionals and juries. This study compared the narrative coherence of witness transcripts produced by autistic and typically developing (TD) children (ages 6-11 years, IQs 70+). Methods and Procedures. Secondary analysis was carried out on interview transcripts from a subset of 104 participants (autism=52, TD=52) who had taken part in a larger study of eyewitness skills in autistic and TD children. Groups were matched on chronological age, IQ and receptive language ability. Coding frameworks were adopted from existing narrative research, featuring elements of ‘story grammar’. Outcomes and Results. Whilst fewer event details were reported by autistic children, there were no group differences in narrative coherence (number and diversity of ‘story grammar’ elements used), narrative length or semantic diversity. Conclusions and Implications. These findings suggest that the narrative coherence of autistic children’s witness accounts is equivalent to TD peers of comparable age and ability

Rates and Equilibria for a Photoisomerizable Antagonist at the Acetylcholine Receptor of Electrophorus Electroplaques

Voltage-jump and light-flash experiments have been performed on isolated Electrophorus electroplaques exposed simultaneously to nicotinic agonists and to the photoisomerizable compound 2,2'-bis-[α-(trimethylammonium)methyl]-azobenzene (2BQ). Dose-response curves are shifted to the right in a nearly parallel fashion by 2BQ, which suggests competitive antagonism; dose-ratio analyses show apparent dissociation constants of 0.3 and 1 µM for the cis and trans isomers, respectively. Flash-induced trans → cis concentration jumps produce the expected decrease in agonist-induced conductance; the time constant is several tens of milliseconds. From the concentration dependence of these rates, we conclude that the association and dissociation rate constants for the cis-2BQ-receptor binding are approximately ~ 10^8 M^(-1) s^(-1) and 60 s^(-1) at 20ºC; the Q_(10) is 3. Flash-induced cis → trans photoisomerizations produce molecular rearrangements of the ligand-receptor complex, but the resulting relaxations probably reflect the kinetics of buffered diffusion rather than of the interaction between trans-2BQ and the receptor. Antagonists seem to bind about an order of magnitude more slowly than agonists at nicotinic receptors

Oxidation = group theory

Dimensional reduction of theories involving (super-)gravity gives rise to sigma models on coset spaces of the form G/H, with G a non-compact group, and H its maximal compact subgroup. The reverse process, called oxidation, is the reconstruction of the possible higher dimensional theories, given the lower dimensional theory. In 3 dimensions, all degrees of freedom can be dualized to scalars. Given the group G for a 3 dimensional sigma model on the coset G/H, we demonstrate an efficient method for recovering the higher dimensional theories, essentially by decomposition into subgroups. The equations of motion, Bianchi identities, Kaluza-Klein modifications and Chern-Simons terms are easily extracted from the root lattice of the group G. We briefly discuss some aspects of oxidation from the E_{8(8)}/SO(16) coset, and demonstrate that our formalism reproduces the Chern-Simons term of 11-d supergravity, knows about the T-duality of IIA and IIB theory, and easily deals with self-dual tensors, like the 5-tensor of IIB supergravity.Comment: LaTeX, 8 pages, uses IOP style files; Talk given at the RTN workshop ``The quantum structure of spacetime and the geometric nature of fundamental interactions'', Leuven, September 200