Ca2+/Calmodulin-dependent Protein Kinase II Is an Essential Mediator in the Coordinated Regulation of Electrocyte Ca2+-ATPase by Calmodulin and Protein Kinase A

Rafael H.F Valverde,Giovane G Tortelote,Thiago Lemos,Elisabeth Mintz,Adalberto Vieyra

doi:10.1074/jbc.m501880200

Abstract

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (approximately 2-fold). The addition of cAMP (5-50 pM), forskolin (10 nM), or cholera toxin (10 or 100 nM) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA alpha-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA.

Highlights

The electrocytes of the electric organs from Electrophorus electricus L. are specialized structures that generate electric potentials and produce electric discharges similar to those of nerve and muscle [1]
Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2؉ pump (Ϸ2-fold)
This study has revealed that the synaptic-like face (IF) of the electrocytes of E. electricus L. has an asymmetrically localized, very high affinity plasma membrane Ca2ϩ-ATPase (Fig. 1A)

Summary

Introduction

The electrocytes of the electric organs from Electrophorus electricus L. are specialized structures that generate electric potentials and produce electric discharges similar to those of nerve and muscle [1]. The plasma membrane Ca2ϩATPase is responsible for fine-tuning of that Ca2ϩ disequilibrium [3,4,5,6,7,8,9], which allows the generation of Ca2ϩ spikes, waves, and oscillations in most of the eukaryotic cells (10 –12). The plasma membrane high affinity Ca2ϩ-ATPase is a member of the P-type class of ATPases [20] and operates as an electrogenic Ca2ϩ/Hϩ exchanger, pumping cytosolic Ca2ϩ to the extracellular space and internalizing Hϩ at a 1:1 stoichiometry [21]. As far as we know, there is no information regarding the localization of the plasma membrane Ca2ϩ-ATPase on the surface of the electrocyte and the mechanisms involved in its regulation. The content of calmodulin (CaM) is especially high in the electric eel (Ϸ1–2% of the total mass) [29], and immunofluorescence studies have demonstrated that this regulatory

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Conclusion