Mon Dec 12th 1:00pm
Mellon Institute Social Room
Final Public Oral Examination for the degree of Doctor of Philosophy
” In vitro and in vivo Studies of BK Channel Trafficking and Localization using Fluorogen-Activating Peptides”
Abstract: BK channels are critical regulators of neuronal activity, involved in regulating spike rate, neurotransmitter release, cerebellar function, circadian rhythms, and seizure disorders. Modulation of BK channel gating is well-characterized, regulated by accessory subunit interactions, intracellular signaling pathways, and membrane potential. In contrast, the role of intracellular trafficking mechanisms in controlling BK channel function, especially in live cells, has been poorly studied .
Fluorogen activating peptides (FAPs) are well-suited for trafficking and physiological studies due to the binding of malachite green (MG) based dyes with sub-nanomolar affinity to the FAP, resulting in bright, photostable, far-red fluorescence. The generation of cell excluded MG dyes enables the selective tagging of surface protein and tracking through endocytic pathways. We developed an N-terminally tagged FAP-BKα construct that exhibits similar properties to the native channel. First, we developed a trafficking assay, GIRO (Green-Inside Red-Outside) in which a pair of fluorogens can elicit spectrally distinct labeling depending on protein localization. Using this assay, we determined that cyclic AMP plays a role to control BKα surface abundance.
In vivo, BKα is subject to extensive alternative splicing and association with β and γ subunits. To observe FAP-tagged BK channels in this context, we generated transgenic mice using CRISPR to insert the FAP into the native BKα locus, where it would be regulated by intrinsic mechanisms and subject to alternative splicing. FAP-BKα does not disrupt BK channel function, as homozygous FAP-BKα transgenic mice showed normal cerebellum-dependent behaviors, including gait and rotorod performance, unlike BKα knockout mice. We identified FAP-tagged BKα expression in brain and smooth muscle, as expected. BKα and HA tag co-staining showed transgene expression in appropriate brain regions, including cerebellum, substantia nigra, hippocampus, and cortex; localization was recapitulated by MG-TCarb staining. In the cerebellum, FAP-BKα formed bright puncta on the plasma membrane of Purkinje cells; this clustering is reminiscent of previous reports using freeze-fracture and electron microscopy. We found that the brain-specific β4 subunit is involved in the formation of these large puncta. With the establishment of this mouse model, future work in FAP-BKα transgenic mice could probe dynamic control of channel trafficking in other brain regions and dissociated cultures in response to plasticity induction, seizure development, or intracellular signaling cascades.
Pittsburgh Center for Pain Research and
University of Pittsburgh Brain Institute
Presents a Special Seminar:
Michael Caterina, M.D., Ph.D.
Solomon H. Snyder Professor,
Director, Neurosurgery Pain Research Institute
Co-Director, Biological Chemistry Graduate Program
Professor of Neurosurgery, Biological Chemistry and Neuroscience
John Hopkins University, School of Medicine
“TRP channels in homeostasis and sensation”
Monday, December 12, 2016
Conference Room 1495, BSTWR
Department of Neuroscience
Presents a Seminar by
Naoshige Uchida, Ph.D.
Professor of Molecular and Cellular Biology
TArithmetic and neural circuits underlying
dopamine prediction errors
Tuesday, December 13, 2016
348 Mellon Institute