title: A sustained increase in the intracellular Ca²⁺ concentration induces proteolytic cleavage of EAG2 channel.
abstract: Voltage-gated EAG2 channel is abundant in the brain and enhances cancer cell growth by controlling cell volume. The channel contains a cyclic nucleotide-binding homology (CNBH) domain and multiple calmodulin-binding motifs. Here we show that a raised intracellular Ca(2+) concentration causes proteolytic digestion of heterologously expressed and native EAG2 channels. A treatment of EAG2-expressing cells with the Ca(2+) ionophore A23187 for 1h reduces the full-length protein by ∼80% with a concomitant appearance of 30-35-kDa peptides. Similarly, a treatment with the Ca(2+)-ATPase inhibitor thapsigargin for 3h removes 30-35-kDa peptides from ∼1/3 of the channel protein. Moreover, an incubation of the isolated rat brain membrane with CaCl2 leads to the generation of fragments with similar sizes. This Ca(2+)-induced digestion is not seen with EAG1. Mutations in a C-terminal calmodulin-binding motif alter the degrees and positions of the cleavage. Truncated channels that mimic the digested proteins exhibit a reduced current density and altered channel gating. In particular, these shorter channels lack a rapid activation typical in EAG channels with more than 20-mV positive shifts in voltage dependence of activation. The truncation also eliminates the ability of EAG2 channel to reduce cell volume. These results suggest that a sustained increase in the intracellular Ca(2+) concentration leads to proteolytic cleavage at the C-terminal cytosolic region following the CNBH domain by altering its interaction with calmodulin. The observed Ca(2+)-induced proteolytic cleavage of EAG2 channel may act as an adaptive response under physiological and/or pathological conditions.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Shimizu
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Natsumi
authors.initials: N
authors.lastname: Sato
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Teppei
authors.initials: T
authors.lastname: Kikuchi
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Ishizaki
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan.
authors.forename: Kazuto
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15261, United States.
authors.forename: Kaori
authors.initials: K
authors.lastname: Kita
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan; Department of Environmental and Occupational Health, University of Pittsburgh, Pittsburgh, PA 15261, United States. Electronic address: koichi@vos.nagaokaut.ac.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Takimoto
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism; D002460:Cell Line; D048429:Cell Size / Q000187:drug effects; D004151:Dipeptides / Q000494:pharmacology; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D059748:Proteolysis / Q000187:drug effects*; D017207:Rats, Sprague-Dawley
chemical_list: D002147:Calmodulin; D004151:Dipeptides; D051638:Ether-A-Go-Go Potassium Channels; C485729:KCNH5 protein, human; D050505:Mutant Proteins; D002118:Calcium; C058076:calpain inhibitor III
keywords: Ca(2+); Cancer; Cell volume; Potassium channel; Proteolysis