Tyrosine decaging leads to substantial membrane trafficking during modulation of an inward rectifier potassium channel

Tyrosine side chains participate in several distinct signaling pathways, including phosphorylation and membrane trafficking. A nonsense suppression procedure was used to incorporate a caged tyrosine residue in place of the natural tyrosine at position 242 of the inward rectifier channel Kir2.1 expressed in Xenopus oocytes. When tyrosine kinases were active, flash decaging led both to decreased K+ currents and also to substantial (15–26%) decreases in capacitance, implying net membrane endocytosis. A dominant negative dynamin mutant completely blocked the decaging-induced endocytosis and partially blocked the decaging-induced K+ channel inhibition. Thus, decaging of a single tyrosine residue in a single species of membrane protein leads to massive clathrin-mediated endocytosis; in fact, membrane area equivalent to many clathrin-coated vesicles is withdrawn from the oocyte surface for each Kir2.1 channel inhibited. Oocyte membrane proteins were also labeled with the thiol-reactive fluorophore tetramethylrhodamine-5-maleimide, and manipulations that decreased capacitance also decreased surface membrane fluorescence, confirming the net endocytosis. In single-channel studies, tyrosine kinase activation decreased the membrane density of active Kir2.1 channels per patch but did not change channel conductance or open probability, in agreement with the hypothesis that tyrosine phosphorylation results in endocytosis of Kir2.1 channels. Despite the Kir2.1 inhibition and endocytosis stimulated by tyrosine kinase activation, neither Western blotting nor 32P labeling produced evidence for direct tyrosine phosphorylation of Kir2.1. Therefore, it is likely that tyrosine phosphorylation affects Kir2.1 function indirectly, via interactions between clathrin adaptor proteins and a tyrosine-based sorting motif on Kir2.1 that is revealed by decaging the tyrosine side chain. These interactions inhibit a fraction of the Kir2.1 channels, possibly via direct occlusion of the conduction pathway, and also lead to endocytosis, which further decreases Kir2.1 currents. These data establish that side chain decaging can provide valuable time-resolved data about intracellular signaling systems

Gating Transitions in Bacterial Ion Channels Measured at 3 µs Resolution

Ion channels of high conductance (>200 pS) are widespread among prokaryotes and eukaryotes. Two examples, the Escherichia coli mechanosensitive ion channels Ec-MscS and Ec-MscL, pass currents of 125–300 pA. To resolve temporal details of conductance transitions, a patch-clamp setup was optimized for low-noise recordings at a time resolution of 3 µs (10–20 times faster than usual). Analyses of the high-resolution recordings confirm that Ec-MscL visits many subconductance states and show that most of the intersubstate transitions occur more slowly than the effective resolution of 3 µs. There is a clear trend toward longer transition times for the larger transitions. In Ec-MscS recordings, the majority of the observed full conductance transitions are also composite. We detected a short-lived (~20 µs) Ec-MscS substate at 2/3 of full conductance; transitions between 2/3 and full conductance did not show fine structure and had a time course limited by the achieved resolution. Opening and closing transitions in MscS are symmetrical and are not preceded or followed by smaller, rapid currents ("anticipations" or "regrets"). Compared with other, lower-conductance channels, these measurements may detect unusually early states in the transitions from fully closed to fully open. Increased temporal resolution at the single-molecule level reveals that some elementary steps of structural transitions are composite and follow several alternative pathways, while others still escape resolution. High-bandwidth, low-noise single-channel measurements may provide details about state transitions in other high-conductance channels; and similar procedures may also be applied to channel- and nanopore-based single-molecule DNA measurements

EQUIPT: protocol of a comparative effectiveness research study evaluating cross-context transferability of economic evidence on tobacco control

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial.This article has been made available through the Brunel Open Access Publishing Fund.Tobacco smoking claims 700 000 lives every year in Europe and the cost of tobacco smoking in the EU is estimated between €98 and €130 billion annually; direct medical care costs and indirect costs such as workday losses each represent half of this amount. Policymakers all across Europe are in need of bespoke information on the economic and wider returns of investing in evidence-based tobacco control, including smoking cessation agendas. EQUIPT is designed to test the transferability of one such economic evidence base-the English Tobacco Return on Investment (ROI) tool-to other EU member states

Proceedings, Pot Chrysanthemum School, 1971

Space management / Robert W. Langhans -- Soils / D. C. Kiplinger -- Nutrition / George L. Staby -- Temperature and photoperiod / Joseph W. Love -- Automated short day control -- R. A. Aldrich -- Growth regulators / James B. Shanks -- Programming for insect-free pot mums / Richard K. Lindquist -- Programming for disease-free pot mums / Lester P. Nichols and Paul E. Nelson -- Where you go wrong / Harry K. Tayam

The global oscillation network group site survey. II. Results

The Global Oscillation Network Group (GONG) Project will place a network of instruments around the world to observe solar oscillations as continuously as possible for three years. The Project has now chosen the six network sites based on analysis of survey data from fifteen sites around the world. The chosen sites are: Big Bear Solar Observatory, California; Mauna Loa Solar Observatory, Hawaii; Learmonth Solar Observatory, Australia; Udaipur Solar Observatory, India; Observatorio del Teide, Tenerife; and Cerro Tololo Interamerican Observatory, Chile. Total solar intensity at each site yields information on local cloud cover, extinction coefficient, and transparency fluctuations. In addition, the performance of 192 reasonable components analysis. An accompanying paper describes the analysis methods in detail; here we present the results of both the network and individual site analyses. The selected network has a duty cycle of 93.3%, in good agreement with numerical simulations. The power spectrum of the network observing window shows a first diurnal sidelobe height of 3 × 10⁻⁴ with respect to the central component, an improvement of a factor of 1300 over a single site. The background level of the network spectrum is lower by a factor of 50 compared to a single-site spectrum