Kinetic Model of Adsorption of Aqueous Solutes onto Lipid Bilayers: Modulation of the Activity of Membrane Proteins.

It has been argued that membrane proteins that are activated by agonist binding and whose activity depends on conformational transitions are sensitive to membrane adsorption of agonists as well as other solutes such as anesthetics. Ligand-gated ion channels such as GABAAR have been observed electrophysiologically to exhibit remarkable temporal complexity, with multiple time scales of desensitization and deactivation that depend on concentration over a very broad range. Earlier theoretical work was able to predict much of this complexity for GABAAR using a model that incorporates a simple Langmuir approximation of adsorption and desorption of neurotransmitters and solutes such as anesthetics, along with classical modeling of ligand binding and conformational transitions among the three canonical protein states. Here, a simple kinetic model is developed that improves on the Langmuir approach by incorporating the energetics of adsorbate-adsorbate (and adsorbate-bilayer) interactions. Predicted equilibrium isotherms are compared to experimental results, along with the time-course of adsorption/desorption, over a range of values of energetic parameters. Initial predictions of effects of adsorbate energetics on ion current traces involving long and short pulses of agonists show remarkably large effects on the time scale(s) of desensitization and deactivation.

Understanding Anesthetic Mechanisms: Analysis of the Complex Kinetics of Ligand-Gated Ion Channels.

Anesthetics modulate the response of ligand-gated ion channels to their neurotransmitter agonists, in a way that is consistent with clinical anesthesia: inhibition of synaptic transmission, by activation of inhibitory receptors and/or inhibition of excitatory receptors. Electrophysiological results for receptors such as GABAAR indicate that this modulation can be remarkably kinetically complex, characterized by concentration-dependent changes in the extent and (multiple) time scales of desensitization and deactivation. The full range of these features cannot be reproduced by a kinetic model in which anesthetic acts only by binding to putative protein sites, without having multiple sites with varying affinities, as well as many additional conformational states beyond the canonical set of three (resting, open, and desensitized). So, we discuss the implementation of a kinetic approach that incorporates only these three states, but accounts for effects of adsorption of anesthetic and agonist to the membrane in which the receptor is embedded, which modulates the conformational free energy landscape of the protein. As a result, the rate constants of conformational transitions become time dependent (non-Markovian), requiring nonstandard methods of kinetic analysis that can readily be implemented using available computational software.

Path to the Desensitized State of Ligand-Gated Ion Channels: Why Are Inhibitory and Excitatory Receptors Different?

The family of pentameric ligand-gated ion channels (pLGICs) includes both inhibitory and excitatory receptors. Electrophysiological methods have explored the time-dependent ion currents induced by their neurotransmitter agonists. Kinetic modeling requires a minimum of three conformational states: resting, active, and desensitized. However, current traces of inhibitory and excitatory pLGICs differ substantially. Reproducing their basic features requires different state connectivity: whether the desensitized state is accessed from the resting or active state. It is surprising that a property as fundamental as state connectivity would differ within the same family. So, we explore the possibility that the connectivity is the same, but corresponding states differ in function: Analogous states on the free energy landscape have similar structure, but differ in ion conductivity, free energies, and agonist binding affinities. This hypothesis is tested using a kinetic model in which agonist and anesthetics modulate the receptor free energy landscape by adsorbing to the membrane in which the receptor is embedded. It was previously shown that even with only three states, the complex behavior observed for GABAAR is reproduced, including its response to anesthetics. It is demonstrated here that this hypothesis accounts for an important difference between inhibitory and excitatory receptors: their opposite responses to inhalation anesthetics.

The evolutionary origin of the need to sleep: an inevitable consequence of synaptic neurotransmission?

It is proposed that the evolutionary origin of the need to sleep is the removal of neurotransmitters (NTs) that escape reuptake and accumulate in brain interstitial fluid (ISF). Recent work suggests that the activity of ionotropic postsynaptic receptors, rapidly initiated by binding of NTs to extracellular sites, is modulated over longer times by adsorption of these NTs to the lipid bilayers in which the receptors are embedded. This bilayer-mediated mechanism is far less molecularly specific than binding, so bilayer adsorption of NTs that have diffused into synapses for other receptors would modulate their activity as well. Although NTs are recycled by membrane protein reuptake, the process is less than 100% efficient; a fraction escapes the region in which these specific reuptake proteins are localized and eventually diffuses throughout the ISF. It is estimated that even if only 0.1% of NTs escape reuptake, they would accumulate and adsorb to bilayers in synapses of other receptors sufficiently to affect receptor activity, the harmful consequences of which are avoided by sleep: a period of efficient convective clearance of solutes together with greatly reduced synaptic activity.

Exploring the mechanism of general anesthesia: kinetic analysis of GABAA receptor electrophysiology.

A kinetic model of the effect of agonist and anesthetics on ligand-gated ion channels, developed in earlier work, is further refined and used to predict traces observed in fast-perfusion electrophysiological studies on recombinant GABAA receptors under a wide range of agonist and/or anesthetic concentrations. The model incorporates only three conformational states (resting, open, and desensitized) but allows for the modulation of the conformational free energy landscape connecting these states resulting from adsorption of agonist and/or anesthetic to the bilayer in which the protein is embedded. The model is shown to reproduce the diverse and complex features of experimental traces remarkably well, including both anesthetic-induced and agonist-induced traces, as well as the modulation of agonist-induced traces by anesthetic, either coapplied or continuously present. The solutions to the kinetic equations, which give the time-dependence of each of the nine protein states (three ligation states for each of the three conformations), describe the flow of probability among these states and thus reveal the kinetic underpinnings of the traces. Many of the parameters in the model, such as the desorption rate constants of anesthetic and agonist, are directly related to model-independent experimental measurements and thus can serve as a definitive test of its validity.

Molecular mechanisms of drug action: an emerging view.

Volatile anesthetics serve as useful probes of a conserved biological process that is essential to the proper functioning of the central nervous system. A kinetic and thermodynamic analysis of their unusual pharmacological and physiological characteristics has led to a general, predictive theory in which small molecules that adsorb to membranes modulate ion channel function by altering physical properties of membrane bilayers. A kinetic model that is both parsimonious and falsifiable has been developed to test this mechanism. This theory leads to predictions about the structure, function, origin, and evolution of synapses, the etiology of several diseases and disease symptoms affecting the brain, and the mechanism of action of several drugs that are used therapeutically. Neuronal membranes may offer an appealing drug target, given the large number of compounds that adsorb to interfaces and hence membranes.

Anesthetic-like modulation of a gamma-aminobutyric acid type A, strychnine-sensitive glycine, and N-methyl-d-aspartate receptors by coreleased neurotransmitters.

INTRODUCTION: A mechanism of anesthesia has recently been proposed which predicts that coreleased neurotransmitters may modulate neurotransmitter receptors for which they are not the native agonist in a manner similar to anesthetics. METHODS: We tested this prediction by applying acetylcholine to a NR1/NR2A N-methyl-d-aspartate receptor, glycine to a wild-type alpha(1)beta(2) and anesthetic-resistant alpha(1)(S270I)beta(2) gamma-amino-butyric acid (GABA) type A receptor, and GABA to a homomeric alpha(1) wild type and anesthetic-resistant alpha(1) S267I glycine receptor. Receptors were expressed in Xenopus laevis oocytes and studied using two-electrode voltage clamping. RESULTS: We found inhibition of N-methyl-d-aspartate receptor function by acetylcholine, enhancement of glycine receptor function by GABA, and enhancement of GABA type A receptor function by glycine. As expected of compounds with anesthetic activity, GABA showed far less potentiation (enhancement) of the function of the anesthetic-resistant S267I glycine receptor than that of the wild-type receptor. Glycine potentiated the function of wild-type GABA type A receptors but inhibited the function of the anesthetic-resistant S270I GABA type A receptor. CONCLUSIONS: These results show that neurotransmitters that are coreleased onto anesthetic-sensitive receptors may modulate the function of receptors for which they are not the native agonist via an anesthetic-like mechanism. These findings lend support to a recent theory of anesthetic action.

Central corneal thickness and visual field loss in fellow eyes of patients with open-angle glaucoma.

PURPOSE: To determine the relationship of central corneal thickness (CCT) and visual field loss between fellow eyes in primary open-angle glaucoma. DESIGN: Retrospective, observational case series. METHODS: Records review of glaucoma patients seen at local Veterans Administration eye clinic. Those with CCT measurements performed within one month of visual field testing were included. Patients were excluded with vision below 20/40 or disease that would affect visual fields. Intrasubject (between fellow eyes) differences in CCT, mean deviation (MD), and pattern standard deviation (PSD) were calculated by subtracting left eye value from right eye value. RESULTS: Of the 100 subjects (94 males), the Spearman correlation coefficient between intrasubject differences in CCT vs intrasubject differences in MD was 0.36 (P = .0003). The Spearman correlation for differences in CCT vs differences in PSD was -0.31 (P = .0019). CONCLUSIONS: Our study suggests that worse visual field changes tend to occur in the eye with the thinner cornea.

AKT activation and response to interferon-beta in human cancer cells.

Significant growth inhibition and induction of apoptosis by IFN-beta in cancer cells including colorectal cancer cells have been observed. We and others have previously reported the Stat 1 induction of TRAIL is a crucial step in the IFN-beta induced apoptosis pathway. However, when evaluating the sensitivity of a panel of colorectal cancer cell lines, we found no clear correlation between activation of the Jak/Stat signaling pathway and response to interferon. In the present study, we have evaluated the interaction of the PI3k/Akt pathway and IFN-beta induced apoptosis in human colorectal cancer cells. The results demonstrate a correlation between Akt activity, phosphorylation of Bad and resistance to interferon-induced apoptosis in these cells. The association of activation of Akt, phosphorylation of Bad and resistance to IFN-beta-induced apoptosis was further supported by the observation that disruption of the pathway in a more resistant cell line led to sensitization, and expression of an activated Akt in a more sensitive cell line led to increased resistance. Taken together, this data indicates that the PI3/Akt kinase pathway may be an important contributor to IFN-beta sensitivity and resistance in colorectal cancer cells. This data demonstrates a potential pathway by which cells may develop resistance to IFN, and further elucidation of this process may allow us to better target IFN therapy.

Anesthetic potency of two novel synthetic polyhydric alkanols longer than the n-alkanol cutoff: evidence for a bilayer-mediated mechanism of anesthesia?

The polyhydroxyalkanes 1,6,11,16-hexadecanetetraol (1) and 2,7,12,17-octadecanetetraol (2) were synthesized utilizing the thiophene ring as a scaffold to affix the hydroxyalkyl chains by lithiation of the acidic alpha-hydrogens and subsequent desulfurization. Both compounds exhibited significant anesthetic potency, individually and in additivity studies with hexanol, using immobility in tadpoles as the phenotypic endpoint. These results, which contradict a protein-binding mechanism in which cutoff results from steric hindrance, are consistent with recent predictions of a membrane-mediated mechanism involving the lateral pressure profile.

Receptor desensitization by neurotransmitters in membranes: are neurotransmitters the endogenous anesthetics?

A mechanism of anesthesia is proposed that addresses one of the most troubling peculiarities of general anesthesia: the remarkably small variability of sensitivity within the human population and across a broad range of animal phyla. It is hypothesized that in addition to the rapid, saturable binding of a neurotransmitter to its receptor that results in activation, the neurotransmitter also acts indirectly on the receptor by diffusing into the postsynaptic membrane and changing its physical properties, causing a shift in receptor conformational equilibrium (desensitization). Unlike binding, this slower indirect mechanism is nonspecific: each neurotransmitter will, in principle, affect all receptors in the membrane. For proteins modeled as having only resting and active conformational states, time-dependent ion currents are predicted that exhibit many characteristics of desensitization for both inhibitory and excitatory channels. If receptors have been engineered to regulate the time course of ion currents by this mechanism, then (a) mutations that significantly alter receptor sensitivity to this effect would be lethal and (b) by design, excitatory receptors would be inhibited, but inhibitory receptors activated, so that their effects are not counterproductive. The wide range of exogenous molecules that affect the physical properties of membranes as do neurotransmitters, but that do not bind to receptors, would thus inhibit excitatory channels and activate inhibitory channels, i.e., they would act as anesthesics. The endogenous anesthetics would thus be the neurotransmitters, the survival advantage conferred by their proper membrane-mediated desensitization of receptors explaining the selection pressure for anesthesic sensitivity.

Size distribution of barrel-stave aggregates of membrane peptides: influence of the bilayer lateral pressure profile.

Some membrane peptides, such as Alamethicin, form barrel-stave aggregates with a broad probability distribution of size (number of peptides in the aggregate). This distribution has been shown to depend on the characteristics of the lipid bilayer. A mechanism for this influence is suggested, in analogy to earlier work on the effects of changes in bilayer composition on conformational equilibria in membrane proteins, that is based on coupling of shifts in the distribution of lateral pressures in the bilayer to depth-dependent changes in the lateral excluded area that accompanies the formation of an aggregate. Thermodynamic analysis is coupled with a simple geometric model of aggregates of kinked cylindrical peptides and with results of previously calculated lateral pressure distributions to predict the effects of changes in bilayer characteristics on aggregate size distributions, in qualitative agreement with experimental results.