ItemThe Regulation of Intracellular Ca2+ and Secretion in the Bag Cell Neurons of Aplysia Californica(2015-08-26) Groten, Christopher; Physiology; Magoski, Neil S.Neurons enter states of heightened excitability and secretory capacity to initiate fundamental behaviours. This is exemplified by the bag cell neurons of the marine mollusc, Aplysia californica. These neuroendocrine cells undergo an afterdischarge to secrete egg-laying hormone (ELH) and initiate reproduction. I examined the role of two cellular signaling pathways that contribute to the afterdischarge: Ca2+ and protein kinase C (PKC). Investigating these systems provides insight into the cellular mechanisms underlying fundamental behaviours. Intracellular Ca2+ modulates excitability and initiates secretion in the bag cell neurons. I determined how Ca2+ sources and removal systems control intracellular Ca2+. My data revealed that the mitochondria strongly influence Ca2+ signaling in cultured bag cell neurons, as they first buffer voltage-gated Ca2+ influx and subsequently release Ca2+ to the cytosol, in a form of Ca2+-induced Ca2+ release (CICR). Moreover, the degree of mitochondrial Ca2+ uptake and release was shown to be dictated by the function of the plasma membrane Ca2+ ATPase. In addition to voltage-gated Ca2+ influx, I characterized the involvement of Ca2+ handling systems with other distinct Ca2+ sources, including CICR and the Na+/Ca2+ exchanger as well as store-operated Ca2+ influx and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA). PKC is implicated in facilitating secretion during the afterdischarge. I tested the impact of PKC on secretion using capacitance tracking under whole-cell voltage-clamp. This technique assays the changes in plasma membrane area that occur during vesicle exocytosis. I demonstrated that PKC activation enhanced Ca2+ influx and potentiated stimulus-evoked secretion. This occurred as a result of the plasma membrane insertion of a covert voltage-gated Ca2+ channel, Apl Cav2, alongside the basal voltage-gated Ca2+ channel, Apl Cav1. iii I provided mechanistic detail of how the interplay between Ca2+ sources and removal systems governs the patterns of free cytosolic Ca2+. These properties have implications for the Ca2+-dependent signaling pathways which mediate long lasting changes in neuronal excitability and secretion. Furthermore, my work demonstrates that protein kinases can dynamically amplify secretory output by rapidly recruiting additional voltage-gated Ca2+ channels to the membrane. This form of facilitation likely enhances secretion during the afterdischarge, and ensures the initiation of reproductive behaviour in Aplysia. ItemRegulation of the Human Ether-a-go-go Related Gene Potassium Channel by Neural Precursor Cell Expressed Developmentally Down-regulated Protein 4-2 Interacting Proteins(2015-08-19) Kang, Yudi; Physiology; Zhang, ShetuanDysfunction of the human ether-a-go-go related gene (hERG)-encoded rapidly activating delayed rectifier K+ channel is a major cause of long QT syndrome (LQTS) due to its critical role in the repolarization of cardiac action potentials. The density of hERG channels on the cell surface, as a key determinant of its regular function, is balanced by channel trafficking to and internalization from the plasma membrane. We have shown that the E3 ubiquitin (Ub) ligase, Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4-2), regulates hERG channel degradation by targeting the PY motif in the C-terminus of hERG channels. Interestingly, although a PY motif exists in both the immature (intracellular) and mature (cell-surface) channels, Nedd4-2 selectively degrades the mature hERG proteins. Moreover, Nedd4-2 is modulated by various proteins, such as protein kinase C (PKC). In this work, I investigated the hypotheses that the selective degradation of the 155-kDa hERG channel by Nedd4-2 is achieved by additional Nedd4 family interacting proteins (Ndfips) and that PKC signalling regulates hERG expression and function through Nedd4-2. Using whole-cell patch-clamp, Western blot, and immunocytochemistry, I demonstrated that Nedd4-2 is directed to specific cellular compartments by Ndfip1 and Ndfip2. Ndfip1 is primarily localized in the Golgi apparatus where it recruits Nedd4-2 to target mature hERG proteins for degradation during channel trafficking to the plasma membrane. Ndfip2 mainly recruits Nedd4-2 to the multivesicular bodies (MVBs), which may impair MVBs function and impede the degradation of internalized hERG proteins. On the other hand, PKA and PKC activations increase hERG proteins on the plasma membrane by distinct mechanisms. While it is possible that PKA enhances hERG protein synthesis, PKC attenuates hERG channel degradation by inactivating Nedd4-2 via phosphorylation. These findings extend our understanding of hERG channel regulation by Nedd4-2 and provide information useful for rescuing impaired hERG function in LQTS. ItemProteolytic Cleavage of the Kv1.5 Channel in the S1-S2 Linker Does Not Affect Channel Function(2015-08-13) Hogan-Cann, Andrew; Physiology; Zhang, ShetuanAtrial fibrillation (AF), the most prevalent human cardiac arrhythmia, is characterized by rapid and disorderly electrical activity in the atria of the heart. Kv1.5 channel mediated ultra-rapidly activating delayed rectifier potassium current (IKur) is critical for timely and adequate atrial repolarization. Since cardiac IKur is specific to the atria, biomedical research surrounding Kv1.5 poses significant promise for developing clinical strategies to treat AF. In fact, loss-of-function mutations in KCNA5 (encoding Kv1.5) have been identified in patients with AF. Importantly, common pathologies, such as selective atrial ischemia, are capable of stimulating the onset of AF. A well-documented consequence of ischemia is a substantial increase in proteolytic enzyme activities. In this regard, it has been reported that cell-surface Kv1.5 channels are sensitive to cleavage by extracellular proteases, such as proteinase K (PK). In this study, we further examined the effects of extracellular proteases on the function and expression profile of Kv1.5 channels stably expressed in HEK 293 cells. Our results demonstrate that PK cleaves membrane-bound mature (75-kDa) Kv1.5 channels at a single locus in the external S1-S2 linker, yielding 42-kDa N-terminal and 33-kDa C-terminal fragments. Contrary to our expectations, whole-cell voltage clamp analysis showed that PK treatment did not affect Kv1.5 current (IKv1.5). Examination of plasma membrane proteins isolated via biotinylation indicated that the N- and C-terminal Kv1.5 fragments were both present and stable on the cell-surface. Co-immunoprecipitation (co-IP) studies following PK cleavage suggest that the two Kv1.5 fragments do not associate. Moreover, the PK-generated N- and C-terminal fragments degraded at different rates. These findings indicate that the C-terminal fragment of Kv1.5 (S2-S6, pore-containing) may be sufficient for current conduction. Our data raises the possibility that cleavage of cell-surface ion channels, assessed by Western blot analysis, does not necessarily result in a loss of channel activities. This novel insight into the Kv1.5 structure-function relationship may be indicative of an inherent protective mechanism for Kv1.5 channel function. ItemScorpion Toxin BeKm-1 Prevents Low K+ Induced Internalization of Cell-Surface hERG Channels(2015-08-07) Han, Xi; Physiology; Zhang, ShetuanThe human ether-à-go-go related gene (hERG) encoded the pore forming subunit of a K+ channel that conducts an important current for the repolarization of the cardiac action potential. Dysfunction of the hERG channel leads to abnormal cardiac activities characterized by prolongation of the QT interval, clinically known as long QT syndrome (LQTS), which can cause lethal tachyarrhythmias and sudden death. We have previously shown that hypokalemia induces a conformational change that leads to degradation of hERG channels from the plasma membrane (Guo et al., 2009). However, the molecular mechanisms for low K+ effect on hERG channels are not known and it is also unknown whether low K+-induced internalization of hERG channels can be prevented. Using various compounds that interact with hERG channels by binding to different regions of channels has been a field of interest to investigate the channel structure-function relationships. Moreover, certain high affinity hERG channel blockers such as E-4031 can rescue trafficking-detective hERG mutants (Gong et al., 2006). The hERG channel has an unusually long extracellular S5-pore linker to which the scorpion toxin BeKm-1 selectively binds. I hypothesized that the S5-pore linker contributes to the unique sensitivity of hERG channels to extracellular K+, and that BeKm-1 prevents the internalization of hERG channels induced by 0 mM [K+]o through binding to S5-pore linker. In the present study, I investigated the protective effects of the scorpion toxin, BeKm-1, on low K+-induced hERG internalization using whole-cell patch-clamp, and Western blot analysis. Our data demonstrate that BeKm-1 effectively prevents low K+-induced hERG current loss and protein degradation. Since BeKm-1 blocks hERG channels, its protective effect on low K+-induced hERG internalization has limited clinical potential. Thus, we designed ten BeKm-1 mutants to screen for peptides that can prevent hERG from low K+-induced internalization without blocking hERG conductance. Our data show that two mutants were able to rescue hERG channels in 0 mM [K+]o but do not block hERG conductance. This study extends our understanding of the structure-function relationship of hERG channels and revealed a potential novel way to protect hERG channels from low K+-induced internalization. ItemThe Effect of Insulin Sensitivity on Corticolimbic Responses to Metabolic and Visual Food Cues(2015-06-02) Alsaadi, Hanin; Physiology; Van Vugt, DeanInsulin is one of several molecules that signals the energy balance state to the brain. This study examined the effect of insulin sensitivity on the responsiveness of appetite regulatory brain regions to visual food cues. Nineteen participants diagnosed with polycystic ovary syndrome (PCOS) were studied. Subjects were divided into insulin-sensitive (n=8) and insulin-resistant (n=11) groups based on the homeostatic model assessment of insulin resistance (HOMA2-IR). Subjects underwent functional magnetic resonance imaging (fMRI) while viewing food pictures following water or dextrose consumption. The corticolimbic Blood Oxygen Level Dependent (BOLD) responses to high-calorie (HC) or low-calorie (LC) food pictures were compared within and between groups. BOLD responses to food pictures were reduced during a glucose challenge in numerous corticolimbic brain regions of insulin-sensitive subjects, but not in insulin-resistant subjects. In addition, a positive interaction was detected between insulin sensitivity and condition. Furthermore, the degree of insulin resistance positively correlated with the corticolimbic BOLD response in the medial prefrontal cortex (mPFC), orbitofrontal cortex (OFC), anterior cingulate and ventral tegmental area (VTA) in response to HC pictures and in the dorsolateral prefrontal cortex (DLPFC), mPFC, anterior cingulate, and insula in response to LC pictures following a glucose challenge. The activity in the OFC, midbrain, hippocampus, and amygdala was positively correlated with HOMA2-IR in response to HC>LC pictures following a glucose challenge. We conclude that the normal inhibition of corticolimbic brain responses to food pictures during a glucose challenge is compromised in insulin-resistant subjects. The increase in brain responsiveness to food pictures during postprandial hyperinsulinemia may lead to greater non-homeostatic eating and perpetuate obesity in insulin-resistant subjects. Understanding how insulin sensitivity affects appetite-regulating brain regions responses to food pictures is necessary for the development of prevention strategies and effective therapeutic targets for the treatment of obesity, particularly obesity related to insulin resistance in PCOS.