Calcium/Calmodulin-dependent Protein Kinase II Phosphorylates and Regulates the Drosophila Eag Potassium Channel

Abstract

Modulation of neuronal excitability is believed to be an important mechanism of plasticity in the nervous system. Calcium/calmodulin-dependent protein kinase II (CaMKII) has been postulated to regulate the ether à go-go (eag) potassium channel in Drosophila. Inhibition of CaMKII and mutation of the eag gene both cause hyperexcitability at the larval neuromuscular junction (NMJ) and memory formation defects in the adult. In this study, we identify a single site, threonine 787, as the major CaMKII phosphorylation site in Eag. This site can be phosphorylated by CaMKII both in a heterologous cell system and in vivo at the larval NMJ. Expression of Eag in Xenopus oocytes was used to assess the function of phosphorylation. Injection of either a specific CaMKII inhibitor peptide or lavendustin C, another CaMKII inhibitor, reduced Eag current amplitude acutely. Mutation of threonine 787 to alanine also reduced amplitude. Moreover, both CaMKII inhibition and the alanine mutation accelerated inactivation. The reduction in current amplitudes and the accelerated inactivation of dephosphorylated Eag channels would result in decreased outward potassium currents and hyperexcitability at presynaptic terminals and, thus, are consistent with the NMJ phenotype observed when CaMKII is inhibited. These results show that Eag is a substrate of CaMKII and suggest that direct modulation of potassium channels may be an important function of this kinase.Numerous studies have suggested that ion channels are an important class of neuronal targets for protein kinases. Modulation of potassium channel activity by kinases has key importance in regulation of neuronal excitability and synaptic plasticity (1). The ether-à -go-go (eag) gene in Drosophila encodes a voltage-gated potassium channel that has a C-terminal cytoplasmic domain with homology to cyclic nucleotide-gated channels (2, 3). This channel is the founding member of a superfamily of vertebrate and invertebrate genes, which include eag, elk, and erg subfamilies (4). In flies, mutation of the eag gene causes shaking after exposure to ether (5) and hyperexcitability at the NMJ 1 (6). These phenotypes have been demonstrated with both hypomorphic and null alleles of eag (see, for example, the NMJ phenotypes of eag 1 , a hypomorph, and eag sc29

Presynaptic N-type Calcium Channels Regulate Synaptic Growth

“…Activated CaMKII phosphorylates the synaptic MAGUK protein DLG, causing release of FAS2 from its synaptic scaffold and subsequent modulation of synaptic growth in Drosophila (32,33). In addition, CaMKII activation also regulates the activity of the ether-a-go-go (eag) family of potassium channels in Drosophila, altering aspects of nerve excitability that could contribute to synaptic growth (48,49). Intracellular calcium levels directly regulate cAMP signaling through the activation of adenylate cyclase by calmodulin (50), enhancing cAMPdependent pathways implicated in synaptic growth (12).…”

Camguk/CASK Enhances Ether-Á-Go-Go Potassium Current by a Phosphorylation-Dependent Mechanism

“…Previous work has shown that EAG is phosphorylated by CaMKII activity endogenous to the oocytes even at basal calcium levels (Wang et al, 2002). Both the increase in current amplitude and the changes in inactivation in the presence of CMG are reminiscent of the changes in current observed as a consequence of phosphorylation.…”

“…eag larvae exhibit spontaneous action potentials in the motor nerve and excitatory junctional potentials in muscle that are broadened compared with those of wild-type larvae (Ganetzky and Wu, 1985). EAG is present in the axons and terminals innervating the larval body-wall musculature (Wang et al, 2002) and is localized with synaptobrevin in the CNS (Sun et al, 2004).…”

“…Intriguingly, there is substantial similarity in the electrophysiological and learning phenotypes of eag mutants and transgenic Drosophila expressing an inhibitory peptide of calcium-and calmodulin-dependent protein kinase II (CaMKII) (Griffith et al, 1994), and phosphorylation of EAG by CaMKII results in a dramatic increase in current (Wang et al, 2002). Recent work has demonstrated that Drosophila CaMKII interacts with the Camguk (CMG) adaptor protein (Lu et al, 2003).…”

Plasticity and Second Messengers During Synapse Development

“…At the NMJ, CaMKII regulates presynaptic excitability and plasticity Park et al, 2002). One mechanism by which excitability can be regulated is by modulation of potassium channels, including the EAG protein Wang et al, 2002). Postsynaptic CaMKII has a role in regulation of Dlg and FasII localization (Koh et al, 2000;Fig.…”