Saturation and Microsecond Gating of Current Indicate Depletion-induced Instability of the MaxiK Selectivity Filter

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed with a high temporal resolution (50-kHz filter) in symmetrical solutions with 50, 150, or 400 mM KCl and 2.5 mM CaCl2 and 2.5 mM MgCl2. At membrane potentials >+100 mV, the single-channel current showed a negative slope resistance, concomitantly with a flickery block, which was not influenced by Ca2+ or Mg2+. The analysis of the amplitude histograms by beta distributions revealed that current in this voltage range was reduced by two effects: rate limitation at the cytosolic side of the pore and gating with rate constants 10–20-fold higher than the cutoff frequency of the filter (i.e., dwell times in the microsecond range). The data were analyzed in terms of a model that postulates a coupling between both effects; if the voltage over the selectivity filter withdraws ions from the cavity at a higher rate than that of refilling from the cytosol, the selectivity filter becomes instable because of ion depletion, and current is interrupted by the resulting flickering. The fit of the IV curves revealed a characteristic voltage of 35 mV. In contrast, the voltage dependence of the gating factor R, i.e., the ratio between true and apparent single-channel current, could be fitted by exponentials with a characteristic voltage of 60 mV, suggesting that only part of the transmembrane potential is felt by the flux through the selectivity filter

The Wooster Voice (Wooster, OH), 1997-10-16

This edition of the College of Wooster student run newspaper was published on October 16 of 1997 and it is twelve pages long. Jilted surgeon general calls for more health education, Dr. Jocelyn Elders presents during the Wooster Forum series about health care and education. Ebert to be dedicated this weekend, the Ebert Art Center is going to be formally dedicated. Renowned musical trio combines classical with new, the Cleveland Duo and saxophonist James Umble perform at the Gault Recital Hall. Athletic updates for the past week are highlighted on pages ten to twelve.https://openworks.wooster.edu/voice1991-2000/1177/thumbnail.jp

Asymmetric Interplay Between K+ and Blocker and Atomistic Parameters From Physiological Experiments Quantify K+ Channel Blocker Release

Modulating the activity of ion channels by blockers yields information on both the mode of drug action and on the biophysics of ion transport. Here we investigate the interplay between ions in the selectivity filter (SF) of K+ channels and the release kinetics of the blocker tetrapropylammonium in the model channel KcvNTS. A quantitative expression calculates blocker release rate constants directly from voltage-dependent ion occupation probabilities in the SF. The latter are obtained by a kinetic model of single-channel currents recorded in the absence of the blocker. The resulting model contains only two adjustable parameters of ion-blocker interaction and holds for both symmetric and asymmetric ionic conditions. This data-derived model is corroborated by 3D reference interaction site model (3D RISM) calculations on several model systems, which show that the K+ occupation probability is unaffected by the blocker, a direct consequence of the strength of the ion-carbonyl attraction in the SF, independent of the specific protein background. Hence, KcvNTS channel blocker release kinetics can be reduced to a small number of system-specific parameters. The pore-independent asymmetric interplay between K+ and blocker ions potentially allows for generalizing these results to similar potassium channels

Influence of Guideline Operationalization on Youth Activity Prevalence in the International Children's Accelerometry Database

Introduction The United Kingdom and World Health Organization recently changed their youth physical activity (PA) guidelines from 60 min of moderate- to vigorous-intensity PA (MVPA) every day, to an average of 60 min of MVPA per day, over a week. The changes are based on expert opinion due to insufficient evidence comparing health outcomes associated with different guideline definitions. This study used the International Children’s Accelerometry Database to compare approaches to calculating youth PA compliance and associations with health indicators. Methods Cross-sectional accelerometer data (n = 21,612, 5–18 yr) were used to examine compliance with four guideline definitions: daily method (DM; ≥60 min MVPA every day), average method (AM; average of ≥60 min MVPA per day), AM5 (AM compliance and ≥5 min of vigorous PA [VPA] on ≥3 d), and AM15 (AM compliance and ≥15 min VPA on ≥3 d). Associations between compliance and health indicators were examined for all definitions. Results Compliance varied from 5.3% (DM) to 29.9% (AM). Associations between compliance and health indicators were similar for AM, AM5, and AM15. For example, compliance with AM, AM5, and AM15 was associated with a lower BMI z-score (statistics are coefficient [95% CI]): AM (−0.28 [−0.33 to −0.23]), AM5 (−0.28 [−0.33 to −0.23], and AM15 (−0.30 [−0.35 to −0.25]). Associations between compliance and health indicators for DM were similar/weaker, possibly reflecting fewer DM-compliant participants with health data and lower variability in exposure/outcome data. Conclusions Youth completing 60 min of MVPA every day do not experience superior health benefits to youth completing an average of 60 min of MVPA per day. Guidelines should encourage youth to achieve an average of 60 min of MVPA per day. Different guideline definitions affect inactivity prevalence estimates; this must be considered when analyzing data and comparing studies.publishedVersionPaid open acces

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

Functional analysis of Pro-inflammatory properties within the cerebrospinal fluid after subarachnoid hemorrhage in vivo and in vitro

<p>Abstract</p> <p>Background</p> <p>To functionally characterize pro-inflammatory and vasoconstrictive properties of cerebrospinal fluid after aneurysmal subarachnoid hemorrhage (SAH) in vivo and in vitro.</p> <p>Methods</p> <p>The cerebrospinal fluid (CSF) of 10 patients suffering from SAH was applied to the transparent skinfold chamber model in male NMRI mice which allows for in vivo analysis of the microcirculatory response to a superfusat. Microvascular diameter changes were quantified and the numbers of rolling and sticking leukocytes were documented using intravital multifluorescence imaging techniques. Furthermore, the pro-inflammatory properties of CSF were assessed in vitro using a monocyte transendothelial migration assay.</p> <p>Results</p> <p>CSF superfusion started to induce significant vasoconstriction on days 4 and 6 after SAH. In parallel, CSF superfusion induced a microvascular leukocyte recruitment, with a significant number of leukocytes rolling (day 6) and sticking (days 2-4) to the endothelium. CSF of patients presenting with cerebral edema induced breakdown of blood vessel integrity in our assay as evidenced by fluorescent marker extravasation. In accordance with leukocyte activation in vivo, significantly higher in vitro monocyte migration rates were found after SAH.</p> <p>Conclusion</p> <p>We functionally characterized inflammatory and vasoactive properties of patients' CSF after SAH in vivo and in vitro. This pro-inflammatory milieu in the subarachnoid space might play a pivotal role in the pathophysiology of early and delayed brain injury as well as vasospasm development following SAH.</p

Device-measured physical activity, adiposity and mortality: a harmonised meta-analysis of eight prospective cohort studies

Background: The joint associations of total and intensity-specific physical activity with obesity in relation to all-cause mortality risk are unclear. Methods: We included 34 492 adults (72% women, median age 62.1 years, 2034 deaths during follow-up) in a harmonised meta-analysis of eight population-based prospective cohort studies with mean follow-up ranging from 6.0 to 14.5 years. Standard body mass index categories were cross-classified with sample tertiles of device-measured total, light-to-vigorous and moderate-to-vigorous physical activity and sedentary time. In five cohorts with waist circumference available, high and low waist circumference was combined with tertiles of moderate-to-vigorous physical activity. Results: There was an inverse dose–response relationship between higher levels of total and intensity-specific physical activity and mortality risk in those who were normal weight and overweight. In individuals with obesity, the inverse dose–response relationship was only observed for total physical activity. Similarly, lower levels of sedentary time were associated with lower mortality risk in normal weight and overweight individuals but there was no association between sedentary time and risk of mortality in those who were obese. Compared with the obese-low total physical activity reference, the HRs were 0.59 (95% CI 0.44 to 0.79) for normal weight-high total activity and 0.67 (95% CI 0.48 to 0.94) for obese-high total activity. In contrast, normal weight-low total physical activity was associated with a higher risk of mortality compared with the obese-low total physical activity reference (1.28; 95% CI 0.99 to 1.67). Conclusions: Higher levels of physical activity were associated with lower risk of mortality irrespective of weight status. Compared with obesity-low physical activity, there was no survival benefit of being normal weight if physical activity levels were low.publishedVersio