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The approach is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger as an example. The assay enables the quantitative study of a wide range of Cl- transporting molecules and proteins whose activity is modulated by ligands, voltage, and membrane composition as well as the investigation of the effects of compounds that directly inhibit or activate the reconstituted transport systems. selleck chemicals llc The present assay is readily adapted to the study of transport systems with diverse substrate specificities and molecular characteristics, and the necessary modifications needed are discussed.Chemical modification of ion channels using the substituted cysteine accessibility method has a rich and successful history in elucidating the structural basis of ion channel function. In this approach, cysteine residues are introduced in regions of interest into the protein and their accessibility to water soluble thiol-reactive reagents is determined by monitoring ion channel activity. Because a wide range of these reagents are available with differing size, charge, and membrane solubility, the physio-chemical environment of the introduced cysteine residue and therefore the protein domain of interest can be probed with great precision. The approach has been widely employed for determining the secondary structure of specific ion channel domains, the location and nature of the channel gate, and the conformational rearrangements in the channel pore that underlie the opening/closing of the pore. In this chapter, we describe the use of these and related approaches to probe the functional architecture and gating of store-operated Orai1 channels.Single molecule Förster Resonance Energy Transfer (smFRET) allows us to measure variation in distances between donor and acceptor fluorophores attached to a protein, providing the conformational landscape of the protein with respect to this specific distance. smFRET can be performed on freely diffusing molecules or on tethered molecules. Here, we describe the tethered method used to study ionotropic glutamate receptors, which allows us to track the changes in FRET as a function of time, thus providing information on the conformations sampled and kinetics of conformational changes in the millisecond to second time scale. Strategies for attaching fluorophores to the proteins, methods for acquiring and analyzing the smFRET trajectories, and limitations are discussed.Combining crosslinking strategies with electrophysiology, biochemistry, and structural in silico analysis is a powerful tool to study transient movements of ion channels during gating. This chapter describes crosslinking in living cells using cysteine and photoactive unnatural amino acids (UAAs) that we have used on glutamate receptor ion channels. Here, we share the protocol for building a perfusion tool to enable rapid chemical modification of glutamate-gated AMPA receptors, optimized for their fast activation. This system can be used to perform state-dependent crosslinking in receptors modified by cysteines or UAA incorporation on the millisecond timescale. Introducing UAAs results in receptors with lower expression levels relative to the introduction of cysteine residues. Reduced expression is principally a challenge for biochemical studies, and we share here our approach to capture the light driven oligomerization of AMPA receptors containing UAA crosslinkers. Finally, we describe strategies for computational analysis to make sense of the crosslinking results in terms of structure and function.Channels and transporters are vital for transmembrane transport of ions and solutes, and also of larger compounds such as lipids and macromolecules. Therefore, they are crucial in many biological processes such as sensing, signal transduction, and the regulation of the distribution of molecules. Dysfunctions of these membrane proteins are associated to numerous diseases, and their interaction with drugs is critical in medicine. Understanding the behavior of channels and transporters requires structural and dynamic information to decipher the molecular mechanisms underlying their function. High-Speed Atomic Force Microscopy (HS-AFM) now allows the study of single transmembrane channels and transporters in action under physiological conditions, i.e., at ambient temperature and pressure, in physiological buffer and in a membrane, and in a most direct, label-free manner. In this chapter, we discuss the HS-AFM sample preparation, application, and data analysis protocols to study the structural and conformational dynamics of membrane-embedded channels and transporters.Biochemical measurements of ligand binding to eukaryotic membrane proteins are challenging because they can require large amounts of purified protein. For this reason, ligand binding is preferentially evaluated on soluble domains rather than on the full length proteins. In this chapter, we describe the use of fluorescence size exclusion chromatography-based thermostability (FSEC-TS) as an assay to monitor ligand binding to the full length mammalian ion channel HCN4. FSEC-TS monitors the effect of the ligand on the thermal denaturation curve of the protein by following the fluorescence of a fused GFP protein. Changes in the melting temperature (Tm) provide a quantitative value for measuring ligand-protein interaction. As a proof of concept, we describe here the protocol for monitoring the binding of the second messenger cAMP and of the known HCN drug Ivabradine to the purified GFP-HCN4 channel. cTMP, a known non-binder of HCN channels, is used as a control. Due to the small amount of protein required, the assay represents a high-throughput screening system for evaluating binding of small molecules to full length proteins.Herding is done predominantly by breeds developed over centuries to millennia specifically for that purpose. Working-level herding breed dogs are intense, high-drive dogs that will work despite severe illness or pain, thereby masking clues that they are ailing or the nature of their problem. The handler should recognize subtle changes that might signal ill health, and veterinarians should take an active role in training handlers on essential skills. Herding dogs typically work entirely outdoors in rural to wilderness environments with continuous exposure to other domestic animals and wildlife and may be affected by trauma, toxin exposure, infectious diseases, and parasitic infections.