The BK channel is a large conductance calcium-activated voltage- dependent potassium channel. This channel plays a key role as a negative feedback mechanism of membrane excitability and cellular Ca2+. There is substantial evidence suggesting that the Ca2+ activation of the BK channel is regulated by localised Ca2+ release from intracellular stores. The aim of the work presented in this thesis was to develop a novel method of measuring the local Ca2+ concentration controlling the BK channel activation. The p2 subunit, an auxiliary protein of the BK channel, was extracted from MG63 cells and cloned. Subsequently, the aequorin sequence was attached to its C-terminus using splicing by overlapping extension. The recombinant protein retained the features of the native proteins emitting light in response to Ca2+ and showed correct targeting to the ceil membrane. The resultant light emission of the new protein was diminished in comparison to the native aequorin. The p2-Aequorin and a cytosolic Luciferase-aequorin were successfully transfected in a HEK293 cell line which stably express the BK channel a subunit. The expression of the aequorin constructs in HEK293 cells in suspension revealed the presence of intracellular mechanosensitive Ca2+ channels. The main finding of this thesis was that the Ca2+ affecting the BK channel is regulated independently of cytosolic Ca2+ in HEK293 cells. Stimulation with agonists such as carbachol, ATP and cyclopiazonic acid demonstrated clear differences in the magnitude of BK channel microdomain and cytosolic Ca2+ signals. Short term exposure to caffeine induced a significant decrease in the Ca2+ signals near the channel. The addition of extracellular Ca2+ led to large Ca2+ transients close to the BK channel suggesting a store-operated Ca2+ mechanism. The Ca2+ effects produced by carbachol, ATP, caffeine and cyclopizaonic acid indicate a coupling between IP3-induced Ca2+ release from the ER and Ca2+- activation of the BK channel.

The present study was undertaken in an attempt to examine the importance of the NaKMgATPase (the 'sodium pump' enzyme) in cation movements in rat skeletal muscle. Though the 'sodium pump' was first postulated in an attempt to explain some of the cation movements recorded in muscle tissue, at the commencement of this study clear evidence of the presence of the NaKMgATPase was still awaited. Several approaches to the problem were made, using physiological and biochemical preparations. Isolated preparations of muscle, maintained in optimum conditions, were subjected to treatments with reagents known to affect the NaKMgATPase and the changes in cation distribution were observed. Similar preparations were studied with respect to their oxygen consumption, to determine whether treatments known to cause changes in NaKMgATPase activity would in turn lead to alterations in oxidative metabolism as had been reported in a variety of other tissues, e.g. kidney, bain. Biochemical approaches consisted largely of attempts to isolate a fraction from skeletal muscle which demonstrated; the properties of the NaKMgATPase that had been isolated from a wide range of tissues, and was especially active in those tissues in which a great deal of active cation movement was known to occur. The studies of cation distribution were largely inconclusive, as though modified cation movements were clearly seen in various conditions known to inhibit NaKMgATPase activity, it was not possible to identify firmly the position of such an enzyme or indeed that it was the sole site of action. Measurements of oxygen uptake failed to reveal any portion of oxidative metabolism which could be ascribed to metabolic activity linked to NaKMgATPase activity. After a variety of isolation techniques had failed to produce a fraction of skeletal muscle containing a clearly demonstrable NaKMgATPase, a procedure involving exposure of the muscle homogenate to 2 M NaI was successful in separating a membrane fraction exhibiting NaKMgATPase activity from rat diaphragm and hind-limb muscle. Whilst isolation of a 'pure' enzyme was not made, the study showed that the properties of the fraction isolated resembled those described for NaKMgATPases isolated from other mammalian tissues.

Large-conductance Ca^2+ -activated K^+ (BKCa) channels play an important role in the regulation of vascular tone. They are activated by membrane depolarization and increases in local Ca^2+ concentration ([Ca^2+]). Their location in the plasma membrane allows them to be activated by transient releases of Ca^2+ from ryanodine receptors (RyR) in the sarcoplasmic reticulum, termed Ca^2+ sparks, leading to the efflux of K^+ known as a spontaneous transient outward current (STOC). Activation of BKCa channels in this manner provides a negative feedback mechanism to regulate vasoconstriction by hyperpolarizing the cell membrane and so reducing Ca^2+ influx through L-type voltage dependent Ca2+ channels. In this thesis I have investigated the relationship between [Ca^2+]i and membrane potential using inside-out patches excised from smooth muscle cells isolated from rat mesenteric artery. Whole-cell BKCa currents in these cells were also investigated both in the form of STOCs and by using voltage pulses to activate BKCa channels. The effects of the vasoconstrictors endothelin-1 (ET- 1) and angiotensin II (Ang II) on both pulse-induced BKCa currents and STOC amplitude and frequency were investigated. Single BKCa channels with a slope conductance of 189 pA were recorded and their activation was shown to be dependent on [Ca^2+]i and membrane potential. Membrane depolarization also increased BKCa whole-cell current and the frequency and amplitude of STOCs. ET-1 and Ang II were found to inhibit pulse-induced BKCa currents and this effect of ET-1 could be inhibited using a peptide PKC inhibitor. ET-1 and Ang II also caused a decrease in both STOC amplitude and frequency, although the decrease in frequency may be the result of the reduction in amplitude. Finally, 1, 2-dioctanoyl-sn-glycerol (DOG), an analogue of the endogenous PKC activator diacylglycerol (DAG), was seen to inhibit both BKCa whole-cell and single channel currents, possibly due to direct inhibition of BKCa channels.

P2X1 receptors are expressed throughout the body and contribute to a range of physiological process, e.g. thrombosis and smooth muscle tone. The intracellular terminals of the P2X1 receptor have been shown to be involved in channel regulation. The aim of this thesis was to explore the contribution of the intracellular amino and carboxy termini to channel regulation, using concatenation, site-directed mutagenesis and biochemical methods. Trimeric concatenated P2X1 receptors were non-functional.The interactions between the intracellular termini were revealed to be essential for subunit aggregation and channel expression. Subsequently, over 30 cysteine point mutations at both the amino and carboxy domains of the P2X1 receptors were generated and examined by electrophysiological studies with methanethiosulfonate modifications. The conserved TXTXK/R at the amino terminal and the residues around the YXXXK motif at the carboxy terminal were highlighted to be important for channel gating and protein trafficking. Residues close to the P2X1 channel pore were shown to be essential in ion conduction and implied interactions between the conserved regulatory motifs and the channel pore of adjacent residues. Roles of the intracellular domains of P2X1 receptor in regulation by protein kinase were revealed by co-expression of minigenes encoding the sequences of amino/carboxy terminus with WT receptors. The results indicated both amino and carboxy termini contribute to the phorbol ester PMA and GPCR mediated response regulations, and demonstrated the important roles of the conserved TXTXK/R motif and the residues around the YXXXK motif. This research sheds light on the possible interactions between the amino and carboxy termini to P2X1 receptor function. The results will be useful for understanding the intracellular topology and signaling modulation mechanism of P2X1 receptor.

In mammalian cells and fungi, early endosomes form a dynamic compartment that undergoes bi-directional motility along microtubules. Previous work has shown that in the model system Ustilago maydis early endosome motility involves the opposing motor proteins dynein and kinesin-3. Here I performed a detailed analysis of the role of the motors in early endosome motility, using quantitative live cell imaging of kinesin-3, dynein and the endosomal GTPase Rab5a. In the first part of my work, I analysed the role of dynein at MT plus-ends, where the motor forms a strong accumulation that was thought to be involved in capturing early endosomes. I could demonstrate that ~55 dynein motors build up the dynein accumulation. In collaboration with Ms. Congping Lin and Prof. Peter Ashwin (Institute for Mathematics, Exeter), I found theoretical evidence that ~25 dynein motors concentrate and leave the plus-ends stochastically. In addition, dynein motors are captured by an interaction of dynactin and the plus-end binding protein EB1. Together both mechanisms increase the number of motors, which ensures that EEs will be loaded onto dynein before they reach the end of their track. In a second project, I provide evidence that loading of dynein is not restricted to the plus-ends. Instead, dynein leaves the plus-ends and is able to bind to kinesin-3 delivered early endosomes, which changes their transport direction from anterograde to retrograde. Kinesin-3 remains bound to these retrograde EEs. When dynein leaves the organelle, it switches back to anterograde motility. Interestingly, a single dynein wins over three to five kinesin-3 motors. I discuss these findings in the light of current motor cooperation concepts. In a third part, I demonstrated that kinesin-3 has an unexpected role in long-range retrograde endosome motility. In contrast, dynein is only responsible for the distal 10-20 µm. This is possible because most of the hyphal cells contain a symmetric and bi-polar MT array. This MT organization is reminiscent of that in dendrites. Kinesin-3-based retrograde motility is required to mix the organelles and might support long-range communication between both cell poles.

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The identification of the biomolecular interaction partners of small bioactive molecules is a fundamental problem in drug discovery and cell biology. This thesis describes the development of fluorescent chemical probes to identify the biomolecular targets of the known organophosphate toxin, phenyl saligenin phosphate (PSP), and the cardioprotective agent diazoxide. PSP is an organophosphate toxin that irreversibly inhibits hydrolase enzymes such as trypsin and chymotrypsin along with the common organophosphate target acetylcholine esterase. PSP is also suspected of affecting many other cell functions and may interact with a large number of cellular proteins. In this work phenyl saligenin phosphate has been synthesised and its inhibitory effect on the action of transglutaminase 2 (TGase2) demonstrated. Analogues of PSP containing an attached dansyl amide fluorescent group have been prepared and incubated with purified enzymes trypsin, chymotrypsin and TGase2. SDS-PAGE analysis demonstrates effective fluorescent labelling and a covalent interaction between the toxin analogue and the enzymes. The KATP channel opener, diazoxide displays marked cardioprotective effects and is reported to bind to mitochondrial KATP channels. However, the molecular structure of these channels is still largely unknown. This thesis describes the design and the synthesis of a chemical tool to covalently attach fluorescently labels to the proteins which will bind diazoxide. Chemical tools for fluorescent labelling of diazoxide targeted proteins have been prepared. Each consists of a photochemically activated reactive ‘barb’ and coupled fluorescent component linked to modified diazoxide bait. In developing these molecules, a range of functionalised diazoxide bait components were prepared and tested for retained biological activity compared to the parent compound. Two active analogues were linked to either benzophenone or diazirine (photoreactive) and dansyl amide (fluorescent) components. The non-specific photochemical reactivity of these labelling compounds with bovine serine albumin was established. The incubation and photolysis with mitochondrial extracts showed selective photo labelling of only three biomolecular components. The identification of these biomolecules is the subject of on-going investigation.

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