A Novel Alcohol-Sensitive Site in the M3 Domain of the NMDA Receptor GluN2A Subunit

Accumulating studies have demonstrated that the N-methyl-D-aspartate receptor is one of the most important targets of ethanol in the central nervous system. Previous studies from this laboratory have found that one position in the third (F637) and two positions in the fourth (M823 and A825) membrane-associated (M) domains of the N-methyl-D-aspartate receptor GluN2A subunit modulate alcohol action and ion channel gating. Using site-directed mutagenesis and whole-cell patch-clamp recording, we have found an additional position in M3 of the GluN2A subunit, F636, which significantly influences ethanol sensitivity and functionally interacts with F637. Tryptophan substitution at F636 significantly decreased the ethanol IC50, decreased both peak and steady-state glutamate EC50, and altered agonist deactivation and apparent desensitization. There was a significant correlation between steadystate: peak current ratio, a measure of desensitization, and ethanol IC50 values for a series of mutants at this site, raising the possibility that changes in ethanol sensitivity may be secondary to changes in desensitization. Mutant cycle analysis revealed a significant interaction between F636 and F637 in regulating ethanol sensitivity. Our results suggest that F636 in the M3 domain of the GluN2A subunit not only influences channel gating and agonist potency, but also plays an important role in mediating the action of ethanol. These studies were supported by grants R01 AA015203-01A1 and AA015203-06A1 from the NIAAA to R.W.P

Reynolds Pressure and Relaxation in a Sheared Granular System

We describe experiments that probe the evolution of shear jammed states, occurring for packing fractions $\phi_S \leq \phi \leq \phi_J$, for frictional granular disks, where above $\phi_J$ there are no stress-free static states. We use a novel shear apparatus that avoids the formation of inhomogeneities known as shear bands. This fixed $\phi$ system exhibits coupling between the shear strain, $\gamma$, and the pressure, $P$, which we characterize by the `Reynolds pressure', and a `Reynolds coefficient', $R(\phi) = (\partial ^2 P/\partial \gamma ^2)/2$. $R$ depends only on $\phi$, and diverges as $R \sim (\phi_c - \phi)^{\alpha}$, where $\phi_c \simeq \phi_J$, and $\alpha \simeq -3.3$. Under cyclic shear, this system evolves logarithmically slowly towards limit cycle dynamics, which we characterize in terms of pressure relaxation at cycle $n$: $\Delta P \simeq -\beta \ln(n/n_0)$. $\beta$ depends only on the shear cycle amplitude, suggesting an activated process where $\beta$ plays a temperature-like role.Comment: 4 pages, 4 figure

Performance Analysis of Hybrid and Full Electrical Vehicles Equipped with Continuously Variable Transmissions

The main aim of this paper is to study the potential impacts in hybrid and full electrical vehicles performance by utilising continuously variable transmissions. This is achieved by two stages. First, for Electrical Vehicles (EVs), modelling and analysing the powertrain of a generic electric vehicle is developed using Matlab/Simulink-QSS Toolkit, with and without a transmission system of varying levels of complexity. Predicted results are compared for a typical electrical vehicle in three cases: without a gearbox, with a Continuously Variable Transmission (CVT), and with a conventional stepped gearbox. Second, for Hybrid Electrical Vehicles (HEVs), a twin epicyclic power split transmission model is used. Computer programmes for the analysis of epicyclic transmission based on a matrix method are developed and used. Two vehicle models are built-up; namely: traditional ICE vehicle, and HEV with a twin epicyclic gearbox. Predictions for both stages are made over the New European Driving Cycle (NEDC).The simulations show that the twin epicyclic offers substantial improvements of reduction in energy consumption in HEVs. The results also show that it is possible to improve overall performance and energy consumption levels using a continuously variable ratio gearbox in EVs

A Site of Alcohol Action at the NMDA Receptor M3-M4 Domain Interface

The N-methyl-D-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The structural model of the NMDA receptor, predicted from the structure of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We investigated possible interactions between the M3 and M4 domain positions of the two subunit types affecting the ethanol sensitivity of the receptor by using dual substitution mutants. In an initial screen of single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(G638) and GluN2A(F636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition were observed at four pairs of positions in GluN1/GluN2A: G638/M823, F639/L824, M818/F636, and L819/F637. Two of these interactions involve a position in M4 of both subunits, GluN1(M818) and GluN2A(L824), that does not by itself alter ethanol sensitivity, and one of the previously identified positions affecting ethanol sensitivity, GluN2A(A825), did not appear to interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, and support the existence of four sites of alcohol action on the NMDA receptor at the M3-M4 domain intersubunit interfaces. These studies were supported by grants R01 AA015203-01A1 and AA015203-06A1 from the NIAAA to R.W.P

Interactions Among Positions in the Third and Fourth Membrane-Associated Domains at the Intersubunit Interface of the N-Methyl-D-Aspartate Receptor Forming Sites of Alcohol Action

The N-methyl-d-aspartate (NMDA) glutamate receptor is a major target of ethanol in the brain. Previous studies have identified positions in the third and fourth membrane-associated (M) domains of the NMDA receptor GluN1 and GluN2A subunits that influence alcohol sensitivity. The predicted structure of the NMDA receptor, based on that of the related GluA2 subunit, indicates a close apposition of the alcohol-sensitive positions in M3 and M4 between the two subunit types. We tested the hypothesis that these positions interact to regulate receptor kinetics and ethanol sensitivity by using dual substitution mutants. In single-substitution mutants, we found that a position in both subunits adjacent to one previously identified, GluN1(Gly-638) and GluN2A(Phe-636), can strongly regulate ethanol sensitivity. Significant interactions affecting ethanol inhibition and receptor deactivation were observed at four pairs of positions in GluN1/GluN2A: Gly-638/Met-823, Phe-639/Leu-824, Met-818/Phe-636, and Leu-819/Phe-637; the latter pair also interacted with respect to desensitization. Two interactions involved a position in M4 of both subunits, GluN1(Met-818) and GluN2A(Leu-824), that does not by itself alter ethanol sensitivity, whereas a previously identified ethanol-sensitive position, GluN2A(Ala-825), did not unequivocally interact with any other position tested. These results also indicate a shift by one position of the predicted alignment of the GluN1 M4 domain. These findings have allowed for the refinement of the NMDA receptor M domain structure, demonstrate that this region can influence apparent agonist affinity, and support the existence of four sites of alcohol action on the NMDA receptor, each consisting of five amino acids at the M3-M4 domain intersubunit interfaces

Two Adjacent Phenylalanines In the NMDA Receptor GluN2A Subunit M3 Domain Interactively Regulate Alcohol Sensitivity and Ion Channel Gating

The N-methyl-d-aspartate (NMDA) receptor is a key target of ethanol action in the central nervous system. Alcohol inhibition of NMDA receptor function involves small clusters of residues in the third and fourth membrane-associated (M) domains. Previous results from this laboratory have shown that two adjacent positions in the M3 domain, F636 and F637, can powerfully regulate alcohol sensitivity and ion channel gating. In this study, we report that these positions interact with one another in the regulation of both NMDA receptor gating and alcohol action. Using dual mutant cycle analysis, we detected interactions among various substitution mutants at these positions with respect to regulation of glutamate EC50, steady-state to peak current ratios (Iss:Ip), mean open time, and ethanol IC50. This interaction apparently involves a balancing of forces on the M3 helix, such that the disruption of function due to a substitution at one position can be reversed by a similar substitution at the other position. For example, tryptophan substitution at F636 or F637 increased or decreased channel mean open time, respectively, but tryptophan substitution at both positions did not alter open time. Interestingly, the effects of a number of mutations on receptor kinetics and ethanol sensitivity appeared to depend upon subtle structural differences, such as those between the isomeric amino acids leucine and isoleucine, as they could not be explained on the basis of sidechain molecular volume or hydrophilicity