| 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 GABA B 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 Galphai/o. However, previous data from the laboratory suggested that the enhancing effect observed was not due to Galphai/o, but a different G protein not previously described in GABAB receptor signaling. Indeed, when the Galphaq 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 Galphaq 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 PKCalpha 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 (IC 50 < 10 mum). 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. |
