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

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

A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction

BACKGROUND: The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. RESULTS: We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, α(s). All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. CONCLUSION: This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs

MSAViewer:interactive JavaScript visualization of multiple sequence alignments

Summary: The MSAViewer is a quick and easy visualization and analysis JavaScript component for Multiple Sequence Alignment data of any size. Core features include interactive navigation through the alignment, application of popular color schemes, sorting, selecting and filtering. The MSAViewer is ‘web ready’: written entirely in JavaScript, compatible with modern web browsers and does not require any specialized software. The MSAViewer is part of the BioJS collection of components. Availability and Implementation: The MSAViewer is released as open source software under the Boost Software License 1.0. Documentation, source code and the viewer are available at http://msa.biojs.net/. Supplementary information: Supplementary data are available at Bioinformatics online. Contact: [email protected]

Immunocytochemical localization of glutamate immunoreactivity in the guinea pig cochlea

The localization of glutamate immunoreactivity was examined in the guinea pig cochlea using affinity purified polyclonal antibodies to glutamate and immunoperoxidase post-embedding staining techniques on one micron plastic sections. Glutamate immunoreactive staining was seen in both inner and outer hair cells and in spiral ganglion cells and auditory nerve fibers. These results support the hypothesis that glutamate may function as the hair cell transmitter or as a precursor to the transmitter and add further support for an excitatory amino acid as the transmitter of the auditory nerve.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27697/1/0000083.pd

Cigarette Smoke-induced Ca 2+ Release Leads to Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Dysfunction

Chronic obstructive pulmonary disease affects 64 million people and is currently the fourth leading cause of death worldwide. Chronic obstructive pulmonary disease includes both emphysema and chronic bronchitis, and in the case of chronic bronchitis represents an inflammatory response of the airways that is associated with mucus hypersecretion and obstruction of small airways. Recently, it has emerged that exposure to cigarette smoke (CS) leads to an inhibition of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel, causing airway surface liquid dehydration, which may play a role in the development of chronic bronchitis. CS rapidly clears CFTR from the plasma membrane and causes it to be deposited into aggresome-like compartments. However, little is known about the mechanism(s) responsible for the internalization of CFTR following CS exposure. Our studies revealed that CS triggered a rise in cytoplasmic Ca2+ that may have emanated from lysosomes. Furthermore, chelation of cytoplasmic Ca2+, but not inhibition of protein kinases/phosphatases, prevented CS-induced CFTR internalization. The macrolide antibiotic bafilomycin A1 inhibited CS-induced Ca2+ release and prevented CFTR clearance from the plasma membrane, further linking cytoplasmic Ca2+ and CFTR internalization. We hypothesize that CS-induced Ca2+ release prevents normal sorting/degradation of CFTR and causes internalized CFTR to reroute to aggresomes. Our data provide mechanistic insight into the potentially deleterious effects of CS on airway epithelia and outline a hitherto unrecognized signaling event triggered by CS that may affect the long term transition of the lung into a hyper-inflammatory/dehydrated environment

Characterization of the Oligomeric Structure of the Ca 2+ -activated Cl − Channel Ano1/TMEM16A

Members of the Anoctamin (Ano)/TMEM16A family have recently been identified as essential subunits of the Ca2+-activated chloride channel (CaCC). For example, Ano1 is highly expressed in multiple tissues including airway epithelia, where it acts as an apical conduit for transepithelial Cl− secretion and helps regulate lung liquid homeostasis and mucus clearance. However, little is known about the oligomerization of this protein in the plasma membrane. Thus, utilizing mCherry- and eGFP-tagged Ano1 constructs, we conducted biochemical and Förster resonance energy transfer (FRET)-based experiments to determine the quaternary structure of Ano1. FRET and co-immunoprecipitation studies revealed that tagged Ano1 subunits directly associated before they reached the plasma membrane. This association was not altered by changes in cytosolic Ca2+, suggesting that this is a fixed interaction. To determine the oligomeric structure of Ano1, we performed chemical cross-linking, non-denaturing PAGE, and electromobility shift assays, which revealed that Ano1 exists as a dimer. These data are the first to probe the quaternary structure of Ano1. Understanding the oligomeric nature of Ano1 is an essential step in the development of therapeutic drugs that could be useful in the treatment of cystic fibrosis

PCR versus Hybridization for Detecting Virulence Genes of Enterohemorrhagic Escherichia coli

We compared PCR amplification of 9 enterohemorrhagic Escherichia coli virulence factors among 40 isolates (21 O/H antigenicity classes) with DNA hybridization. Both methods showed 100% of the chromosomal and phage genes: eae, stx, and stx2. PCR did not detect 4%–20% of hybridizable plasmid genes: hlyA, katP, espP, toxB, open reading frame (ORF) 1, and ORF2