Interactions between the skeletal dihydropyridine receptor {u03B2} subunit, the {u03B1}{u2081}s II-III loop and the ryanodine receptor

Excitation-contraction (EC) coupling in skeletal muscle requires a physical coupling between the dihydropyridine receptor (DHPR) Ca2+ channel in the surface membrane and the ryanodine receptor (RyRl) Ca2+ release channel in the sarcoplasmic reticulum (SR) Ca2+ store. However, the exact molecular mechanism of this interaction remains unresolved. Both the {u03B1}{u2081}s and {u03B2}{u2081}a subunits of the DHPR are essential for the skeletal BC coupling process and a central critical region of the {u03B1}{u2081}s II-III loop has been shown to be important for this interaction. The {u03B2} subunit plays an essential role in the targeting of the pore-forming {u03B1}{u2081} subunit to the t-tubular membrane and in the modulation of the DHPR Ca2+ channel. In addition the skeletal isoform, {u03B2}{u2081}a, supports tetrad formation of the DHPRs opposite the ryanodine receptors. {u03B2}{u2081}a has a modular structure consisting of N, C termini and SH3/guanylate kinase (OK) domains separated by a Hook region. A high affinity interaction between the {u03B1}{u2081}s I-II loop and the {u03B2}{u2081}a-OK domain is responsible for the targeting function of the {u03B2} subunit. However the functional significance of the SH3 domain remains unclear. SH3 domains are protein interaction domains that typically bind to proline rich motifs in their interacting partners. The critical region of the {u03B1}{u2081}s II-III loop contains at least two such proline rich motifs. Therefore this study investigated the possibility of an interaction between the {u03B2}{u2081}a-SH3 domain and the {u03B1}{u2081}s II-III loop. The {u03B2}{u2081}a subunit and its SH3 domain bound to the critical region of the {u03B1}{u2081}s II-III loop with an affinity of ~2 {u03BC}M. One of these interactions was narrowed down to the first proline-rich motif of the critical region which encompasses four skeletal specific residues (A739, F741, P742 and D744) that have been previously shown to be important for skeletal type ECcoupling in-vivo. Mutation of these residues to their cardiac counterparts showed residues P742 and D744 to be important for the binding of the {u03B2}{u2081}a-SH3 domain to the critical region of the {u03B1}{u2081}s II-III loop. The C-terminus of the {u03B2}{u2081}a subunit binds to RyRl in vitro and the end 35 residues of the {u03B2}{u2081}a C-terminus is important for skeletal type BC coupling. This study investigated the structure of a peptide corresponding to this region by NMR and identified a nascent helical region extending from residues L493 to G506 . Three hydrophobic residues (L496, L500 and W503 ) within this helical region form a hydrophobic surface which could be a putative binding surface for the skeletal ryanodine receptor. Mutation of these residues to alanines partially disrupts the helical surface and decreases the ability of the mutant peptides to activate the ryanodine receptor. In conclusion this study shows that the SH3 domain of the skeletal {u03B2} subunit is able to bind to the critical region of the {u03B1}{u2081}s II-III loop in-vitro. This study also identifies a quasi-structured helical region in the C terminal tail of the {u03B2}{u2081}a subunit that affects its interaction with the skeletal ryanodine receptor. Based on these findings, a model is proposed where the {u03B2}{u2081}a subunit acts as a conduit in the transformation of the BC coupling signal from the skeletal DHPR to RyRl

Determination of descriptors by liquid-liquid partition and chromatographic methods

ABSTRACT DETERMINATION OF DESCRIPTORS BY LIQUID-LIQUID PARTITION AND CHROMATOGRAPHIC METHODS by THUSHARA KARUNASEKARA September 2012 Advisor: Prof. Colin F. Poole Major: Chemistry (Analytical) Degree: Doctor of Philosophy Partition coefficients for varied compounds were determined for several totally organic biphasic systems using formamide, propylene carbonate, ethylene glycol or dimethyl sulfoxide as the base solvent and n-heptane, 1,2-dichloroethane, 1-octanol or isopentyl ether as the counter solvent. These partition coefficient databases are analyzed using the solvation parameter model facilitating a quantitative comparison of these systems with other totally organic and water-based partition systems. These totally organic biphasic solvent systems offer a complementary approach to water-based partition systems for sample preparation and the determination of descriptors for compounds virtually insoluble in water or unstable in water. A new method of solvent classification is proposed based on the five system constants of the solvation parameter model for transfer of neutral compounds from the gas phase to solvent and hierarchical cluster analysis for identifying solvents with similar properties and for organizing them into selectivity groups. This method resulted in the classification of 36 common solvents used in chromatography into seven selectivity groups with four solvents (2,2,2-trifluoroethanol, N,N-dimethylformaide, dimethyl sulfoxide and water) behaving independently. The classification scheme provides a logical approach for solvent selection as the first step in chromatographic method development. A similar approach for the transfer of neutral compounds between condensed phases and hierarchical cluster analysis was used to classify 19 aqueous and 17 totally organic biphasic partition systems for liquid-liquid extraction. The totally organic biphasic partition systems exhibit an almost continuous range of properties with minimal group formation demonstrating a wider and complementary range of selectivity to the aqueous biphasic systems. The classification of the liquid-liquid partition systems provides a suitable method of identifying suitable systems for sample preparation based on liquid-liquid extraction and for the simulation of extractions for target compound isolation. Retention factors on a minimum of eight stationary phases at various temperatures by gas-liquid chromatography and liquid-liquid partition coefficients for eight totally organic biphasic systems were combined to estimate descriptors for 28 fragrance compounds with an emphasis on compounds that are known or potential allergens. Similar aproch with additional liquid chromatographic measurements was used to estimate descriptors for 24 esters widely used as plasticizers and solvents in industry. The descriptors facilitated the estimation of several properties of biological and environmental interest (sensory irritation threshold, odor detection threshold, nasal pungency threshold, skin permeability from water, skin-water partition coefficients, octanol-water partition coefficients, absorption by air particles, adsorption by diesel soot particles, air-water partition coefficients, and adsorption by film water). A combination of gas chromatography and liquid-liquid partition in totally organic biphasic systems is used to determine descriptor values for compounds of low volatility suitable for characterizing open tubular columns at high temperatures. The descriptor database of varied compounds includes several difficult to determine by conventional techniques due to their low water solubility or stability. The descriptor database facilitates an expansion of the descriptor space and compound variation for characterizing separation systems. As an application the descriptor database is used to determine the system constants for SPB-Octyl, HP-5, Rxi-5Sil MS, Rtx-440, and Rtx-OPP for the temperature range 200-300,,aC. As an example of the broader affect of temperature on column selectivity the variation of the system constants for Rtx-440 over the temperature range 60-300,,aC is described in detail. These studies demonstrate the persistence of polar interactions to the highest temperature studied and that at high temperatures selectivity differences persist for moderately polar stationary phases

A Profile of Biomass Stove Use in Sri Lanka

A large body of evidence has confirmed that the indoor air pollution (IAP) from biomass fuel use is a major cause of premature deaths, and acute and chronic diseases. Over 78% of Sri Lankans use biomass fuel for cooking, the major source of IAP in developing countries. We conducted a review of the available literature and data sources to profile biomass fuel use in Sri Lanka. We also produced two maps (population density and biomass use; and cooking fuel sources by district) to illustrate the problem in a geographical context. The biomass use in Sri Lanka is limited to wood while coal, charcoal, and cow dung are not used. Government data sources indicate poor residents in rural areas are more likely to use biomass fuel. Respiratory diseases, which may have been caused by cooking emissions, are one of the leading causes of hospitalizations and death. The World Health Organization estimated that the number of deaths attributable to IAP in Sri Lanka in 2004 was 4300. Small scale studies have been conducted in-country in an attempt to associate biomass fuel use with cataracts, low birth weight, respiratory diseases and lung cancer. However, the IAP issue has not been broadly researched and is not prominent in Sri Lankan public health policies and programs to date. Our profile of Sri Lanka calls for further analytical studies and new innovative initiatives to inform public health policy, advocacy and program interventions to address the IAP problem of Sri Lanka

Cyclization of the intrinsically disordered α 1S dihydropyridine receptor II-III loop enhances secondary structure and in vitro function

A key component of excitation contraction (EC) coupling in skeletal muscle is the cytoplasmic linker (II-III loop) between the second and third transmembrane repeats of the α1S subunit of the dihydropyridine receptor (DHPR). The II-III loop has been pre

Understanding Neural Population Coding: Information Theoretic Insights from the Auditory System

In recent years, our research in computational neuroscience has focused on understanding how populations of neurons encode naturalistic stimuli. In particular, we focused on how populations of neurons use the time domain to encode sensory information. In this focused review, we summarize this recent work from our laboratory. We focus in particular on the mathematical methods that we developed for the quantification of how information is encoded by populations of neurons and on how we used these methods to investigate the encoding of complex naturalistic sounds in auditory cortex. We review how these methods revealed a complementary role of low frequency oscillations and millisecond precise spike patterns in encoding complex sounds and in making these representations robust to imprecise knowledge about the timing of the external stimulus. Further, we discuss challenges in extending this work to understand how large populations of neurons encode sensory information. Overall, this previous work provides analytical tools and conceptual understanding necessary to study the principles of how neural populations reflect sensory inputs and achieve a stable representation despite many uncertainties in the environment

Thorough QT study of the effect of intravenous amisulpride on QTc interval in Caucasian and Japanese healthy subjects.

AIM: The D2 /D3 antagonist amisulpride has shown promising efficacy against postoperative nausea and vomiting (PONV) at low doses. We investigated whether intravenous amisulpride has an effect on the QTc interval in a formal Thorough QT study (TQT). METHODS: This was a randomized, double-blind, placebo and positive-controlled, four-way crossover study. Forty healthy Caucasian and Japanese subjects were included to receive a single administration of 5 mg and 40 mg of i.v. amisulpride or a single oral dose of moxifloxacin or placebo per period. RESULTS: The therapeutic dose of 5 mg amisulpride was associated with a slight, transient increase in mean ΔΔQTcF, from 2.0 ms prior to dosing to a peak of 5 ms (90% CI: 2.8, 7.1 ms) at 8 min, decreasing to 2.1 ms at 30 min after dosing. The supra-therapeutic dose of 40 mg given at twice the infusion rate was associated with prolongation in ΔΔQTcF peaking at 23.4 ms (90% CI: 21.3, 25.5 ms) at the end of infusion (8 min), returning below 10 ms within 1.5 h. Assay sensitivity was confirmed; ΔΔQTcF had increased by 12.3 ms (90% CI 10.1, 14.6 ms) at 4 h post-dose. The PK-PD relationship revealed no differences between Caucasian and Japanese subjects (p-value > 0.5). CONCLUSIONS: Amisulpride has a plasma concentration-dependent effect on the QTc interval. The proposed therapeutic dose for management of PONV does not lead to a prolongation of QTcF above the threshold of regulatory concern, while such effect could not be excluded for the supratherapeutic dose

Determination of solvation descriptors for terpene hydrocarbons from chromatographic measurements

This article discusses the determination of solvation descriptors for terpene hydrocarbons from chromatographic measurements