S-palmitoylation is a reversible post-translational lipid modification of proteins by adding a 16-carbon palmitate onto a cysteine residue. Palmitoylation has been shown to control the trafficking and function of many signalling proteins including ion channels. In this Thesis, palmitoylation is shown to control both the plasma membrane expression and gating properties of large conductance calcium- and voltage- dependent potassium (BK) channels. The BK channel is assembled from four pore-forming α-subunits. Each α-subunit contains seven transmembrane domains (S0-S6), with an extracellular N-terminus and a large intracellular C-terminus. BK channel α-subunit is encoded by a single gene Kcnma1 that undergoes extensive pre mRNA splicing at various splice sites, thus there are a number of alternatively spliced variants of α-subunits. Using quantitative imaging assays, palmitoylation of the intracellular S0-S1 loop controlled trafficking of full length ZERO variant BK channels to the plasma membrane in HEK293 cells as well as neuronal N2a cells. Importantly, all four α-subunits need to be palmitoylated for robust surface expression. Thus, palmitoylation of the S0-S1 loop of the α-subunit is important for surface expression of BK channels. The BK channel may also assemble with auxiliary β-subunits (β1-4) that regulate surface expression and gating properties of BK channels. The N-terminus of the β1- subunit and the C-terminus of the β4-subunit were shown to be palmitoylated using [3H]-palmitate incorporation, respectively. However, mutation of the palmitoylated cysteine (C18 in β1 and C193 in β4) to alanine to generate depalmitoylated β- subunits had no significant effects on the electrophysiological properties resulting from co-expression with the ZERO variant of the BK channel. However, although palmitoylation of the S0-S1 loop does not affect the electrophysiological properties of the ZERO channels alone, it is important for the shift in the V0.5max of ZERO channel when co-expressed with the β1-subunit, but not β4-subunit. These data suggest that palmitoylation of the S0-S1 loop controls the functional coupling between the ZERO α-subunit and β1-subunit. Although palmitoylation of C18 in the N-terminus of the β1-subunit was not required for functional coupling to α-subunits, we identified other critical residues within the short intracellular N-terminus of the β1-subunit that are essential. The functional coupling between BK α- and β1-subunit was predicted to be controlled by the interaction between a non-classic amphipathic α-helix in the β1 subunit N-terminus and the plasma membrane. Deletion, or mutations predicted to disrupt the interaction significantly decreased the β1-subunit induced left shift in the BK channel V0.5max. This suggests that the amphipathic in-plane anchor is critical for functional coupling of β1-subunits with BK channel α-subunits. In this Thesis, we demonstrated: i) palmitoylation of the α-subunit S0-S1 loop controls surface membrane expression of BK channels, and also controls functional regulation by β1, but not β4-subunits; and ii) a potential non-classical amphipathic in-plane anchor in the β1 N-terminus is essential for functional coupling with α- subunits. These studies help us further understand the regulation of BK channels and suggest potential therapeutic targets for various diseases related to dysfunctional BK channels, such as hypertension.

Tolerance to anesthetic drugs is mediated partially by homeostatic mechanisms that attempt to restore normal neural excitability. The BK-type Ca2+-activated K+ channel, encoded by the slo gene, plays an important role in this neural adaptation. In Drosophila, a single sedative dose of the organic solvent anesthetic benzyl alcohol induces dynamic spatiotemporal changes in histone H4 acetylation across the slo regulatory region and leads to slo induction and tolerance. Mutations ablating the expression of slo also block the acquisition of tolerance, whereas activating the expression of a slo transgene results in resistance to drug sedation. Moreover, artificially inducing histone acetylation with the histone deacetylase inhibitor causes similar acetylation changes, slo induction, and functional tolerance to the drug. Histone acetylation changes occur over two highly conserved non-coding DNA elements, 6b and 55b, of the slo control region. To investigate the function of these two elements, I generated individual knockout mutants by gene targeting. Both knockout alleles are backcrossed into the CS wild type background. The 6b element seems to repress slo induction after drug sedation, because the 6b knockout allele overreacts to the drug. Compared to the wild type, 6b knockout allele shows a much greater slo message induction after drug sedation, it also displays stronger enhancements in seizure susceptibility and following frequency. In addition, the 6b deletion causes a persistent tolerance for at least a month, while tolerance only lasts about 10 days in wild type flies. My investigation also indicates that the 55b element limits basal slo expression in muscle. Finally, to investigate if the particular histone acetylation spikes are required for drug-induced slo induction and tolerance, I tether the histone-modifying enzymes, HDAC or HAT, to the 6b and 55b DNA elements, respectively. I observe that the positioning of an HDAC on these two elements blocks drug-induced slo induction and the development of tolerance. Therefore, histone acetylation across slo control region is required for the activation of slo and the acquisition of tolerance. / text

Large conductance Ca2+-activated K+ channels (BK channels) are expressed ubiquitously in both excitable and non-excitable cells and are important for a range of physiological functions. BK channels gate K+ efflux in response to concurrent depolarized membrane voltage and increased intracellular Ca2+ to modulate action potential shape and duration in neurons, regulate contractility in smooth muscle, and control fluid secretion by epithelial cells in the airway and gut. In addition, mutations in the human BK channel gene (KCNMA1) are linked to neurological disease, such as epilepsy and paroxysmal dyskinesia. Thus, BK channel modulators may provide treatment avenues for BK channelopathies. It will be important to expand our arsenal of BK channel-selective activators and inhibitors and to grow our understanding of their molecular mechanisms of action. Discovery of new channel modulators will further lead to a greater understanding of BK channel structure and function. To better understand the basic structure-function relationship of BK channel gating in response to increased intracellular Ca2+ concentration, in this work I initially investigate structural determinants of BK channel activation in response to conformational changes following Ca2+ binding. I analyze crystal structures of the BK channel cytosolic Ca2+-sensing domain (CSD), also known as the “gating ring”, formed by the C-terminal domains of each of the four identical pore-forming subunits. In the Ca2+-bound state, N449 from the adjacent subunit contacts the bound Ca2+ ion, forming a “Ca2+ bridge.” Mutating N449 to alanine eliminates this coordinate interaction, and using electrophysiology, I found that BK channels with the N449A mutation exhibit a shift in the voltage required for half maximal activation (V1/2) towards more positive voltages. Using size-exclusion chromatography, I observed that the purified BK channel CSD with the N449A mutation shows reduced gating ring oligomerization in response to Ca2+ compared to the wild-type CSD. To further probe molecular determinants of BK channel gating and increase our arsenal of BK channel gating modulators, I optimized a fluorescence-based high throughput screening approach to discover compounds with BK channel inhibitor activity with 99.73% confidence. Through this approach I discovered that the -opioid receptor agonist, loperamide, is a potent BK channel inhibitor. Loperamide (LOP) reduced the open probability of channels at depolarized voltages, but not at very negative voltages when the voltage-sensor is at rest. I observed a weak voltage dependence of loperamide inhibition, consistent with loperamide binding shallow within the inner cavity to block the channel pore. I quantified the inhibitory effect of LOP using an allosteric model in which LOP blocks conduction through open channels and binds with 45-fold higher affinity to the open state over the closed state. These data suggest that loperamide may represent a new class of “use-dependent,” open channel blockers. Together this work describes an approach to understanding BK channel structure and function with the goal of identifying and developing novel therapeutics for the treatment of BK-related diseases. / Biochemistry

Corticotroph cells from the anterior pituitary are an integral component of the hypothalamic-pituitary-adrenal (HPA) axis, which controls the neuroendocrine response to stress. Following stressful stimuli, corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus act synergistically to stimulate adrenocortiotrophin hormone (ACTH) secretion from corticotroph cells. ACTH is released into the circulation where it stimulates the secretion of glucocorticoids from the adrenal cortex. The HPA axis is kept in fine balance through an elegant negative feedback system where elevation of plasma glucocorticoids results in inhibition at the level of both the pituitary and the hypothalamus. During acute stress, glucocorticoids can be beneficial however chronic elevation of glucocorticoids can have many adverse effects on health. Corticotroph cells are electrically excitable and have been shown to fire single-spike action potentials as well as complex bursting patterns. Stimulation of corticotrophs with physiological concentrations of CRH/AVP results in a robust increase in firing frequency and a transition from spiking to bursting. Intracellular Ca2+ increases to a greater extent during bursting which has been proposed to drive hormone secretion. There is evidence to suggest that large conductance calcium- and voltage-gated potassium (BK) channels promote bursting behaviour in anterior pituitary cells. Glucocorticoids have been shown to regulate ACTH secretion and also modulate BK channel activity. However, the effects of glucocorticoids on native corticotroph excitability are currently unknown. The aim of this study was to first characterise the electrical properties of corticotrophs under basal conditions and following exposure to CRH/AVP. Secondly, to investigate the regulation of corticotroph excitability by glucocorticoids. Finally, establish the role of the BK channel in regulating bursting behaviour and CORT regulation in corticotroph cells. Corticotroph cells were acutely isolated by trypsin digestion from mice aged 2-5 months constitutively expressing GFP under control of the POMC promoter (POMC-GFP). Mice used for pituitary cell culture were male unless otherwise stated. Cells were maintained in a serum free media and electrophysiological recordings obtained 24-96 hours post-isolation. Current clamp recordings were obtained from corticotrophs using the perforated patch technique. Although spontaneous activity of corticotroph cells was variable, they displayed predominantly single-spike action potentials under basal conditions. Stimulation with physiological concentrations of CRH and AVP (0.2 nM and 2 nM respectively) resulted in a membrane depolarisation accompanied by an increase in firing frequency and a transition to bursting. Individually, CRH and AVP were able to increase corticotroph excitability. However, only CRH was able to drive an increase in bursting suggesting that bursting is primarily regulated through the cAMP/PKA pathway. Experiments were performed to investigate the modulation of corticotroph activity by glucocorticoid negative feedback. Acute exposure (< 10 min) to corticosterone resulted in a decrease in spontaneous activity as well as shortening the response to CRH/AVP. Pretreatment of corticotrophs with 100 nM corticosterone (90 min) resulted in a membrane hyperpolarisation and a decrease in spontaneous firing frequency. Following corticosterone pretreatment, CRH/AVP failed to induce a significant transition from spiking to bursting. Increasing the pretreatment time to 150 minutes resulted in a further suppression of both spontaneous and CRH/AVPevoked activity. Fast activation of BK channels during the upstroke of an action potential has been proposed to promote bursting behaviour in other pituitary cells. Corticotrophs treated with a BK channel blocker (1 μM paxilline) or isolated from BK-/- mice showed no significant difference in basal activity but displayed a reduction in CRH/AVPevoked bursting activity. In both cases, bursting was significantly reduced but not completely abolished. Corticosterone treatment of BK-/- cells resulted in a further decrease in both firing frequency and bursting behaviour. Taken together, these results suggest that although BK channels play an important role in bursting, they are not the only component. Comparisons of male and female corticotrophs revealed subtle differences in their properties. Following CRH/AVP stimulation, male cells displayed a high degree of bursting activity whereas female cells exhibited predominantly an increase in singlespike action potential frequency. Treatment of female corticotrophs with corticosterone (150 min) resulted in a significant reduction in firing frequency but no measurable change in bursting behaviour. BK-/- cells from female mice showed no difference in bursting activity following CRH/AVP compared to wild types. This data suggests that modulation of firing frequency is the more important component in female corticotroph cells. In conclusion, CRH/AVP is proposed to drive ACTH secretion in male corticotroph cells through an increase in bursting activity. Corticosterone pretreatment suppresses both spontaneous and CRH/AVP-evoked activity. It is possible that corticosterone regulates corticotroph excitability through two mechanisms. Corticosterone suppresses bursting activity following CRH/AVP stimulation through multiple targets which might include the BK channel. Additionally, corticosterone reduces firing frequency through a mechanism independent of BK channels. It is important to further characterise the physiology of corticotroph cells and how ACTH secretion is regulated through their electrical excitability. This would lead to a greater understanding of the role of corticotrophs in the HPA axis. Further study of corticotrophs could potentially lead to pharmacological manipulation of the stress response and novel treatments for stress-related disorders.

No description available.

No description available.

No description available.

Alcohol produces changes in behavior through molecular effects on ion channels, enzymes and transporters. Many proteins have been elucidated that at least in part mediate behavioral changes induced by alcohol. However, it has been difficult thus far to uncover key amino acid residues within a protein that are necessary for the effects of alcohol. This information is critical, potentially leading to effective pharmacological treatments for alcohol use disorders (AUD) and identification of allelic variations that predispose an individual for AUD. The big conductance voltage- and calcium-activated potassium (BK) channel has recently emerged as a critical protein for the effects of alcohol across species. In this dissertation, we study the molecular action of alcohol on the BK channel, and how this action contributes to behavioral intoxication. To accomplish this, we first provide credence for using the nematode C. elegans for studying the behavioral effects of ethanol. We demonstrate how behavioral intoxication and internal ethanol concentration in C. elegans is altered by the osmolarity of the ethanol-solution, reconciling results from previous conflicting reports in the literature. We then identify the amino acid residue T381 on the BK channel in C. elegans is critical for behavioral intoxication, but not other BK channel-dependent behaviors. These results suggest a functional BK channel resistant to ethanol. By knocking-in the human BK channel, we then demonstrate that the equivalent residue, T352 is also critical for behavioral intoxication in C. elegans, but not other BK channel-dependent behaviors. Using single-channel recordings, we find that the T352 residue is critical for the potentiating effects of ethanol on the human BK channel, without being critical for basal-function. Finally, we investigate the role of calcium-sensing residues on the worm BK channel for behavioral intoxication in C. elegans. We find that these residues are non-essential for intoxication, in contrast to in vitro reports in the mammalian channel suggesting the calcium-sensing residues are critical for ethanol-activation of the BK channel. / text

Humans are approximately 99% similar with inter-individual differences caused in part by single-nucleotide polymorphisms (SNPs), which poses a challenge for the effective treatment of disease. Bioinformatics resources can help to store and analyse gene and protein information to address this challenge, however these resources have limitations, so the collation and biocuration of gene and protein information is required. Using the large conductance calcium- and voltage-activated potassium channel, also known as the Big Potassium (BK) channel as an example, due to its ubiquitous expression and widespread varied role in human physiology, this study aimed to prioritise SNPs with the potential to affect the function of the channel. Using a BK channel resource created with bioinformatics tools and published literature, mSlo SNPs H55Q and G57A, located in the S0-S1 linker, were prioritised and selected for lab-based verification. These SNPs flank three cysteine residues proven to modulate channel cellular distribution via palmitoylation, a reversible process shown to increase protein association with the cell membrane. The SNPs alter the predicted palmitoylation status of C56, one of the cysteine residues located in the S0-S1 linker. The cellular distribution of BK channels incorporating the SNPs was assessed using confocal microscopy and revealed that the direction and magnitude of SNP mimetic cell membrane expression was closely related to the C56 predicted palmitoylation score; a 'C56 palmitoylation pattern' was observed. It was shown that exposure to metabolic substrates glucose, palmitate and oleate modulated SNP-mimetic cellular distribution and could invert the 'C56 palmitoylation pattern', indicating that there is interplay between the metabolic status of the cell and the amino-acid composition of the channel via palmitoylation. The creation of a novel BK channel resource in this thesis highlighted the limitations, and inter-dependency of bioinformatics and lab based experimentation, whilst SNP verification experiments solidified the link between S0-S1 cysteine residues and BK cellular distribution. BK channel function is linked with a number of physiological processes; thus, the potential clinical consequences of the SNPs prioritised in this thesis require further research.

No description available.