Date of Award
Spring 2014
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biological Sciences
First Advisor
Mynlieff, Michelle
Second Advisor
Buchanan, James
Third Advisor
Downs, Stephen M.
Abstract
Activation of the metabotropic GABAB receptor has most commonly been demonstrated to produce inhibitory effects on neurons, including the attenuation of voltage-dependent calcium current. However, during the early neonatal period in mammalian development, activation of GABAB receptors leads to an enhancement of calcium current through a specific class of calcium channels, termed L-type channels, (because they conduct Long-lasting current) . This response peaks at 7 days postnatal, and is only demonstrated in a subset of cells. In the work presented here, the signal transduction pathway of GABAB receptor-mediated increase of L-type current is described.
GABAB receptors couple to G proteins, traditionally believed to be Gαi/o. However, previous data from the laboratory suggested that the enhancing effect observed was not due to Gαi/o, but a different G protein not previously described in GABAB receptor signaling. Indeed, when the Gαq G protein was knocked down in cell culture, the enhancement of L-type channels was no longer observed. These data suggest that GABAB receptors couple to Gαq(/sub> G proteins to mediate calcium current enhancement.
Protein kinase C (PKC) had previously been demonstrated as a requisite member of this pathway. Furthermore, there was precedence for PKC to work through calcium/calmodulin-dependent kinase II (CaMKII) to enhance L-type current. However, the isozyme of PKC was not known, nor was the involvement of CaMKII on L-type current enhancement. Confocal imaging analysis suggests PKCα is the isozyme that is activated by GABAB receptor activation, and pharmacological studies indicate CaMKII is not a participant in this pathway.
In seeking to inhibit CaMKII signaling, highly specific pharmacological inhibitors are often required. However, several inhibitors that were thought to be specific initially demonstrate nonspecific effects. A newly synthesized molecule, CK59, has been described to potently inhibit CaMKII activity (IC50 < 10 μm). However, data presented here describe off-target effects of CK59, specifically its ability to inhibit voltage-gated calcium channels. Treatment of cells with CK59 significantly reduced calcium influx in depolarized neurons, whereas other CaMKII inhibitors did not change calcium influx. Thus, CK59 is not a useful tool when studying the interplay between voltage-caged calcium channels and CaMKII signaling.