Biophysical journal
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Cys-loop receptors are molecular targets of general anesthetics, but the knowledge of anesthetic binding to these proteins remains limited. Here we investigate anesthetic binding to the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), a structural homolog of cys-loop receptors, using an experimental and computational hybrid approach. Tryptophan fluorescence quenching experiments showed halothane and thiopental binding at three tryptophan-associated sites in the extracellular (EC) domain, transmembrane (TM) domain, and EC-TM interface of GLIC. ⋯ Tryptophans were within reach of both intra- and intersubunit binding sites. Multiple molecular dynamics simulations on GLIC in the presence of halothane at different sites suggested that anesthetic binding at the EC-TM interface disrupted the critical interactions for channel gating, altered motion of the TM23 linker, and destabilized the open-channel conformation that can lead to inhibition of GLIC channel current. The study has not only provided insights into anesthetic binding in GLIC, but also demonstrated a successful fusion of experiments and computations for understanding anesthetic actions in complex proteins.
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Biophysical journal · Aug 2010
Mitochondrial free [Ca2+] increases during ATP/ADP antiport and ADP phosphorylation: exploration of mechanisms.
ADP influx and ADP phosphorylation may alter mitochondrial free [Ca2+] ([Ca2+](m)) and consequently mitochondrial bioenergetics by several postulated mechanisms. We tested how [Ca2+](m) is affected by H2PO4(-) (P(i)), Mg2+, calcium uniporter activity, matrix volume changes, and the bioenergetic state. We measured [Ca2+](m), membrane potential, redox state, matrix volume, pH(m), and O2 consumption in guinea pig heart mitochondria with or without ruthenium red, carboxyatractyloside, or oligomycin, and at several levels of Mg2+ and P(i). ⋯ Carboxyatractyloside prevented the ADP-induced [Ca2+](m) increase. Adding CaCl2 had no effect on bioenergetics, except for a small increase in state 2 and state 4 respiration at 485 nM [Ca2+](e). These data suggest that matrix ADP influx and subsequent phosphorylation increase [Ca2+](m) largely due to the interaction of matrix Ca2+ with ATP, ADP, P(i), and cation buffering proteins in the matrix.
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Biophysical journal · Mar 2010
Monte Carlo simulation of buffered diffusion into and out of a model synapse.
Buffered diffusion occurs when ligands enter or leave a restricted space, such as a chemical synapse, containing a high density of binding sites. This study used Monte Carlo simulations to determine the time and spatial dependences of buffered diffusion without a priori assumptions about kinetics. The synapse was modeled as a box with receptors on one inner face. ⋯ Simulations with irreversible ligands showed larger, persistent spatial gradients. These simulations identify characteristics that can be used to test whether a synaptic process is governed by buffered diffusion. They also indicate that fundamental differences in synapse function may occur with irreversible ligands.
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Kinesin is a dimeric motor with twin catalytic heads joined to a common stalk. Kinesin molecules move processively along microtubules in a hand-over-hand walk, with the two heads advancing alternately. Recombinant kinesin constructs with short stalks have been found to "limp", i.e., exhibit alternation in the dwell times of successive steps. ⋯ Limping was equally unaffected by mutations that produced 50-fold changes in stalk stiffness, ruling out models where limping arises from an asymmetry in torsional strain. However, limping was enhanced by perturbations that increased the vertical component of load on the motor, including increases in bead size or net load, and decreases in the stalk length. These results suggest that kinesin heads take different vertical trajectories during alternate steps, and that the rates for these motions are differentially sensitive to load.
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Biophysical journal · Oct 2008
In vivo mitochondrial oxygen tension measured by a delayed fluorescence lifetime technique.
Mitochondrial oxygen tension (mitoPO(2)) is a key parameter for cellular function, which is considered to be affected under various pathophysiological circumstances. Although many techniques for assessing in vivo oxygenation are available, no technique for measuring mitoPO(2) in vivo exists. Here we report in vivo measurement of mitoPO(2) and the recovery of mitoPO(2) histograms in rat liver by a novel optical technique under normal and pathological circumstances. ⋯ The results demonstrate mitoPO(2) values of approximately 30-40 mmHg. mitoPO(2) was highly sensitive to small changes in inspired oxygen concentration around atmospheric oxygen level. Ischemia-reperfusion interventions showed altered mitoPO(2) distribution, which flattened overall compared to baseline conditions. The reported technology is scalable from microscopic to macroscopic applications, and its reliance on an endogenous compound greatly enhances its potential field of applications.