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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.

An important requirement of the patch clamp technique, by which the functional properties of a single ion channel in a cell membrane may be studied, is a tight seal between the membrane and the glass pipette electrode. Hitherto, the properties of ionic channels in mammalian skeletal muscle have been studied mostly in cultured cells which present a relatively clean surface membrane. Here a method is described by which the surface membrane (sarcolemma) of adult skeletal muscle, which is normally covered by a basement membrane, is made accessible for patch clamping. It is a simplified version of a procedure previously used for the production of membrane vesicles from frog muscle by treatment with a high KCl solution containing collagenase. The method has proved applicable to human muscle biopsy samples and so may be useful in clinical investigations. In exploring sarcolemmal vesicles from rat and human muscles the following ion channels could be readily demonstrated in high KCl solution: (i) ATP-sensitive K channels, (ii) delayed rectifier K channels, (iii) Ca-activated K channels. In addition, the following channels could be found though more rarely: (iv) inwardly rectifying K channels, (v) a channel of small conductance, (vi) a chloride channel. In subsequent work, attention was focused on the inwardly rectifying K channel as that channel had hitherto been little studied. The role of Mg2+ in producing inward rectification was demonstrated by comparing the properties of the channel in patches left attached to vesicles and detached into Mg2+-free solution. In the presence of internal Mg2+ no current passes when an outward driving force is applied and the inward current flowing in response to a small inside negative potential shows frequent interruptions (flickery block). In the absence of internal Mg2+, flickery block disappears and current flows instantaneously as readily in outward as in inward direction. When Mg2+ does not block the channel the existence of an intrinsic gating process which operates so as to close the channel on depolarization becomes evident. The time and voltage dependence of this gating process is described. The relationship between channel open probability (Po) and voltage is a steep one with a slope factor of 4.13mV. These results are compared with the already known properties of inwardly rectifying K channels in cardiac muscle. With the patch clamp technique, membrane patches may be detached from cells or vesicles so as to allow access to the inner surface of the membrane. The effect of changes in the composition of the "intracellular" solution on channel activity may therefore be studied. To change the composition of the internal solution, detached patches were exposed to a stream of flowing solution of variable composition. Under such conditions it was found that the kinetic properties of the inwardly rectifying K channel were influenced by flow itself. This entirely new phenomenon was found also in patches detached from dispersed muscle fibres from rat flexor digitorum brevis. Analysis of the new phenomenon showed that as a result of flow, the maximum open probability is decreased and the midpoint of the Po-voltage relation is shifted to the right by more than 20mV. No evidence could be found for the existence of a local concentration gradient sensitive to flow. Application of suction to the patch pipette showed the inwardly rectifying channels not be sensitive to membrane stretch. The possibility is contemplated that shear stress upon the inner face of the patch modulates the kinetic behaviour of the channel. From experiments on whole cells, it is known that the inwardly rectifying K conductance is sensitive to changes in intracellular pH (pHi). This effect was studied at the single channel level on patches excised from rat sarcolemmal vesicles and subjected to changes in pHi under conditions of constant flow. When pHi was lowered from 7.4 to 6.9, 6.5 or 6. 0, the channel continued to show transitions between open and short closed states, but this activity became interrupted by long lasting closures, the more so the lower pHi On exposure to pHi 5.0, the channel soon closed down until pHi was returned to 7.4. During the periods of channel activity at pHi below 7.4, Po decreased in graded fashion the lower pHi. This reduction in Po during the periods of channel activity was not obviously voltage dependent and cannot therefore be attributed to a shift in the Po-voltage relation.

Bone turnover is regulated by a cocktail of hormones and signalling factors controlling key cell processes such as proliferation, differentiation, mineralisation and apoptosis. Disruption to the overall mineralisation-resorption balance leads to bone disorders, such as osteoporosis - a 'silent' disease affecting around 7 million people in England and Wales. Ion channels that are presumed targets for bone signalling factors include voltage-gated K channels, ATP-dependent K channels and transient receptor potential TRP channels, and several of these channel-types reportedly have roles in cell proliferation, apoptosis, and differentiation in various tissues. This Thesis shows that human osteoblasts express a number of channels in these families, including maxi-K, ATP-dependent K channels, TRPV1 and TRPM7. The maxi-K channel, displaying characteristic electrophysiological hallmarks, is abundant in patch-clamp recordings of primary human osteoblasts implying a functional role, and the Katp agonist pinacidil is shown to promote osteoblast proliferation. Electrophysiological evidence for the TRPVI channel is not found, although the mRNA signal for a TRPVI splice variant TRPVlb may provide an answer, as it renders the channel less sensitive to capsaicin and protons. However, Ca imaging indicates that osteoblastic TRPV1 channels allow Ca2 influx, and are sensitive to 1 µM capsaicin and protons. In functional studies the TRPVI ligands capsaicin and capsazepine do not influence mineralisation, but interestingly the TRPVI agonists capsaicin, resiniferatoxin and anandamide appear to prevent differentiation of osteoblastic pre-cursor cells to adipocytes, and instead encourage maturation along the osteoblast pathway, whilst TRPV1 antagonists do not affect adipocyte differentiation. In conclusion, a number of K channels and the TRPV1 channel are expressed in osteoblasts and may have important putative roles in osteoblast cell function. Further steps are required to confirm this before the channels can be considered targets for drug development to treat bone disorders.

Mechanisms underlying relaxation in response to inhibitory NANC nerve stimulation and putative neurotransmitters of these nerves have been examined in the guinea-pig internal anal sphincter (IAS) and compared with those in the bovine retractor penis muscle (ERP) and guinea-pig taenia caeci. Two types of techniques were employed. One which measured the effects of nerve stimulation and drugs on electrical membrane properties where intracellular microelectrode and simultaneous mechanical recording techniques were used. Drugs, for example ATP or cromakalim were applied by perfusion in the Krebs' solution, microinjection into the bath, or by hydrostatic pressure ejection. A second method assessed the underlying biochemical changes accompanying relaxation by measuring alterations in second messenger systems, for example cyclic AMP and cyclic GMP using radiomimmunoassay techniques. Electrical events were clearly an important accompaniment to mechanical inhibition in the IAS. Field stimulation (single pulse and 5 pulses at 5, 10 and 20 Hz; 0.5ms; supramaximal voltage) produced large inhibitory junction potentials of up to 15mV in amplitude which accompanied relaxation of 80% of muscle tone. Indeed, hyperpolarising electrotonic current passed into the IAS produced relaxation. The neurotransmitter which is released by field stimulation of the inhibitory nerves is probably ATP since exogenous application of purine by hydrostatic pressure ejection (5.8x10-4M; 10-55ms) produced a dose-dependent hyperpolarisation. The membrane potential change was similar in size, rate of decline and duration to the ijp. Neither hyperpolarisation nor relaxation could be achieved with the P2x-purinergic agonist, betaMeATP (10-5-10-3M) or the P2-purinergic agonist adenosine (10-3M) thus ATP was acting on the P2y-purinergic receptor. Inhibitory NANC neurotransmission was not peptidergic since VIP (10-7-10-5M), bradykinin (10-3M), neuropeptide Y (10-5M), bombesin (10-5M), leu-enkephalin (1.8x10-4M), met-enkephalin (1.8x10-5M), somatostatin (10-6-10-3M) and substance P (7.6x10-6 - 7.6x10-4M) each had no effect on the membrane potential of the IAS. There is also evidence that stimulation of -adrenoceptors by isoprenaline (10^-9 - 10^-5M) produced relaxation which was accompanied hyperpolarisation of the IAS. In all cases where hyperpolarisation and relaxation are associated in the IAS, the mechanism underlying the electrical change appeared to be an increase in K^+ conductance. Apamin (4.5 x 10^-6M) which blocks certain Ca^2+-mediated K^+ channels, antagonised the electrical and mechanical responses produced by field stimulation and ATP. Similarly, TEA (8x10^-2M), which blocks most K^+ channels, antagonised the hyperpolarisations and relaxations produced by field stimulation, ATP and isoprenaline. Indeed, the K^+ channel activator cromakalim (10^-9-10^-5M) produced hyperpolarisation and relaxation of the IAS suggesting that an increase in K^+ conductance is important in the mediation of mechanical inhibition of the IAS. Relaxation of the IAS was also produced without a significant change in membrane potential by altering the levels of cyclic nucleotides within the smooth muscle cells of the IAS. Forskolin (10^-9-10^-5M), which activates adenylate cyclase with a subsequent increase in cyclic AMP, relaxed the IAS. Similarly, sodium nitroprusside (10^-9-10^-4M) - a cyclic GMP phosphodiesterase inhibitor, and 8-bromo-cyclic GMP (10^-4M) each increased cyclic GMP and produced relaxation of the IAS. Direct measurement of cyclic nucleotide levels of the IAS showed that field stimulation (80 pulses at 8H_Z; 0.5ms; supramaximal voltage) and ATP (10^-4) elevated the cyclic AMP and cyclic GMP contents of the IAS. All other stimuli which produced slow, prolonged electrical and mechanical changes increased the level of only one cyclic nucleotide. Isoprenaline (10^-4M), cromakalim (10^-5M) and forskolin (10^-5) increased cyclic AMP content while sodium nitroprusside (10^-5M) increased the cyclic GMP content. Further investigation of other second messenger systems involved in relaxation of the IAS showed that increase in inositol phosphate turnover was not associated with stimulation of inhibitory P_2y-purinoceptors by ATP (10^-2M) in the IAS. However, an increase in inositol phosphate accumulation was produced by noradrenaline (10^-4M) and associated with contraction. A method was devised to measure the intraluminal pressure changes of the internal anal spincter in the anaesthetised guinea-pig using a Millar pressure transducer. Using this method the in vitro results were largely confirmed by this in vivo study. Basal intraluminal sphincter pressure was increased by noradrenaline acting on -adrenoceptors and decreased by isoprenaline acting on -adrenoceptors, ATP on P_2y-purinoceptors and 2-chloroadenosine on P_1-purinoceptors.

Application of 5-hydroxytryptamine induces a voltage-dependent inward current in voltage clamped C1 neurones of Helix aspersa. This response has been shown to be the result of a decrease in K conductance and was studied using patch clamp and voltage clamp techniques. Single channel K currents were recorded from cell-attached patches of the C1 neurone. Two sizes of unitary outward currents were commonly observed. The I-V relationships of both these unitary currents could be fitted by the Goldman-Hodgkin-Katz equation for a K current, having slope conductances of around 14pS and 54pS at +10mV, patch potential. Experiments, altering the K concentration in the patch pipette, or on the outer surface of isolated outside-out patches, suggested that these unitary currents were due to the flow of K+ ions. Application of 5-hydroxytryptamine onto the C1 neurone, from out with the patch pipette, reduced the activity of the larger K channels, recorded in the cell-attached patch. Both Ca-dependent, and Ca-independent K channels were observed on isolated inside-out membrane patches. It was unclear which of these types of channel corresponded to the 5-hydroxytryptamine sensitive channel in the cell-attached patch. Voltage clamp experiments also gave confusing results regarding the Ca-dependency of the 5-hydroxytryptamine response. However, in some C1 neurones 5-hydroxytryptamine caused a flattening of the "N" shaped I-V relationship, suggesting a decrease in the Ca-dependent outward current. The possibility that more than one type of K current was suppressed by 5-hydroxytryptamine was considered. The effect of phosphodiesterase inhibitors was consistent with a mediation of the 5-hydroxytryptamine response by cyclic nucleotides. Injection of cAMP induced an inward current in the C1 neurone. Single channel outward currents, which reversed at -50mV, were recorded from the A neurone. The activity of these channels was increased by 5-hydroxytryptamine, but their ionic nature was uncertain. Unitary outward currents of the M neurone were also recorded.

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis and pathogen defense. A subset of SNAREs, including the Arabidopsis SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K+ channels and to moderate their gating at the plasma membrane. R-SNAREs, also known as vesicle-associated membrane proteins (VAMPs), are most commonly associated with trafficking vesicles. In guard cells of Arabidopsis, ABA-dependent stomatal closure was inhibited when the expression of VAMP71 family genes were suppressed by an antisense VAMP711 construct. Such results reveal that VAMPs play an important role in plant response to stress, especially in the regulation of stomatal closure under ABA treatment. Two R-SNAREs, VAMP721 and VAMP722, are known to assemble in SNARE core complexes with SYP121 and with its closest homolog SYP122, which raises the question whether the channel interaction might extend to the R-SNAREs leading to VAMP regulating channel gating. To answer this question, I investigated the interaction between the VAMP7 proteins with KC1 and KAT1 K+ channels by mating based Split-Ubiquitin System (mbSUS) assay, and verified these interactions by ratiometric bimolecular fluorescence complementation (rBiFC) assay. I found VAMP721 and VAMP722, but not VAMP723, interacted with the channels. The selective binding was associated with the VAMP longin domain, notably with Tyr57. What is the effect of the VAMP-K+ channel interaction on transmembrane ion transport and vesicle traffic? I found VAMP721 affected K+ channel gating and suppressed the K+ current within the physiological voltage range by electrophysiological analysis in Xenopus oocytes and in VAMP over-expression wild type Arabidopsis. The effect of VAMP721 on K+ channel regulation was opposite to the action of SYP121 on K+ channel. From localization, interaction and electrophysiological studies, I was able to show that Tyr57 is a key residue required both for VAMP-dependent gating of the K channels and for localization of the VAMPs at the plasma membrane. For vesicle traffic, I found overexpression of full length VAMP721 in Arabidopsis seedlings blocked the secretion of secYFP while coexpressing with KC1 K+ channel rescued the traffic block, demonstrating a potential action of VAMP-K+ channel interaction on secretory traffic. These results add to understanding the interaction between SNARE and K+ channels that link membrane traffic with osmotically active solute transport in the plant.

Background Ovarian cancer is the second most common cancer of the female genital tract in the United Kingdom (UK), accounting for 6% of female deaths due to cancer. This cancer is associated with poor survival and there is a need for new treatments in addition to existing chemotherapy to improve survival. Potassium (K) channels have shown to be promising therapeutic targets in the treatment of various cancers. We sought to determine the expression of EAG and HERG potassium channels in ovarian cancer and establish their role in cell proliferation. Material and Methods The Trent Research and Ethics Committee granted initial approval for the study. Informed consent was obtained from all patients undergoing surgery for ovarian cancer and oopherectomy for benign causes for their participation in the study. The tissues were prospectively collected and analysed anonymously. Immunoflurescence. Immunohistochemistry, Western blotting and Reverse transcriptase Polymerase chain reaction experiments were used to determine the expression of EAG and HERG potassium channels in ovarian cancer and normal ovaries. The effect of the EAG blockers (imipramine and clofilium) and HERG blockers (E-4031 and ergtoxin) on SK~OV-3 ovarian cancer cell line proliferation was assessed using the MTS assay with further investigation of their role in the cell cycle and apoptosis determined by flow cytometry. Results EAG and HERG potassium channels have significant (P<0.001) higher expression in patients with ovarian cancer compared to normal ovarian cells and high expression of EAG channels is significantly associated with poor survival (P=0.016) unlike HERG channel expression where there was no correlation with survival. There was also a significant association of EAG staining with high tumour grade (p=0.014) and presence of residual disease (p=0.011). Proliferation of SK-OV-3 cells was significantly (p<0.001) inhibited after treatment with voltage gated K+ channel blockers. There was significant inhibition of proliferation of SK-OV- 3 ovarian cancer cells by imipramine (p<0.001) and ergtoxin (p<0.05) at 72 hours of culture. Incubation of cells with ergtoxin led to the accumulation of cells in the S and G2IM phase while cells accumulated in S phase after incubation with E-403 1, with no effect on apoptosis.imipramine did not affect the cell cycle but increased the proportion ofSK-OV-3 cells undergoing early apoptosis. Conclusion Both EAG and HERG channels are expressed in ovarian cancer and have a role in cell proliferation. Higher expression of EAG channel is associated with poor prognosis suggesting its role as a poor prognostic marker in patients with ovarian cancer. HERG channels affect the cell cycle while EAG channels are implicated in the inhibition of apoptosis of ovarian cancer cells. EAG channels have the potential to be used as new therapeutic targets in patients with ovarian cancer with use of anti-EAG monoclonal antibody.

Huntington’s disease (HD) has classically been categorized as a neurodegenerative disorder. However, the expression of the disease-causing mutated huntingtin gene in skeletal muscle may contribute to the symptoms of HD, namely those that involve involuntary muscle contraction. In the R6/2 transgenic mouse model of HD, we previously observed ion channel defects that could contribute to involuntary muscle contraction. Here, in R6/2 muscle we investigated the consequence of these ion channel defects on action potentials (APs), the first step in excitation-contraction (EC) coupling. We found that the ion channel defects were associated with depolarizing the baseline membrane potential during AP trains. We also observed changes in the AP waveform in R6/2 muscle, including a prolonged falling phase, which was associated with reduced K + channel expression (another ion channel defect). Next, we investigated the consequence of prolonged APs on intracellular Ca 2+ release flux, the second step in EC coupling. We observed an increase in Ca 2+ release flux duration, which compensated for a reduction in peak Ca 2+ release flux, resulting in normal levels of Ca 2+ available for contraction in R6/2 muscle. Finally, we investigated the consequence of prolonged APs and normal levels of Ca 2+ available for contraction on muscle force generation, the final step in EC coupling. We found that, when accounting for muscle atrophy, the force generated by one AP (twitch) was normal in R6/2 mice. This could be explained by the reduced parvalbumin and normal levels of Ca 2+ available for contraction we observed in R6/2 muscle. We conclude that downregulation of K + channels to prolong APs is a compensatory mechanism for muscle weakness that leads to increased Ca 2+ release duration and force production in R6/2 muscle. This is the first study to examine the entire EC coupling sequence in HD muscle, revealing the importance of the AP waveform in contributing to muscle force generation.

Inorganic arsenic and elevated glucose concentrations increase endothelial production of superoxide, which impairs endothelium-derived nitric oxide bioavailability and associated nitric oxide-dependent arterial relaxations. However, there is now evidence in animal models of diabetes mellitus that relaxations attributed to endothelium-dependent smooth muscle hyperpolarization or endothelium-derived hyperpolarizing factor (EDHF) may be augmented and serve to compensate for the loss of nitric oxide, thereby maintaining arterial responsiveness. The effects of arsenite and elevated glucose concentrations on "EDHF-type" relaxations were thus investigated in isolated rabbit iliac artery rings using the G-protein-coupled agonist acetylcholine and the sarcoendoplasmic reticulum Ca2+ ATPase inhibitor cyclopiazonic acid. Arsenite and elevated glucose both potentiated EDHF-type relaxations evoked by cyclopiazonic acid. Differential effects of arsenite and glucose against EDHF-type responses to acetylcholine were identified in that arsenite attenuated relaxation, whereas glucose potentiated relaxation. Further experiments showed that the arsenite- and glucose-augmented components of relaxation were reversed to control levels by the hydrogen peroxide scavenger catalase and the NADPH oxidase inhibitor apocynin. Arsenite-augmented responses were also reversed by the cell-permeable superoxide dismutase/catalase mimetic manganese porphyrin. It follows that hydrogen peroxide derived from NADPH oxidase may augment EDHF-type relaxations in diabetes mellitus and arsenic toxicity, thus maintaining endothelial control of arterial tone when nitric oxide bioavailability is impaired by oxidative stress. These results are consistent with the demonstrations that hydrogen peroxide augments EDHF-type relaxations in the rabbit iliac artery by promoting endothelial Ca2+ mobilization and enhancing the opening of endothelial Ca2+-activated K + channels, and that the increased activity of these channels underpins augmented EDHF-type arterial relaxations in animal models of diabetes.

Beyond the linear sequence of the genome, the epigenome dynamically controls chromatin remodeling and transcriptional poise without inducing changes in DNA sequence. These epigenetic mechanisms, particularly modifications to the histone proteins which mediate chromatin compaction, play critical roles in health and disease. To empower further successes in epigenetic therapies, current tools for understanding these systems must be improved. In particular, Chromatin Immunoprecipitation (ChIP) offers the gold standard technique for probing DNA associations with modified histones. This protocol isolates chromatin complexes (nucleosomes) for affinity purification of only those displaying the modification of interest. Final analysis of captured DNA can identify novel pathways or provide a quantitative view of current transcriptional state. Unfortunately, this assay’s large sample requirements and laborious, user-dependent protocol have generally proven prohibitive to widespread clinical deployment. This doctoral dissertation presents the development of a semi-automated, miniaturized platform for performing the ChIP assay using droplet microfluidics, capitalizing on an assay format in which pL-scale encapsulation in immiscible oil limits sample loss and accelerates mass transfer. A wide array of supporting technologies empower dynamic control over droplet composition and reaction conditions, making each droplet analogous to a reaction vessel (but for handling up to thousands of discrete volumes every second). Finally, these platforms serially process dynamic samples sizes by adjusting the total number of droplets handled, not individual reagent conditions, suggesting the potential for dynamic scalability of ChIP across a range of sample sizes. First, the dissertation informs the direction of the work in the context of epigenetic and microfluidic challenges. Chapter I motivates opportunities and hindrances in the analytical characterization of the epigenome from the perspective of precision medicine, providing a survey of both conventional methods and emerging microfluidic techniques. Then, it outlines strategies to address technical difficulties of the ChIP protocol using droplet-based methods. Importantly, it identifies points-of-need in droplet technologies, especially for affinity purification capabilities essential to ChIP (and to other important bioassays). Next, the dissertation describes fundamental advances in droplet microfluidic technologies. Chapter II describes the K-channel, a multifunctional, switchable, and scalable approach to improve droplet handling and chemical manipulation. K-channels performed reagent injection (0-100% of droplet volume) and magnetically biased droplet splitting (1:1 daughter droplet ratio, 96% magnetic particles retained), among other operations. Chapter III presents translation of droplet techniques into mass manufacturable thermoplastic materials, anticipating future needs in practically deploying these technologies. Chapter IV describes the Counter-Current Continuous Phase Extraction module for determinant control over droplet packing fraction (50 - 85%) and applies it with the K-channel for high temporal resolution analysis (~100 ms) of flow behavior for confined droplets (example incubation time dispersions reduced by up to 50%). Lastly, the dissertation describes a critical innovation in droplet microfluidic purifications and, empowered by it, the full development and characterization of the droplet-based ChIP technology. Chapter V introduces the Coalesce-Attract-Resegment Washing (CAR-Wash) platform for efficient droplet-mediated particle washing (greater than 100-fold dilutions achieved at 500 Hz processing with 98% particle capture) and demonstrates its efficacy in the bioanalytical context of affinity separations. Chapter VI fully realizes the droplet microfluidic ChIP platform with automated cell lysis, chromatin digestion, immunoprecipitation, and particle washing, achieving successful application to two modified histone targets (H3K4me3 and H3K27me3). Finally, Chapter VII concludes the work and offers future directions in both technical improvements and general directions for droplet bioassay development.

Protein kinase inhibition by staurosporine causes apoptotic volume decrease involving potassium (K) channels in immortalized human B3 lens epithelial cells (LECs). Here, the effect of two pro-apoptotic protein kinase C (PKC) inhibitors [12-O-tetradecanoyl-phorbol-13-acetate (TPA) and chelerythrine (CET)] were studied on membrane K transport in a fetal human LEC line (FHL124) by western blotting, immunofluorescence, ion flux, ATP, apoptosis, and biotinylation assays. Long term TPA exposure (0-6 h) inhibited 75% of Na-K-2Cl cotransport (NKCC). In contrast, short term (0-20 min) exposure to 50 µM CET reduced Na/K pump and NKCC by >90% and >70%, respectively, without retrieval from the membrane into the cytosol, loss of ATP and early signs of apoptosis. CET (10-30 µM) decreased cell K by 33%. Results suggest PKC modulation through the use of pro-apoptotic agents caused major early membrane changes independently affecting K channels, the Na/K pump and NKCC function, prior to rise of any significant apoptosis.

In this thesis, the patch clamp technique has been used to examine single channels of cultured human retinal epithelial (RPE) cells. Three main channel types were detected in the cell-attached recording mode. They had mean conductances of 24 pS, 59 pS and 96 pS with physiological solutions present. All three channel types were primarily permeable to potassium. Under physiological conditions the activity of all three channel types was voltage dependant, and increased as the patches were depolarized. However, all channel types had low levels of activity at the membrane potential. The activity of the 96 pS channel type was time dependent; the channel inactivated in response to depolarizing steps. This channel type was also sensitive to the concentration of calcium; when the cells were bathed in low calcium solution the channel open probability fell towards zero. The 96 pS channel is thus similar to the maxi-K channels in other epithelia. The presence of microvilli on the top surface of these cells supports a comparison to the apical membrane. Single channel recordings on fresh bovine RPE were also attempted, but most of the channel openings were obscured by a large, noisy conductance. The voltage-sensitivity of channel activity found in cultured cells suggests that these channels may provide a mechanism for integrating dynamic transport across the RPE.

The effects of potassium channel opening drugs (KCOs) were investigated in an in vitro vagally innervated tube preparation of the guinea-pig trachea. It has previously been reported that croraakalim inhibits transmission in pulmonary cholinergic nerves, (Hall & Maclagan, 1988) . This thesis further investigates the action of KCOs on pulmonary nerves using cromakalim (racemate), lemakalim ((-) isomer), BRL38226 ((+) isomer) and SDZPC0400 (an unrelated (-) isomer). Cromakalim, lemakalim and SDZPC0400 did not significantly affect the dose related rise in intraluminal pressure elicited by the spasmogens acetylcholine, histamine or the thromboxane mimetic U46619. Therefore the KCOs do not appear to have any significant antispasmodic action nor do they inhibit postjunctional responses elicited by muscarinic receptor activation. Contractions of the trachea evoked by preganglionic stimulation of the cholinergic vagus nerve (PCS) or transmural stimulation in the presence of hexamethonium (TMS) were both inhibited by cumulative doses of cromakalim, lemakalim or SDZPC0400. The KCOs act prejunctionally on pulmonary parasympathetic cholinergic nerves since they have no action on responses to exogenous acetylcholine which contracts the trachea by acting on postjunctional muscarinic receptors. As the inhibitory effect of cromakalim was greater on responses to PCS than TMS, this suggests that cromakalim has an inhibitory action at the ganglia as well as at the final nerve terminals. The specific blocker of ATP-operated potassium channels, glibenclamide significantly reduced the inhibitory action of cromakalim, lemakalim and SDZPC0400 on contractions of the guinea-pig trachea elicited by PCS and TMS suggesting that ATP operated K"*" channels are opened by these KCOs in the pulmonary nerves, The (+) isomer of cromakalim, BRL38226 caused only a slight inhibition of responses to nerve stimulation, but its presence significantly reduced the effect of lemakalim on responses to both PCS and TMS. It is possible that BRL38226 is blocking K+ channels opened by lemakalim in the pulmonary nerves. Transmitter release in the pulmonary cholinergic nerves could be inhibited by prejunctional muscarinic M2 autoreceptors. An investigation was made into a possible interaction of muscarinic autoreceptors and potassium channels using methoctramine and pilocarpine. However results obtained did not support this theory. Inhibition of nerve-induced contractions (PGS and IMS) by cromakalim was unaffected by the presence of the M2 muscarinic antagonist, methoctramine. The M2 receptor agonist, pilocarpine caused an inhibition of contractions of the trachea to PGS and TMS which was unaffected by the presence of glibenclamide When tracheal tone was raised by the spasmogen histamine, NANC mediated relaxations were seen to transmural stimulation (in the presence of atropine, 1-propranolol and hexamethonium). Cromakalim and lemakalim facilitated these NANC mediated relaxations of the guinea-pig trachea. Experiments were performed to see if this facilitatory effect was due to a potentiation of the effect of the inhibitory NANC transmitter (possibly VIP) on the trachealis muscle. Relaxations of the trachea to VIP were facilitated by cromakalim and lemakalim to the same extent as NANCergic relaxations indicating a postjunctional action of KCOs on facilitation of NANCergic transmission. In summary, in the isolated guinea-pig trachea, opening of potassium channels inhibits cholinergic neurotransmission but facilitates NANC mediated relaxations. The in vivo bronchodilator effect of cromakalim and lemakalim in vivo is probably due to a combination of inhibition of cholinergic bronchoconstriction, a facilitation of NANC mediated relaxation and a direct effect on airway smooth muscle. These properties suggest that potassium channel opening drugs should have potential for clinical use in asthma therapy.

Obesity is an epidemic in today's society. Weight loss requires negative energy balance, which can induce adaptive thermogenesis-the reduction of energy expenditure (EE) beyond that accounted for by the weight lost. Adaptive thermogenesis varies between individuals. Here, I investigated the underlying mechanisms responsible for the interindividual variation in adaptive thermogenesis. First, I examined EE and physical activity before and after 21 days of 50% calorie restriction in male and female rats with lean and obesity prone phenotypes-rat strains selectively bred for high and low intrinsic aerobic capacity (HCR and LCR, respectively). My data suggest that calorie restriction decreased EE more than was predicted by loss of weight and lean mass, demonstrating adaptive thermogenesis. Both groups showed similar suppression in resting EE. The calorie restriction-induced suppression in non-resting EE, which includes activity EE, was significantly greater in HCR than in LCR in both male and female rats. Calorie restriction also significantly suppressed physical activity in HCR but not LCR, but physical activity was still higher in HCR than in LCR even after calorie restriction. Weight loss did not differ between groups. These results suggest that individual differences in calorie restriction-induced adaptive thermogenesis may be accounted for by variation in aerobic capacity. Moreover, it is likely activity EE, not resting or basal metabolism, accounts for the individual variation in adaptive thermogenesis. Next, I examined how calorie restriction impacts the molecular mechanisms of skeletal muscle work efficiency and how the suppression in activity EE relates to these changes in molecular pathways in skeletal muscle at the gene and protein levels. I tested the hypotheses that (1) muscle molecular pathways important for thermogenesis decrease after calorie restriction, (2) calorie restriction enhances pathways important for energy conservation and lipid handling, and (3) these muscle responses are dependent on aerobic capacity. These muscle pathways were compared between HCR and LCR at baseline and after 2 and 21 days of calorie restriction. The data suggest potential roles of UCPs, ATP-gated K-channels and MED1 in the differential adaptive thermogenesis observed between HCR and LCR. One or more of the changes observed in the HCR may contribute to increased muscle efficiency and therefore suppressed non-resting EE. There were differences seen between short-term (2-day) vs. long-term (21-day) expression levels suggesting possible adaptations leading to restricted weight loss. No one particular protein stands out that may play a singular role in the muscle adaptive thermogenesis induced by calorie restriction.Finally, using micropunched brain samples collected from the same animals, I observed region-specific changes in brain MC3R, MC4R, and AgRP. Calorie restriction-induced changes in melanocortin vs. AgRP signaling may serve to counteract energy shortage by inducing adaptive thermogenesis and suppressing physical activity, promoting energy conservation during food shortages. In conclusion these results suggest that differential adaptive thermogenesis depends more on activity (non-resting) energy expenditure and varies with changes seen in molecular mechanisms in the skeletal muscle and opposing MCR-AgRP signaling during calorie restriction.

In addition to acting in synaptic transmission, neurotransmitters have been shown to play roles in the development of nervous system. Developing neurons extend neurites to connect to their target cells, and growth cones at the tip of growing neurites are critical for pathfinding. Although evidence for the regulation of axonal growth and growth cone guidance by neurotransmitters and neuromodulators is emerging, less is known about the mechanisms by which neurotransmitters affect developing neurons. Here, I focus on three neurotransmitters/ neuromodulators and describe their actions (a) at the level of growth cone, especially on filopodia, which serve as sensors that allow growth cones to probe the environment they are traversing, and (b) on how neurotransmitters modulate neuronal electrical properties, which, in itself, have been shown to affect neurite extension. The goals of this dissertation are to investigate 1) the cholinergic modulation of neuronal activity and its effects on growth cone motility; 2) the excitatory modulation of neuronal excitability by nitric oxide (NO); and 3) the inhibitory modulation of neuronal activity by dopamine (DA). The work uses a well-established model system to investigate growth cone motility and neuronal activity: identified neurons from the pond snail Helisoma trivolvis studied in cell culture or in the intact ganglion in situ. The study of B5 neurons demonstrates that acetylcholine (ACh) induces filopodial elongation, which is mediated by opening of nicotinic ACh receptors, membrane depolarization, and elevation of intracellular Ca level in growth cones. This dissertation also shows that NO inhibits two types of Ca-activated K channels to depolarize the membrane potential of B19 neurons. Additionally, the study reveals that DA serves as an inhibitory neurotransmitter to hyperpolarize and silence the electrical activity of firing B5 neurons via a D2-like receptor/PLC/K channel pathway. Taken together, this dissertation elucidates novel cellular mechanisms through which neurotransmitters can regulate growth cone motility and neuronal electrical properties, further supporting evidence for potential roles of neurotransmitters in axon pathfinding and synaptic transmission in vivo.

This dissertation is dedicated to understanding the dynamical mechanisms underlying early afterdepolarization (EAD)-mediated arrhythmias. EADs are a richly mined area of research that has resulted in little therapeutic benefit at the bedside since their first description in the early 1980's. In an effort to provide a fresh perspective on EADs, I take an experimental and computational non-linear dynamics approach to studying these arrhythmias that plague millions worldwide and are associated with significant cost burden and high mortality. Here, I show that the dual Hopf-homoclinic bifurcation mechanism for generating EADs may be responsible for EAD-mediated arrhythmias in cardiac disease, providing new therapeutic targets and expanding the current concept of reduced repolarization reserve. Also, I provide evidence that certain pathological cardiac tissue substrates may exhibit the unique dynamical property of bistability (ie. switch-like behavior) in the form of biexcitability, the ability to switch between INa- and ICa-mediated propagation. This unique property of biexcitability may provide significant mechanistic insight into the seemingly paradoxical properties of certain EAD-mediated arrhythmias such as Torsades de Pointes (TdP), that is uniquely characterized by a slow rate and undulating QRS complexes on ECG. Lastly, I show that activation of KATP channels (inward rectifying K channels that open when cellular ATP levels fall) "flips the switch" from ICa- to INa-mediated propagation. Consistent with clinical observations of TdP and polymorphic ventricular tachycardia (PVT), the majority of ICa-dependent reentries spontaneously terminate following KATP channel activation, with a minority of ICa-dependent reentries converting to INa-dependent reentries. These results suggest that in the clinical setting, KATP channels may serve as an "emergency repolarization reserve" to spontaneously terminate most episodes of ICa-dependent arrhythmias such as PVT and TdP, but may also facilitate the occasional degeneration of these arrhythmias to INa-dependent VT/VF. These findings provide a fresh perspective on EAD-mediated arrhtyhmias that may lead to novel therapeutics at the bedside.

Living cells maintain constant volume in response to physiological stresses by altering trans-membrane ion, solute and water flow. In the present study, early and late membrane transport changes in human lens epithelial (HLE-B3) cells under hyposmotic and apoptotic stress were compared. Cell potassium (Ki), rubidium (Rbi)-uptake and water content were measured by atomic absorption spectrophotometry and gravimetry, respectively. Intracellular chloride concentration [Cl]i was determined with the fluorescence dye N- (ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE), during regulatory volume decrease (RVD) after hyposmotic stress and apoptotic volume decrease (AVD) induced by staurosporine (STP), a protein-kinase inhibitor. Cell water increased in hyposmotic balanced salt solution (BSS) as compared to isosmotic BSS by > 2-fold at 5 min and decreased within 30 min to baseline values, accompanied by K efflux commensurate with cell swelling and RVD. Clotrimazole (CTZ) reduced Ki and water loss in hyposmotic BSS, and Rbi uptake, suggesting a role for intermediate conductance K (IK) channels during RVD. This was verified by reverse transcriptase polymerase chain reaction (RT-PCR), Western blot and immunochemistry of HLE-B3 cells. Apoptosis measured 2 h after 2 μM STP treatment was significant and accompanied by 20% and 41% water loss after 30 min and 2 h, respectively. During apoptosis, Ki fell at a rate of 0.007/min until 30 min and decreased to 42% by 2 h. [Cl]i decreased by 25% and 19% below baseline values at 30 min and 60 min, respectively. High extracellular K, and 2 mM 4-aminopyridine (4-AP) significantly reduced apoptosis whereas the presence of CTZ did not. Annexin labeling appeared at 15 min suggesting lipid changes preceded the water and K loss initiating AVD. In contrast, loss of K, water, and most likely Cl differed by an order of magnitude between RVD and AVD at early time points. Quantitative PCR revealed decreased expression of IK channels during prolonged hyposmotic and apoptotic stress. These studies suggest that in HLE-B3 cells, IK channels partake in RVD, but cannot yet be discounted in AVD, which in part commences through 4-AP-sensitive voltage-gated K channels

My research focuses on understanding the mechanisms of eukaryotic inwardly rectifying potassium channels (Kir), which are responsible for creating membrane potential stability. N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion- permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1L) and the G protein-sensitive Kir3.1 (Kir3.1S) channels in the absence of PIP2 show the cytoplasmic ion- permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1L and Kir3.1S suggest a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structure comparison reveals a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1L that is important for modulating inward rectification. The Kir2.1L structural allows us the first structural look at the potential causes of Andersen's Syndrome, which is caused by defects in the inwardly rectifying potassium channel 2.1 (Kir2.1). We have characterized individual Andersen's Syndrome mutants R218Q, G300V, E303K, and [delta]314-315 and have shown that they have multiple affects on the ability of Kir2.1 channel's cytoplasmic domains to assemble into proper tetrameric assemblies. The 2.0 Å x-ray crystal structure of the N- and C-terminal cytoplasmic domains of Kir2.1 R218Q combined with T309K, reveals the G-loop and CD loop disruptions by the R218Q mutation. We show that E303 plays an important role in gating. Moreover, we demonstrate a novel DE loop stabilizing 2-methyl-2,4- pentanediol binding site, and cytoplasmic bound potassium ion, the first identified for this class of K channels. We have also conducted NMR studies on the last 58 residues of Kir2.1 to show the flexibility of this region and binding footprint of PSD95 PDZ domains, which tethers the channels to the cell's cytoskeleton. Taken together, these results demonstrate that cytoplasmic domains of Kir channels are structurally significant components of the channel and undergo structural changes to modulate gating and inward rectification

This thesis describes studies of unitary currents flowing through two different potassium (K) channels present in sarcolemmal vesicles of the frog, Rana temporaria. The ATP-regulated K-channel is described first and the results are divided into three parts. Firstly, ATP applied to the cytoplasmic face of a membrane patch closes the channels in a dose-dependent fashion. Different nucleotides and other metabolic substances are used in order to find chemicals which can substitute for ATP or which regulate its effect. Secondly, the permeability properties of the channel are described. Ion flux is non-independent. Rubidium (Rb) is permeant, and anomalous mole-fraction behaviour is demonstrated in mixtures of K+and Rb+. The final part investigates the kinetic properties of the channel. Both voltage and ATP affect the rate constants regulating transitions between closed and open states of the channel. In particular, ATP causes the channel to occupy a very long-lived closed state. Block of the channel by tetraethylammonium (TEA) ions applied to either membrane surface is described as well. Block by external TEA+ is very fast and it is suggested that the channel cannot close when blocked. The block by internal TEA+ is slower and some evidence of voltage dependency is seen. The delayed rectifier K-channel is investigated. The first of two parts describes Rb+ permeability of the channel and its effect on open and closed times. The Hodgkin-Huxley model of the channel is questioned by the very different times of occupancy of closed states and differing voltage dependencies of the steps leading to opening of the channel. The second part describes block of the channel by externally applied TEA+ and the blocking reaction is shown to be very fast and voltage dependent.

In large coronary arteries the physiological response to a decrease in oxygen tension (hypoxia) is vasodilation, increasing blood flow to the heart. The mechanism(s) underlying hypoxia-induced vasorelaxation remains elusive. Our laboratory has previously investigated prevailing theories of oxygen sensing in the porcine coronary artery (Circ. Research 86:862-870, 2000). In that study we examined the effects of hypoxia on isometric force, intracellular Ca 2+ , pH, and energetics. None of these theories applied in the porcine coronary artery. In addition, the specific blockade of various ion channels did not affect the relaxation response to acute hypoxia. We observed an unperturbed hypoxia-induced relaxation in the presence of K ATP , K Ca , and K IR inhibition as well as during inhibition of protein kinase G and C activity. This suggested that there is a novel mechanism of hypoxic relaxation in this tissue not explained by the current theories. However, we did observe relaxation to hypoxia with and without changes in intracellular Ca 2+ . We describe this as Ca 2+ -dependent and -independent mechanisms of hypoxic relaxation. We discovered that organ culture at 37 (C for 24 hr specifically inhibits relaxation to hypoxia in porcine coronary without significantly affecting isometric force generation. I utilize this organ culture model to identify changes in other pathways that could be involved in hypoxia-induced relaxation. Both Ca 2+ -dependent and -independent mechanisms of hypoxic relaxation are inhibited by organ culture, but A- and G-kinase relaxation pathways remain intact. This suggests that inhibition by organ culture is specific to hypoxic relaxation and is a novel tool for investigating mechanisms of oxygen sensing. I also examined changes in gene and protein expression after organ culture using differential display and proteomics. Results indicate a possible role for voltage-dependent K + channels and ryanodine receptors in the Ca 2+ -dependent component of hypoxic relaxation. Additionally, I identify changes smooth muscle protein expression following organ culture that could contribute to an inhibition of hypoxic relaxation and have a role in the Ca 2+ -independent component. Thus, organ culture offers a unique approach to elucidate the mechanism of hypoxic relaxation. Together these results offer new unique targets for the mechanism of hypoxia-induced vasodilation in the porcine coronary artery.

The object of this study was to investigate the mechanism and control of the basolateral potassium conductance of turtle colon epithelial cells. Basolateral K currents were measured directly in isolated epithelial tissues which had been treated with the poreforming antibiotic, amphotericin B, to functionally eliminate the apical membrane as a barrier to K flow. Initially, it was found that the properties of the basolateral K conductance exhibited a striking dependence on the anionic composition of the serosal bathing solution. When the major serosal anion was benzene-sulfonate, the K current was completely inhibited by quinidine (a blocker of calcium-activated K channels in a variety of cell types). When chloride was the major serosal anion, however, the current was unaffected by quinidine. Other experiments showed that the K conductances under these conditions could be distinguished physically as well a pharmacologically. Experiments involving cation substitutions showed that the quinidine-sensitive conductance was markedly more selective for K over Rb than was the quinidine-insensitive conductance. Tracer flux measurements showed that the kinetics of ion flow through the quinidine-insensitive conductance were consistent with simple diffusion, while ion flow through the quinidine-sensitive conductance was anomalous. These results are consistent with the idea that differences in pharmacological sensitivity relected two separate populations of K channel in the basolateral membrane. Additional experiments involving mucosal anion substitutions and permeant non-electrolytes showed that the quinidine-sensitive conductance could be activated by osmotic cell swelling. These observations led to the working hypothesis that the quinidine-insensitive conductance is present in the basolateral membrane under normal conditions, while the quinidine-sensitive conductance is activated by increases in cell volume--possibly playing a role in cell volume regulation.