The rockefeller university

The stable structural conformations that occur along the complete reaction coordinate for ion channel opening have never been observed. In this study, we describe the equilibrium ensemble of structures of Slo2.2, a neuronal Na+-activated K+ channel, as a function of the Na+ concentration. We find that Slo2.2 exists in multiple closed conformations whose relative occupancies are independent of Na+ concentration. An open conformation emerges from an ensemble of closed conformations in a highly Na+-dependent manner, without evidence of Na+-dependent intermediates. In other words, channel opening is a highly concerted, switch-like process. The midpoint of the structural titration matches that of the functional titration. A maximum open conformation probability approaching 1.0 and maximum functional open probability approaching 0.7 imply that, within the class of open channels, there is a subclass that is not permeable to ions.

We establish a link between classical heterotic strings and the groups of the magic square associated with Jordan algebras, allowing for a uniform treatment of the bosonic and superstring sectors of the heterotic string.

This paper proposes a new iterative solution method for nonlinear stochastic optimal control problems based on path integral analysis. First, we provide an iteration law for solving a stochastic Hamilton-Jacobi-Bellman (SHJB) equation associated to this problem, which is a nonlinear partial differential equation (PDE) of second order. Each iteration procedure of the proposed method is represented by a Cauchy problem for a linear parabolic PDE, and its explicit solution is given by the Feynman-Kac formula. Second, we derive a suboptimal feedback controller at each iteration by using the path integral analysis. Third, the convergence property of the proposed method is investigated. Here, some conditions are provided so that the sequence of solutions for the proposed iteration converges, and the SHJB equation is satisfied. Finally, numerical simulations demonstrate the effectiveness of the proposed method.

Membrane transport is an essential component of pathogenesis for most infectious organisms. In African trypanosomes, transport to and from the plasma membrane is closely coupled to immune evasion and antigenic variation. In mammals and fungi an octameric exocyst complex mediates late steps in exocytosis, but comparative genomics suggested that trypanosomes retain only six canonical subunits, implying mechanistic divergence. We directly determined the composition of the Trypanosoma brucei exocyst by affinity isolation and demonstrate that the parasite complex is nonameric, retaining all eight canonical subunits (albeit highly divergent at the sequence level) plus a novel essential subunit, Exo99. Exo99 and Sec15 knockdowns have remarkably similar phenotypes in terms of viability and impact on morphology and trafficking pathways. Significantly, both Sec15 and Exo99 have a clear function in endocytosis, and global proteomic analysis indicates an important role in maintaining the surface proteome. Taken together these data indicate additional exocyst functions in trypanosomes, which likely include endocytosis, recycling and control of surface composition. Knockdowns in HeLa cells suggest that the role in endocytosis is shared with metazoan cells. We conclude that, whilst the trypanosome exocyst has novel components, overall functionality appears conserved, and suggest that the unique subunit may provide therapeutic opportunities.

The interaction of glutamate and dopamine in the striatum is heavily dependent on signaling pathways that converge on the regulatory protein DARPP-32. The efficacy of dopamine/D1 receptor/PKA signaling is regulated by DARPP-32 phosphorylated at Thr-34 (the PKA site), a process that inhibits protein phosphatase 1 (PP1) and potentiates PKA action. Activation of dopamine/D1receptor/PKA signaling also leads to dephosphorylation of DARPP-32 at Ser-97 (the CK2 site), leading to localization of phospho-Thr-34 DARPP-32 in the nucleus where it also inhibits PP1. In this study the role of glutamate in the regulation of DARPP-32 phosphorylation at four major sites was further investigated. Experiments using striatal slices revealed that glutamate decreased the phosphorylation states of DARPP-32 at Ser-97 as well as Thr-34, Thr-75, and Ser-130 by activating NMDA or AMPA receptors in both direct and indirect pathway striatal neurons. The effect of glutamate in decreasing Ser-97 phosphorylation was mediated by activation of PP2A. In vitro phosphatase assays indicated that the PP2A/PR72 heterotrimer complex was likely responsible for glutamate/Ca2+-regulated dephosphorylation of DARPP-32 at Ser-97. As a consequence of Ser-97 dephosphorylation, glutamate induced the nuclear localization in cultured striatal neurons of dephospho-Thr-34/dephospho-Ser-97 DARPP-32. It also reduced PKA-dependent DARPP-32 signaling in slices and in vivo. Taken together, the results suggest that by inducing dephosphorylation of DARPP-32 at Ser-97 and altering its cytonuclear distribution, glutamate may counteract dopamine/D1 receptor/PKA signaling at multiple cellular levels.

Hypertension in male and aging female rodents is associated with glutamate-dependent plasticity in the hypothalamus, but existing models have failed to capture distinct transitional menopausal phases that could have a significant impact on the synaptic plasticity and emergent hypertension. In rodents, accelerated ovarian failure (AOF) induced by systemic injection of 4-vinylcyclohexane diepoxide mimics the estrogen fluctuations seen in human menopause including the perimenopause transition (peri-AOF) and postmenopause (post-AOF). Thus, we used the mouse AOF model to determine the impact of slow-pressor angiotensin II (Ang ll) administration on blood pressure and on the subcellular distribution of obligatory N-methyl-D-aspartate (NMDA) receptor GIuN1 subunits in the paraventricular hypothalamic nu-cleus (PVN), a key estrogen-responsive cardiovascular regulatory area. Estrogen-sensitive neuronal profiles were identified in mice expressing enhanced green fluorescent protein under the promoter for estrogen receptor (ER) beta, a major ER in the PVN. Slow-pressor Angll increased arterial blood pressure in mice at peri-and post-AOF time points. In control oil-injected (nonhypertensive) mice, Angll decreased the total number of GIuN1 in ERR-containing PVN dendrites. In contrast, Angll resulted in a reapportionment of GIuN1 from the cytoplasm to the plasma membrane of ER beta-containing PVN dendrites in peri-AOF mice. Moreover, in post-AOF mice, Angll increased total GIuN1, dendritic size and radical production in ERR-containing neurons. These results indicate that unique patterns of hypothalamic glutamate receptor plasticity and dendritic structure accompany the elevated blood pressure in peri-and post-AOF time points. Our findings suggest the possibility that distinct neurobiological processes are associated with the increased blood pressure during perimenopausal and postmenopausal periods.

The multiprotein Mediator coactivator complex functions in large part by controlling the formation and function of the promoter-bound preinitiation complex (PIC), which consists of RNA polymerase II and general transcription factors. However, precisely how Mediator impacts the PIC, especially post-recruitment, has remained unclear. Here, we have studied Mediator effects on basal transcription in an in vitro transcription system reconstituted from purified components. Our results reveal a close functional interplay between Mediator and TFIIH in the early stages of PIC development. We find that under conditions when TFIIH is not normally required for transcription, Mediator actually represses transcription. TFIIH, whose recruitment to the PIC is known to be facilitated by the Mediator, then acts to relieve Mediator-induced repression to generate an active form of the PIC. Gel mobility shift analyses of PICs and characterization of TFIIH preparations carrying mutant XPB translocase subunit further indicate that this relief of repression is achieved through expending energy via ATP hydrolysis, suggesting that it is coupled to TFIIH's established promoter melting activity. Our interpretation of these results is that Mediator functions as an assembly factor that facilitates PIC maturation through its various stages. Whereas the overall effect of the Mediator is to stimulate basal transcription, its initial engagement with the PIC generates a transcriptionally inert PIC intermediate, which necessitates energy expenditure to complete the process. (C) 2016 Elsevier Ltd. All rights reserved.

The superfamily of G protein-coupled receptors (GPCRs) mediates a wide range of physiological responses and serves as an important category of drug targets. Earlier biochemical and biophysical studies have shown that GPCRs exist temporally in an ensemble of interchanging conformations. Single-molecule techniques are ideally suited to understand the dynamic signaling and conformational complexity of G protein-coupled receptors (GPCRs). Here, we review the progress in single-molecule studies on GPCRs. We introduce the fundamental technical aspects of single-molecule fluorescence. We also survey the methodologies for labeling GPCRs with biophysical probes, particularly fluorescent dyes, and highlight the relevant chemical biology innovations that can be instrumental for studying GPCRs. Finally, we illustrate how the optical techniques and the labeling schemes have been combined to investigate GPCR signaling and dynamics at the single-molecule level.