TRPC channels: Structure, function, regulation and recent advances in small molecular probes.

Transient receptor potential canonical (TRPC) channels constitute a group of receptor-operated calcium-permeable nonselective cation channels of the TRP superfamily. The seven mammalian TRPC members, which can be further divided into four subgroups (TRPC1, TRPC2, TRPC4/5, and TRPC3/6/7) based on their amino acid sequences and functional similarities, contribute to a broad spectrum of cellular functions and physiological roles. Studies have revealed complexity of their regulation involving several components of the phospholipase C pathway, Gi and Go proteins, and internal Ca2+ stores. Recent advances in cryogenic electron microscopy have provided several high-resolution structures of TRPC channels. Growing evidence demonstrates the involvement of TRPC channels in diseases, particularly the link between genetic mutations of TRPC6 and familial focal segmental glomerulosclerosis. Because TRPCs were discovered by the molecular identity first, their pharmacology had lagged behind. This is rapidly changing in recent years owning to great efforts from both academia and industry. A number of potent tool compounds from both synthetic and natural products that selective target different subtypes of TRPC channels have been discovered, including some preclinical drug candidates. This review will cover recent advancements in the understanding of TRPC channel regulation, structure, and discovery of novel TRPC small molecular probes over the past few years, with the goal of facilitating drug discovery for the study of TRPCs and therapeutic development.

Tethered agonist analogs as site-specific probes for domains of the human α7 nicotinic acetylcholine receptor that differentially regulate activation and desensitization.

Homomeric α7 nicotinic acetylcholine receptors represent an important and complex pharmaceutical target. They can be activated by structurally diverse agonists and are highly likely to enter and remain in desensitized states at rates determined by the structures of the agonists. To identify structural elements regulating this function, we introduced reactive cysteines into the α7 ligand-binding domain allowing us to bind sulfhydryl-reactive (SH) agonist analogs or control reagents onto specific positions in the ligand binding domain. We identified four α7 mutants (S36C, L38C, W55C, and L119C) in which the tethering of the SH reagents blocked further acetylcholine-evoked activation of the receptor. However, after selective reaction with SH agonist analogs, the type II allosteric modulator N-(5-chloro-2,4-dimethoxyphenyl)-N'-(5-methyl-3-isoxazolyl-3-isoxazolyl)-urea (PNU-120596) could reactivate L119C and W55C mutants and receptors with a reduced or modified C-loop. Modified S36C and L38C mutants were insensitive to reactivation by PNU-120596, whether they were reacted with agonist analogs or alternative SH reagents. Molecular modeling showed that in the W55C and L119C mutants, the ammonium pharmacophore of the agonist analog methanethiosulfonate-ethyltrimethylammonium would be in a similar but nonidentical position underneath the C-loop. The orientation assumed by the ligand tethered to 119C was approximately 3-fold more sensitive to PNU-120596 than the alternative pose at 55C. Our results support the hypothesis that a single ligand can bind within the receptor in different ways and, depending on the specific binding pose, may variously promote activation or desensitization, or, alternatively, function as a competitive antagonist. This insight may provide a new approach for drug development.

Lipid bilayer regulation of membrane protein function: gramicidin channels as molecular force probes.

Membrane protein function is regulated by the host lipid bilayer composition. This regulation may depend on specific chemical interactions between proteins and individual molecules in the bilayer, as well as on non-specific interactions between proteins and the bilayer behaving as a physical entity with collective physical properties (e.g. thickness, intrinsic monolayer curvature or elastic moduli). Studies in physico-chemical model systems have demonstrated that changes in bilayer physical properties can regulate membrane protein function by altering the energetic cost of the bilayer deformation associated with a protein conformational change. This type of regulation is well characterized, and its mechanistic elucidation is an interdisciplinary field bordering on physics, chemistry and biology. Changes in lipid composition that alter bilayer physical properties (including cholesterol, polyunsaturated fatty acids, other lipid metabolites and amphiphiles) regulate a wide range of membrane proteins in a seemingly non-specific manner. The commonality of the changes in protein function suggests an underlying physical mechanism, and recent studies show that at least some of the changes are caused by altered bilayer physical properties. This advance is because of the introduction of new tools for studying lipid bilayer regulation of protein function. The present review provides an introduction to the regulation of membrane protein function by the bilayer physical properties. We further describe the use of gramicidin channels as molecular force probes for studying this mechanism, with a unique ability to discriminate between consequences of changes in monolayer curvature and bilayer elastic moduli.

Esterified trityl radicals as intracellular oxygen probes.

Triarylmethyl (trityl) radicals exhibit high stability and narrow linewidth under physiological conditions which provide high sensitivity and resolution for the measurement of O2 concentrations, making them attractive as EPR oximetry probes. However, the application of previously available compounds has been limited by their poor intracellular permeability. We recently reported the synthesis and characterization of esterified trityl radicals as potential intracellular EPR probes and their oxygen sensitivity, redox properties, and enzyme-mediated hydrolysis were investigated. In this paper, we report the cellular permeability and stability of these trityls in the presence of bovine aortic endothelial cells. Results show that the acetoxymethoxycarbonyl-containing trityl AMT-02 exhibits high stability in the presence of cells and can be effectively internalized. The intracellular hydrolysis of AMT-02 to the carboxylate form of the trityl (CT-03) was also observed. In addition, this internalized trityl probe was applied to measure intracellular O2 concentrations and the effects of menadione and KCN on the rates of O2 consumption in endothelial cells. This study demonstrates that these esterified trityl radicals can function as effective EPR oximetry probes measuring intracellular O2 concentration and consumption.

Principal component analysis of CYP2C9 and CYP3A4 probe substrate/inhibitor panels.

Cytochrome P450 (P450) inhibition often occurs in a strongly substrate- and inhibitor-dependent manner, with a given inhibitor affecting the metabolism of different substrates to differing degrees and with a given substrate responding differently to different inhibitors. Traditionally, patterns of functional similarity and dissimilarity among substrates and inhibitors have been studied using clustering analysis of pair-wise correlation coefficients. Principal component analysis (PCA) is a widely used statistical technique that identifies the globally most significant independent trends in a set of data. Here, we show that PCA can be usefully applied to study the differential effects on a panel of P450 probe substrates by a panel of inhibitors, using published data on CYP3A4 (Kenworthy et al., 1999) and CYP2C9 (Kumar et al., 2006). PCA can detect functional similarities among substrates and inhibitors that are not readily apparent using pair-wise clustering analysis. PCA also allows identification of the functionally typical and atypical substrates that might be used in combination to fully explore the P450 functional landscape.

A comparison of microarray and MPSS technology platforms for expression analysis of Arabidopsis.

BACKGROUND: Several high-throughput technologies can measure in parallel the abundance of many mRNA transcripts within a sample. These include the widely-used microarray as well as the more recently developed methods based on sequence tag abundances such as the Massively Parallel Signature Sequencing (MPSS) technology. A comparison of microarray and MPSS technologies can help to establish the metrics for data comparisons across these technology platforms and determine some of the factors affecting the measurement of mRNA abundances using different platforms. RESULTS: We compared transcript abundance (gene expression) measurement data obtained using Affymetrix and Agilent microarrays with MPSS data. All three technologies were used to analyze the same set of mRNA samples; these samples were extracted from various wild type Arabidopsis thaliana tissues and floral mutants. We calculated correlations and used clustering methodology to compare the normalized expression data and expression ratios across samples and technologies. Abundance expression measurements were more similar between different samples measured by the same technology than between the same sample measured by different technologies. However, when expression ratios were employed, samples measured by different technologies were found to cluster together more frequently than with abundance expression levels.Furthermore, the two microarray technologies were more consistent with each other than with MPSS. We also investigated probe-position effects on Affymetrix data and tag-position effects in MPSS. We found a similar impact on Affymetrix and MPSS measurements, which suggests that these effects were more likely a characteristic of the RNA sample rather than technology-specific biases. CONCLUSION: Comparisons of transcript expression ratios showed greater consistency across platforms than measurements of transcript abundance. In addition, for measurements based on abundances, technology differences can mask the impact of biological differences between samples and tissues.

Discovery of a small molecule antagonist of the parathyroid hormone receptor by using an N-terminal parathyroid hormone peptide probe.

Once-daily s.c. administration of either human parathyroid hormone (PTH)-(1-84) or recombinant human PTH-(1-34) provides for dramatic increases in bone mass in women with postmenopausal osteoporosis. We initiated a program to discover orally bioavailable small molecule equivalents of these peptides. A traditional high-throughput screening approach using cAMP activation of the PTH/PTH-related peptide receptor (PPR) as a readout failed to provide any lead compounds. Accordingly, we designed a new screen for this receptor that used a modified N-terminal fragment of PTH as a probe for small molecule binding to the transmembrane region of the PPR, driven by the assumption that the pharmacological properties (agonist/antagonist) of compounds that bound to this putative signaling domain of the PPR could be altered by chemical modification. We developed DPC-AJ1951, a 14 amino acid peptide that acts as a potent agonist of the PPR, and characterized its activity in ex vivo and in vivo assays of bone resorption. In addition, we studied its ability to initiate gene transcription by using microarray technology. Together, these experiments indicated that the highly modified 14 amino acid peptide induces qualitatively similar biological responses to those produced by PTH-(1-34), albeit with lower potency relative to the parent peptide. Encouraged by these data, we performed a screen of a small compound collection by using DPC-AJ1951 as the ligand. These studies led to the identification of the benzoxazepinone SW106, a previously unrecognized small molecule antagonist for the PPR. The binding of SW106 to the PPR was rationalized by using a homology receptor model.

Redistribution of lipid probes at early stages of bilayer lipid membrane fusion.

Redistribution of hydrophobic probes (R18 and dipicrylamine DPA-) through a water gap between two bilayer lipid membranes (BLM) was studied. Both probes are widely used for monitoring lipid redistribution in different cell systems. The donor membrane loaded with the probe was brought into a close contact with the acceptor membrane, and the redistribution of the probe, was recorded as changes in the permittance of the acceptor membrane. It was shown that DPA- easily penetrated the water gap, whereas R18 was not detected in the acceptor membrane 30 min after the close contact formation. After fusion, R18 rapidly spread into the acceptor membrane; hence, only R18 reflects the behaviour of membrane lipids in the fusion process.

Diphtheria toxin as a molecular tool in the study of acidic fibroblast growth factor signalling.

In eukaryotic cells proteins are translocated across a number of cellular membranes into various intracellular organelles such as the endoplasmatic reticulum, mitochondria, peroxisomes and chloroplasts. In all these cases the proteins are translocated away from the cytosol. However, certain proteins are also translocated in the opposite direction, from the exterior to the cytosol. Well established examples are some bacterial and plant protein toxins, that exert their effect in the cytosol. A common property of protein toxins with intracellular action is that they contain two functionally different moieties, in many cases consisting of two. disulfide-linked polypeptides. Relatively little is known about how these proteins cross the membrane. The translocation process is best understood in the case of diphtheria toxin, which binds to cell surface receptors, is then taken up by endocytosis and is subsequently translocated to the cytosol, where it inactivates elongation factor 2. Recently it has been recognized that diphtheria toxin as well as a few other protein toxins can be used to carry passenger peptides or proteins into cells (in addition to other usefull roles which the toxins have begun to play in understanding many cellular processes and in certain prophylactic and therapeutic purposes). Here, the approach of using diphtheria toxin as a translocation vehicle in the study of new aspects of signal transduction mechanisms activated by acidic fibroblast growth factor is discussed and the possibility that some proteins have distinct functions in more than one cellular compartment is considered. Finally, this article focuses on the role of the toxins as tools in cell biology and experimental medicine.