Fish naturally experience fluctuating levels of O2 and CO2 in their environment. To cope with the deleterious effects of lowered O2 (hypoxia) or elevated CO2 (hypercapnia), fish exhibit an array of cardiorespiratory adjustments aimed at preserving homeostasis. One of the most significant of these responses is reflex hyperventilation. In zebrafish (Danio rerio), hyperventilation during hypoxia or hypercapnia is thought to be initiated by the activation of chemoreceptor cells, termed neuroepithelial cells (NECs) which detect changes in ambient levels of O2 or CO2. The NECs of larval zebrafish are found throughout the integument and recent studies have shown that these NECs likely mediate the ventilatory responses to hypoxia and the cardiac responses to hypercapnia. However, no study has yet examined the ventilatory response of larval zebrafish to hypercapnia and regardless of developmental stage, the signalling pathways involved in CO2 sensing remain unclear. In the mouse, a background potassium channel (TASK-2) was shown to contribute to the sensitivity of chemoreceptor cells to CO2. Zebrafish have two specific TASK-2 channel paralogs encoded by kcnk5a and kcnk5b. The purpose of this thesis was to determine whether TASK-2 channels are expressed in NECs of larval zebrafish and whether they are involved in CO2 sensing. Immunohistochemical approaches were used to visualize TASK-2 protein (encoded by kcnk5a) within NECs of larvae and adult gill. TASK-2 protein was observed on NECs in both larvae and adult gill. Exposure of larvae to hypercapnia caused an increase in cardiac and breathing frequencies; these responses were blunted in fish experiencing either TASK-2 and/or TASK-2b knockdown. The results of these experiments suggest that TASK-2 has a role in activating NECs thus eliciting cardiorespiratory responses, when larvae are exposed to hypercapnia.

Master of Science / Department of Anatomy and Physiology / Bruce D. Schultz / Epithelial cells lining the vas deferens modify the luminal contents to which sperm are exposed in response to neuroendocrine, autocrine and lumicrine transmitters. The role and identity of vas deferens epithelial potassium channels that provide the correct luminal environment for sperm maturation and delivery have not yet been determined. Cultures of vas deferens epithelial cells isolated from adult pigs were employed to investigate contributions of selected ion channels to net flux. A two-pore potassium channel, TASK-2, was identified on the apical membrane of cultured primary porcine vas deferens epithelial cells (1°PVD). Bupivacaine, a known TASK-2 inhibitor, when added to the apical bathing solution, inhibited forskolin- stimulated short circuit current, Isc, in a concentration dependent manner with a maximum inhibition of 72 ± 6% and an IC50 of 7.4 ± 2.2 µM. Apical exposure of 1°PVD cells to quinidine, lidocaine, and clofilium (other known TASK-2 blockers) inhibited forskolin-stimulated Isc in a concentration dependent manner. Fitting a modified Michalis-Menten function to the data revealed IC50 values of 274 µM, 531 µM, and 925 µM, respectively. Riluzole, a two-pore potassium channel activator, stimulated bupivacaine-sensitive Isc, further confirming the contribution of TASK-2 to net ion flux. Western blotting demonstrated the presence of TASK-2 immunoreactivity in 1°PVD cell lysates, while immunocytochemistry demonstrated apical localization of the targeted epitope in virtually all cells lining native porcine vas deferens. These results suggest that TASK-2 likely plays a role in vas deferens epithelial ion transport that may account for the reportedly high concentration of potassium in the male reproductive duct lumen. TASK-2 likely contributes to male fertility as an integral member of the regulated transport processes that account for the luminal environment to which sperm are exposed.