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P2X7 receptors form non-selective cation channels at the plasma membrane and are gated by the binding of extracellular ATP. Prolonged P2X7 receptor activation also leads to formation of a pore that allows permeation of large molecules. Many cell types express P2X7 receptors. Expression is particularly high in cells of hematopoietic origin, where they have important roles in many biological processes, including release of pro-inflammatory cytokines and apoptosis. Furthermore, the· properties of P2X7 receptors, including their functional expression and pharmacology, exhibit striking species differences. However, the underlying molecular bases for these differences are not fully understood. The gene encoding human P2X7 receptors contains numerous single nucleotide polymorph isms (SNP), several of which are non-synonymous (ns- SNP). Two ns-SNP mutations, H155Y and A348T, were of particular interest due to their gain-of function effect on P2X7 receptors. Furthermore, a previous study found ATP-induced secretion of cytokines was enhanced in immune cells from individuals harbouring the A348T mutation. These two mutations were therefore examined in detail. Whole-cell patch-clamp current recordings clearly showed both mutations increased agonist-induced maximal currents, with no or very mild effect on agonist sensitivity. Cells expressing human P2X7 receptors had lower ATP-evoked maximal currents in comparison to the rat receptor. Mutations H155Y and A348T change the residues to those in the corresponding positions of the WT rat P2X7 receptor. Introduction of reciprocal mutations, Y155H and T348A into the rat P2X7 receptor reduced agonist-evoked current amplitudes. Substitution of residues surrounding His 155 and Ala348 in the human P2X7 receptor with the corresponding residues of the rat P2X7 receptor did not result in gain-of-function, with the exception F353L. However, the reciprocal mutation L353F in the rat P2X7 receptor had no effect. Taken together, these results strongly indicate that residues 155 and 348 are important in determining the functional expression of P2X7 receptors. In addition, they reveal that the residues at these two positions contribute to the differences in functional expression of human and rat P2X7 receptors. Further investigations into the functional roles of His 155 and Ala348 were performed by studying the effects of mutating to residues with side chains of distinct properties. Substitution of His 155 with leucine and aspartic acid reduced ATP-induced current amplitudes, whilst phenylatanine, arginine and alanine had no significant effect. Mutation of Ala348 revealed a discernible effect on the human P2Xy receptor in that substitution with residues with larger side chains reduced, whereas changes to residues with small side chains increased, the amplitude of ATP-evoked currents. Immunostaining and biotin labelling revealed the H155Y mutation of human P2Xy receptors increased, whilst Y155H of rat receptors decreased surface expression. No such effect on surface expression resulted from reciprocal mutations at position 348. A human P2Xy receptor model, based on the recently determined crystal structure of the zebrafish P2X4 receptor, indicates that His 155 is in the extracellular region, distant from the agonist-binding site and ion-permeating pore, whilst Ala348 is located immediately intracellular to the narrowest part of the ion-conducting pathway. Therefore, the simple and most consistent explanation for the effects of mutating residues at 155 and 348 is that the residue at 155 is important in determining receptor surface expression, and the residue at 348 is involved in single channel function. The monkey P2Xy receptor, which shares 96% sequence homology with the human P2Xy receptor, has been functionally characterised, and its pharmacological properties were similar to that of the human receptor. The monkey P2Xy receptor had a 14-fold higher sensitivity to BzATP over ATP. Furthermore, the sensitivity to ATP and BzATP was slightly lower than in comparison to human P2Xy receptors (2.5- and 2-fold, respectively). The sensitivity of the monkey P2Xy receptor to the P2Xy receptor antagonists KN-62, AZ11645373 and A-438079 was virtually indistinguishable from that of the human P2Xy receptor. Therefore the five amino acids in the extracellular domain that differ between human and monkey P2Xy receptors do not critically interact with these antagonists. The similar pharmacological profiles of human and monkey P2Xy receptors suggests the monkey provides a suitable model for to investigate P2Xy receptor involvement in human diseases.

Ion channels control the movement of ions allowing specific ions to pass across biological membranes. This apparently simple process is used to control many essential physiological processes including the generation and propagation of neuronal excitation, regulation of insulin secretion, heartbeat and cell volume. Ion channels are able to do this by having multiple means of controlling the opening and closing of the ion conduction pathway. Several recent ion channel structures have shed light on the important process of ion selectivity as well as providing clues on channel gating. However, it is not yet completely clear how ion channels control the passage of ions across the membrane. My work is centred on understanding the gating process through structural analysis mostly using X-ray crystallography. In order to get to this point, crystals of the target ion channels need to be grown. This thesis presents four projects that were aimed at developing the techniques for over-expression, purification and crystallisation of ion channels or transporters making them amenable for structural studies. One of the difficult steps in the structure determination pathway is obtaining well-ordered crystals of the target membrane protein. Crystals of the EcCLC channel (now found to be a transporter) from E. coli were initially unacceptable for structure determination. It was shown that the process of dehydration improved the quality and diffraction limit of the EcCLC crystals. This simple method provides a possible procedure for improving the resolution limit of soluble and membrane protein crystals. Inward rectifier potassium channels (Kir channels) are a distinct subfamily that preferentially allows K⁺ ions to move into the cell. Two bacterial homologues of Kir channels, KirBac1.1 and KirBac3.1, were crystallised in 3-D and 2-D forms, respectively. As the crystal structure revealed the full-length model provided an excellent starting point for understanding the gating process in these channels. The 3-D structure of KirBac1.1 was captured in the closed state. This K⁺ channel prevents ion movement by using hydrophobic residues to occlude the ion conduction pathway, altering the conformation of selectivity filter, misaligning the pore helices and decreasing the volume of the cavity. The 2-D projection maps of KirBac3.1 were obtained in both closed and a potentially open conformations. These two maps provided evidence as to how the structural elements of the inner, outer, slide helices and the C-terminal domains change relative to one another as the channel moves between these two gating states. These motions were modelled as rigid body movements. They included bending of the inner helices, a rotation of the outer helices as they straighten in the membrane, translocation of the slide helices and C-terminal domain rotations. It is hoped that the information gained from these projects, the plasmids construction, the expression systems and the protocols that were established for crystallisation of the ion channels and transporter will provide guidelines for future structural studies of membrane proteins.

Solid-supported membranes present a biomimetic platform that can be adapted for bio-physical, biochemical and electrophysiological studies. In addition to this they offer an environment to host membrane proteins for the purposes of biosensing. This thesis examines the use of such a system and the possibilities it presents for the studies of ion channels and their potential applications in biosensing. Electrochemical impedance spectroscopy (EIS) is a powerful technique in the study of solid-supported membranes giving access to capacitance and resistance data, and as such was employed as the main method of characterisation. Electrodes were designed for this purpose in conjunction with Philips Research, and the suitability of the surface for the formation of insulating tethered bilayer lipid membranes (tBLMs). The development of these electrodes led to the incorporation of a SiO2 insulating layer, however its addition resulted in diculties with the formation of self-assembled monolayers (SAMs). However, further refinement of the manufacturing process should resolve these issues. Despite these diffculties, studies were performed using first generation electrodes (P1). Two ionophores, valinomycin and gramicidin, were employed in the characterisation of the ion transport properties of the tBLM system. These studies yielded important information about the structure of the tBLM system under investigation, as well as the ways ion transport can be presented in EIS. Using the work on ionophores as a foundation, an investigation into the incorporation and characterisation of ion channels in tBLM was conducted. Three channels were studied - a ligand-gated eukaryotic Ca2+-permeant channel (TRPC5), a voltage-gated prokaryotic Na+-permeant channel (NavCbt), and a pH-gated K+-permeant channel (KcsA). The success of these studies varied, but provided strong evidence that ion channel incorporation is possible. Further investigation of channel function in the tBLM is required as measured activity is lower than that suggested by literature.

Class 5 myosins are motor proteins involved in the transport and localisation of diverse cargoes in eukaryotic cells. Molecules have two heads and a tail and can move along actin filaments, by making step-like movements driven by MgATP hydrolysis. Each head comprises an actin-binding motor and a lever, an extended α-helix carrying six light chains bound to IQ motifs. The lever is an essential mechanical element in myosin 5 function, but an understanding of its mechanical properties and how these derive from its substructure is lacking. To address this, the first aim of this study was to look for and characterise flexibility within the heads of myosin 5. A second aim was to test the effect of altering the IQ motif spacing on the properties and behaviour of the molecule. The structure and flexibility of the head region of mammalian myosin 5a was studied using single-particle image processing of images from negative stain electron microscopy (EM). Image averaging revealed features of the motor and lever and provided evidence of independent rotational freedom of the motor about the lever axis, and thermally-driven flexing at the motor-lever junction. A stiffness of 32-51 pN·nm·rad-2 for the flexing was inferred, which represents a significant compliance in the unattached head. Variation in light chain orientations at each IQ motif suggested torsional flexibility. Lever bending analysis, by decomposition of average lever shapes into Fourier modes, yielded a persistence length of 50.9 ± 18.6 nm. All these results indicate that, under EM conditions, the unattached head is more flexible than has previously been measured for the actinbound complex in optical trap experiments. Lever mutants with altered IQ motif spacings were constructed and initial characterisations by negative stain EM, MgATPase assay and single-molecule fluorescence microscopy were made. For the latter, two calmodulin point mutants were made, for site-specific labelling and attachment to the lever as a fluorescent probe. One lever mutant, with closerspaced IQ motifs, resembled wild-type and had similar actin-activated MgATPase activity. However, its motile properties remain undetermined. Another mutant, with wider-spaced IQ motifs did not bind calmodulin and it had no actin-activated MgATPase. The molecule appeared misfolded but retained residual actin-binding properties. These contrasting results highlight the importance of the lever’s structure in the function of the molecule.

Cellular membrane dynamics has been subject to an ever-growing research interest since the introduction of the fluid mosaic model in the early seventies. The recognition that individual components of a cell membrane are able to diffuse in a two-dimensional matrix led to the crucial questioning of the structure-function relationship. The stunning diversity of lipid or proteins making up the plasma membrane of mammalian cells prevents theoretical treatment to apprehend membrane organization and dynamics. For this reason, membrane dynamics has remained up to now predominantly an experimental field of study. The presence of membrane micro-domains including lipid rafts and the co-existence of several phases has for instance been recently confirmed using single-molecule fluorescence detection methods. These domains as well as overall membrane fluidity are thought to be essential in many key cellular processes such as signal transduction, pathogen entry or trafficking. This thesis focuses on the development, characterization and applications of novel microfluidic tools for probing cellular plasma membrane structure and dynamics. We successfully demonstrated dielectrophoretic trapping of single mammalian cells (typically 10μm in diameter) as a means to facilitate time-resolved studies on living cell membranes for timescales of minutes. Firstly, microfluidic devices embedding micro-electrodes have been fabricated. These dielectrophoretic (DEP) traps were characterized to assess their potential as a tool for performing in-vitro membrane bio-assays. DEP traps have been subsequently used to trap single-cells near a defined surface and reagents were introduced via microfluidic channels. Incorporation of a Förster Resonance Energy transfer (FRET) acceptor dye within a donor labelled cellular membrane allowed for time-resolved observation of colocalization events using a scanning confocal microscope and fluorescence lifetime imaging. The presence of cholesterol was shown to influence probes localization. Such microfluidic devices coupled with high-resolution imaging of single cells can potentially be used to study the organization dynamics of individual molecules on the membrane of live cells.

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No description available.

The body is continuously exposed to a variety of toxins and metabolic waste products but is able to rid itself of these by using various detoxification mechanisms such as enzymes and transmembrane transporters. A large number of the transporters which play an essential role in the detoxification mechanisms are found in the liver, kidney and intestines. The largest family of transporters is the Solute Carrier (SLC) Superfamily with 255 members in humans. While most SLC transporters are highly specialized, a number of these transporters are polyspecific, generalized transporters that play a major role in the elimination process. Accordingly, the aim of this research programme was to develop novel methodology to enable the study of the interaction of drugs and related compounds with these transporters, in a rapid and facile manner. In the initial studies the application of affinity chromatography was carried out with the use of α1-acid glycoprotein (AGP) column to achieve enantioselective separation of the drug ketamine and its metabolites. The enantioselective separation of ketamine and norketamine from plasma samples was achieved and the assay conducted was sensitive and reproducible. Building upon the experience gained in this work on affinity chromatography and LC-MS, these useful tools for the determination of chiral drugs in biological fluids were also used in the study of drug-protein interactions. With the nicotinic acetylcholine receptors it was possible to explore the use of immobilized liquid chromatographic stationary phases containing drug transporters in on-line high throughput screening (HTS). I Thus having demonstrated that a stationary phase containing immobilized membranes can be used to identify substrate/inhibitors of an expressed receptor/transporter the next and principal phase of the programme was to adapt the methodology to other target biopolymers such as P-glycoprotein which over the last decade has been focused upon for its role in drug resistance in cancer treatments. In particular liquid chromatographic columns containing the drug transporters, P-glycoprotein (Pgp) and human organic cation transporter 1 (hOCT1) were prepared, evaluated and exploited. Initial studies were conducted to confirm the functionality of a stably transfected cell line expressing Pgp (Pgp-(+), LCC6/MDR1 cell line) through a comparison with the non-transfected cell line (Pgp-(-), LCC6 cell line). Initially membranes from the Pgp(+) and Pgp(-) cell lines were then immobilized on immobilized artificial membranes. However, although the resulting column could actively bind the Pgp substrates, strong non-specific interactions with the IAM backbone led to large retention times and peak tailing. A more successful approach was to immobilize on the surface of open tubular capillaries. Such columns were used to sort compounds with or without affinity for Pgp, by comparing the differential retention time on the Pgp (+) and Pgp (-) columns. In this way the non-specific interactions with the constituents of the cellular membrane were compensated for. The results from the sorting by differential chromatography were compared with the behavior of the same compounds in Caco-2 monolayers cultured in 96-well transwell plates, the standard method for the determination of substrates for Pgp. A group of 14 compounds II previously characterized as substrates or non-substrates of Pgp were studied using the chromatographic and Caco-2 methods. In the parallel chromatographic screen, the value of

Bibliography: p. 159-171. / xviii, 171 p., [61] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 1996?