No abstract

Ionotropic glutamate receptors mediate the majority of vertebrate excitatory synaptic transmission and are therapeutic targets for cognitive enhancement and treatment of schizophrenia. The binding domains of these tetrameric receptors consist of two dimers, and the dissociation of the dimer interface of the ligand-binding domain leads to desensitization in the continued presence of agonist. Positive allosteric modulators act by strengthening the dimer interface and reducing desensitization, thereby increasing steady-state activation. Removing the desensitized state for simplified analysis of receptor activation is commonly achieved using cyclothiazide (CTZ), the most potent modulator of the benzothiadiazide class, with the flip form of the AMPA receptor subtype. IDRA-21, the first benzothiadiazide to have an effect in behavioral tests, is an important lead compound in clinical trials for cognitive enhancement as it can cross the blood-brain barrier. Intermediate structures between CTZ and IDRA-21 show reduced potency suggesting that these two compounds have different contact points associated with binding. To understand how benzothiadiazides bind to the pocket bridging the dimer interface, we generated a series of crystal structures of the GluR2 ligand-binding domain complexed with benzothiadiazide derivatives (IDRA-21, hydroflumethiazide, hydrochlorothiazide, chlorothiazide, trichlormethiazide, and althiazide) for comparison with an existing structure for cyclothiazide. The structures detail how changes in the substituents in the 3- and 7-positions of the hydrobenzothiadiazide ring shift the orientation of the drug in the binding site and, in some cases, change the stoichiometry of binding. All derivatives maintain a hydrogen bond with the Ser754 hydroxyl, affirming the partial selectivity of the benzothiadiazides for the flip form of AMPA receptors.

The lifetimes of the unitary currents from ion channels, as revealed from single-channel recording, are traditionally thought to follow exponential or multiexponential distributions. The interpretation of these event-time distributions is that the gating process follows Markov kinetics among a small number of states. There is recent evidence, however, that certain systems exhibit distributions that follow power laws or functions related to power laws. Likewise, it has been suggested that data sets that appear to be multiexponential may be fit to simple power laws as well. In this paper we propose a different view of ion-channel-gating kinetics that is consistent with these recent experimental observations. We retain the Markovian nature of the kinetics, but, in contrast to the traditional models, we suggest that ion-channel proteins have a very large number of states all of similar energy. Gating, therefore, resembles a diffusion process. We show that our simplest one-dimensional model exhibits single-channel distributions that follow power laws of the form tea, where 1/2 c a c 3/2. Exponents determined from recent experiments approximately fall within this range. We believe that this model is consistent with modern views of protein dynamics and, thus, may provide a key to the molecular details of the gating process.Since its development over a decade ago, the patch-clamp experiment has provided enormous insight into the molecular basis of ion-channel behavior (1). Ion channels exhibit unitary currents that rise and fall between a conducting and a nonconducting state, and the time-dependent record appears as a stochastically modulated square wave. Analysis of these records is performed by collecting together events of a particular conducting state (i.e., the open state or the closed state) as indexed by their temporal length and then constructing an event-time histogram. A closed-time distribution, for example, is modeled by assuming a small number of underlying closed states connected by first-order (or pseudofirst-order) rate constants. With such a model, the number of exponentials required to fit the distribution is equal to the number of states. This approach has been used to explain a wide variety of single-channel phenomena including inactivation in voltage-gated channels (2), desensitization in the acetylcholine receptor (3), and brief openings in the acetylcholine receptor (4).When modeling patch-clamp data, the point of view usually taken is that the simplest kinetic scheme that fits the data is the correct scheme; and the simplest kinetic scheme is interpreted to mean that with the fewest conformational states. However, as we learn more about the molecular details of ion channels, we must address the issue of whether these descriptions are realistic from the molecular perspective. For instance, molecular dynamics (5), NMR (6), and crystallographic studies (7) have suggested that proteins have many local energy minima and that the internal motion is liquidlike in nature; i.e., structural fluctua...

Environmental concerns surrounding drilling for gas are intense due to expansion of shale gas drilling operations. Controversy surrounding the impact of drilling on air and water quality has pitted industry and lease-holders against individuals and groups concerned with environmental protection and public health. Because animals often are exposed continually to air, soil, and groundwater and have more frequent reproductive cycles, animals can be used as sentinels to monitor impacts to human health. This study involved interviews with animal owners who live near gas drilling operations. The findings illustrate which aspects of the drilling process may lead to health problems and suggest modifications that would lessen but not eliminate impacts. Complete evidence regarding health impacts of gas drilling cannot be obtained due to incomplete testing and disclosure of chemicals, and nondisclosure agreements. Without rigorous scientific studies, the gas drilling boom sweeping the world will remain an uncontrolled health experiment on an enormous scale.

Proteins of the rho subfamily of ras GTPases have been shown to be crucial components of pathways leading to cell growth and the establishment of cell polarity and mobility. Presented here is the solution structure of one such protein, Cdc42Hs, which provides insight into the structural basis for specificity of interactions between this protein and its effector and regulatory proteins. Standard heteronuclear NMR methods were used to assign the protein, and approximately 2100 distance and dihedral angle constraints were used to calculate a set of 20 structures using a combination of distance geometry and simulated annealing refinement. These structures show overall similarity to those of other GTP-binding proteins, with some exceptions. The regions corresponding to switch I and switch II in H-ras are disordered, and no evidence was found for an alpha-helix in switch II. The 13-residue insertion, which is only present in rho-subtype proteins and has been shown to be an important mediator of binding of regulatory and target proteins, forms a compact structure containing a short helix lying adjacent to the beta4-alpha3 loop. The insert forms one edge of a "switch surface" and, unexpectedly, does not change conformation upon activation of the protein by the exchange of GTP analogs for GDP. These studies indicate the insert region forms a stable invariant "footrest" for docking of regulatory and effector proteins.