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

Zheng Wang,Gisela F Wilson,Leslie C Griffith

doi:10.1074/jbc.m201949200

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.

Highlights

Modulation of neuronal excitability is believed to be an important mechanism of plasticity in the nervous system
These results show that Eag is a substrate of calmodulin-dependent protein kinase II (CaMKII) and suggest that direct modulation of potassium channels may be an important function of this kinase
Peak amplitudes of Eag-T787A currents elicited by test pulses to ϩ40 mV were 1.1 Ϯ 0.1 ␮A (n ϭ 9) and 1.0 Ϯ 0.1 ␮A (n ϭ 7) for control and lavendustin C-treated oocytes, respectively. These results suggest that CaMKII endogenous to oocytes phosphorylates Eag channels and that phosphorylation at Thr-787 produces a substantial increase in Eag current

Summary

EXPERIMENTAL PROCEDURES

GST Fusion Protein Constructs—Fragments of the Eag cDNA were cloned into pGEX-2T (Amersham Biosciences) to make GST fusion proteins. 25 ␮l of SDS sample loading buffer (9% SDS, 25% glycerol, 0.186 M Tris pH 8.9, 5% 2-mercaptoethanol) was added to the reaction mixture and heated at 100 °C for 3 min. The reactions were allowed to proceed at 30 °C for 10 min and terminated by adding 200 ␮l of solubilization buffer and 5 ␮l of the anti-Eag antibody. The beads were washed, and proteins were boiled off in SDS sample loading buffer and separated on SDS gels. Preparations were washed three times (15 min each) with PBT (PBS, 0.3% Triton X-100, 0.1% bovine serum albumin) and incubated in 1:50 affinity-purified anti-Thr(P)-787 in PBT overnight at 4 °C, followed by staining with a rhodamine-conjugated anti-rabbit IgG secondary antibody (Cappel, 1:200, 60 min at 37 °C).

Phosphorylation of Eag by CaMKII

RESULTS

DISCUSSION

TABLE I Eag kinetics

WT control WT plus lavendustin C