G-protein–gated TRP-like Cationic Channel Activated by Muscarinic Receptors: Effect of Potential on Single-channel Gating

There is little information about the mechanisms by which G-protein–coupled receptors gate ion channels although many ionotropic receptors are well studied. We have investigated gating of the muscarinic cationic channel, which mediates the excitatory effect of acetylcholine in smooth muscles, and proposed a scheme consisting of four pairs of closed and open states. Channel kinetics appeared to be the same in cell-attached or outside-out patches whether the channel was activated by carbachol application or by intracellular dialysis with GTPγS. Since in the latter case G-proteins are permanently active, it is concluded that the cationic channel is the major determinant of its own gating, similarly to the KACh channel (Ivanova-Nikolova, T.T., and G.E. Breitwieser. 1997. J. Gen. Physiol. 109:245–253). Analysis of adjacent-state dwell times revealed connections between the states that showed features conserved among many other ligand-gated ion channels (e.g., nAChR, BKCa channel). Open probability (PO) of the cationic channel was increased by membrane depolarization consistent with the prominent U-shaped I-V relationship of the muscarinic whole-cell current at negative potentials. Membrane potential affected transitions within each closed-open state pair but had little effect on transitions between pairs; thus, the latter are likely to be caused by interactions of the channel with its ligands, e.g., Ca2+ and Gαo-GTP. Channel activity was highly heterogeneous, as was evident from the prominent cycling behavior when PO was measured over 5-s intervals. This was related to the variable frequency of openings (as in the KACh channel) and, especially, to the number of long openings between consecutive long shuttings. Analysis of the underlying Markov chain in terms of probabilities allowed us to evaluate the contribution of each open state to the integral current (from shortest to longest open state: 0.1, 3, 24, and 73%) as PO increased 525-fold in three stages

Voltage- and cold-dependent gating of single TRPM8 ion channels

Transient receptor potential (TRP) channels play critical roles in cell signaling by coupling various environmental factors to changes in membrane potential that modulate calcium influx. TRP channels are typically activated in a polymodal manner, thus integrating multiple stimuli. Although much progress has been made, the underlying mechanisms of TRP channel activation are largely unknown. The TRPM8 cation channel has been extensively investigated as a major neuronal cold sensor but is also activated by voltage, calcium store depletion, and some lipids as well as by compounds that produce cooling sensations, such as menthol or icilin. Several models of TRPM8 activation have been proposed to explain the interaction between these diverse stimuli. However, a kinetic scheme is not yet available that can describe the detailed single-channel kinetics to gain further insight into the underlying gating mechanism. To work toward this goal, we investigated voltage-dependent single-channel gating in cell-attached patches at two different temperatures (20 and 30°C) using HEK293 cells stably expressing TRPM8. Both membrane depolarization and cooling increased channel open probability (Po) mainly by decreasing the duration of closed intervals, with a smaller increase in the duration of open intervals. Maximum likelihood analysis of dwell times at both temperatures indicated gating in a minimum of five closed and two open states, and global fitting over a wide range of voltages identified a seven-state model that described the voltage dependence of Po, the single-channel kinetics, and the response of whole-cell currents to voltage ramps and steps. The major action of depolarization and cooling was to accelerate forward transitions between the same two sets of adjacent closed states. The seven-state model provides a general mechanism to account for TRPM8 activation by membrane depolarization at two temperatures and can serve as a starting point for further investigations of multimodal TRP activation

Laminar and Dorsoventral Molecular Organization of the Medial Entorhinal Cortex Revealed by Large-scale Anatomical Analysis of Gene Expression

Neural circuits in the medial entorhinal cortex (MEC) encode an animal's position and orientation in space. Within the MEC spatial representations, including grid and directional firing fields, have a laminar and dorsoventral organization that corresponds to a similar topography of neuronal connectivity and cellular properties. Yet, in part due to the challenges of integrating anatomical data at the resolution of cortical layers and borders, we know little about the molecular components underlying this organization. To address this we develop a new computational pipeline for high-throughput analysis and comparison of in situ hybridization (ISH) images at laminar resolution. We apply this pipeline to ISH data for over 16,000 genes in the Allen Brain Atlas and validate our analysis with RNA sequencing of MEC tissue from adult mice. We find that differential gene expression delineates the borders of the MEC with neighboring brain structures and reveals its laminar and dorsoventral organization. We propose a new molecular basis for distinguishing the deep layers of the MEC and show that their similarity to corresponding layers of neocortex is greater than that of superficial layers. Our analysis identifies ion channel-, cell adhesion- and synapse-related genes as candidates for functional differentiation of MEC layers and for encoding of spatial information at different scales along the dorsoventral axis of the MEC. We also reveal laminar organization of genes related to disease pathology and suggest that a high metabolic demand predisposes layer II to neurodegenerative pathology. In principle, our computational pipeline can be applied to high-throughput analysis of many forms of neuroanatomical data. Our results support the hypothesis that differences in gene expression contribute to functional specialization of superficial layers of the MEC and dorsoventral organization of the scale of spatial representations

Liquidity and price discovery on the London stock exchange

The London Stock Exchange is constantly changing as the global financial landscape evolves. By aggregating detailed intraday trading data, I analyse its liquidity for a period spanning the introduction of a pure electronic order book platform for the most liquid stocks in 1997, its expansion to include less liquid stocks with market maker participation, the structural and environmental changes brought about by MiFID regulation in 2007, and the beginning of the global financial crisis. By all measures, liquidity has increased over the years, although recently intensified competition from alternative trading venues may be limiting further improvement. The most liquid stocks are the constituents of the FTSE 100 index, which are picked by largest market capitalization. When a new stock is added to this index there is a temporary price effect which I ascribe to the closing auction just before the index is revised. This is a natural time for passive investors who track the index by replicating its composition to adjust their portfolio holdings. The auction trade is facilitated by a build-up of liquidity on the opposite side of the order book in advance, as limit orders are placed in competition to take the other side of this information-free order flow at a premium. Naturally, ordinary trading in index constituents does contain information about individual stocks, groups of stocks and the entire market. Conveniently, another liquid security trades on the market which can be used as a conduit for the latter information: the FTSE 100 exchange-traded fund. Due to arbitrage opportunities its price is closely related to the index, and in fact I determine that they are cointegrated, even intraday. According to the eo integration analysis the fund makes a significant contribution to the index price discovery process, and this is especially evident when order flows are incorporated into the model.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

G-protein control of voltage dependence as well as gating of muscarinic metabotropic channels in guinea-pig ileum.

1. Voltage-dependent properties of muscarinic receptor cationic current activated by carbachol in single smooth muscle cells have been studied using patch-clamp recording techniques. Cells were obtained by enzymic digestion from the longitudinal muscle layer of guinea-pig small intestine. 2. The inward cationic current showed a pronounced U-shaped current-voltage relationship (inward current negative). The relationship of cationic conductance to voltage could be described by a Boltzman distribution which was shifted 36 mV in the negative direction on the voltage axis by increasing fractional receptor occupancy (by increasing agonist concentration from 3 to 300 microM), and in the positive direction by desensitization during prolonged application of agonist. Cationic channels opened by low and high concentrations of carbachol at the same potential do not have identical properties. 3. Release of GTP within the cell, by flash photolysis of an inert caged precursor, had the same effect on the current-voltage relationship as increasing receptor occupancy by the agonist. Release of GDP beta S by flash photolysis had the opposite effect. 4. These various results could be explained if cationic channel opening upon receptor activation required binding of at least one alpha-GTP subunit, but the position of the activation curve on the voltage axis depended critically on the concentration of activated G-protein alpha-subunits in the cell