Molecular diversity and physiological contributions of neuronal trek channels

University of Minnesota Ph.D. dissertation. October 2012. Major: Pharmacology. Advisor:Kevin D. Wickman. 1 computer file (PDF); xi, 180 pages.Two-pore domain K+ (K2P) channels underlie leak or background potassium conductances in many cells. The Trek subfamily consists of three members: Trek1/Kcnk2, Trek2/Kcnk10, and the more distantly-related family member Traak/Kcnk4. Trek family members exhibit complex regulation and can modulate cell excitability in response to a wide array of stimuli, and have been implicated in depression, nociception, and cognition. Furthermore, a number of studies support a role for the Trek family in neuroprotection during ischemia. Despite their identification more than one decade ago, they are still relatively understudied, which is especially true for Trek2. To further our understanding of molecular diversity within the Trek family, we identified and characterized the murine alternative splice variants of Trek2. We identified two novel exons, which contribute to the production of six alternative splice variants; three N-terminal two-pore variants and three non-functional C-terminal one-pore variants. Of the N-terminal variants, the previously unreported isoform, Trek2b, displayed significantly greater whole-cell current amplitudes when expressed heterologously, a phenomenon ultimately attributable to greater surface expression. While characterizing the Trek2 splice variants, we observed significant cell-to-cell variability in current amplitudes in HEK293 cells heterologously expressing Trek channels. We hypothesized this was the result of changes in Trek channel expression during the cell cycle. Indeed, changes in current amplitudes and Trek protein expression correlated with cell cycle stage. Ultimately, we concluded this was likely a non-Trek specific effect of cell cycle progression on the artificial CAG promoter used to drive heterologous Trek expression. To further our understanding of the physiological contributions of Trek channels to animal behavior, specifically Trek2, we probed the effect of constitutive Trek2 ablation, as well as the simultaneous constitutive ablation of all three Trek family genes, in paradigms that assess motor activity, coordination, anxiety-related behavior, learning and memory, and drug-induced reward-related behavior. Few behavioral differences were observed between Trek2–/–, Trek1/2/Traak–/– and wild-type mice, which argue that despite their broad distribution in the CNS, Trek channels exert a minimal influence on a wide-range of behaviors. Instead, we propose that Trek channel activity may be low under normal conditions, where they become impactful only under certain circumstances, such as during ischemia