Vol. 63, Issue 5, 1125-1136, May 2003

Destabilization of Na_v1.7 Sodium Channel -Subunit mRNA by Constitutive Phosphorylation of Extracellular Signal-Regulated Kinase: Negative Regulation of Steady-State Level of Cell Surface Functional Sodium Channels in Adrenal Chromaffin Cells

Toshihiko Yanagita, Hideyuki Kobayashi, Yasuhito Uezono, Hiroki Yokoo, Takashi Sugano, Tomokazu Saitoh, Shin-Ichi Minami, Seiji Shiraishi, and Akihiko Wada

Department of Pharmacology, Miyazaki Medical College, Miyazaki, Japan

In cultured bovine adrenal chromaffin cells expressing Na_v1.7 isoform of voltage-dependent Na⁺ channels, treatment (6 h) with serum deprivation, PD98059, or U0126 increased cell surface [³H]saxitoxin ([³H]STX) binding by ~58% (t_1/2 = 12.5 h), with no change in the K_d value. Immunoblot analysis showed that either treatment attenuated constitutive phosphorylation of extracellular signal-regulated kinase (ERK) 1 and ERK2 but not of p38 mitogen-activated protein kinase and c-Jun N-terminal kinase (JNK) 1 and JNK2. The increase of [³H]STX binding and the attenuated phosphorylation of ERK1 and ERK2 returned to the control nontreated levels after the addition of serum or the washout of PD98059- or U0126-treated cells. Simultaneous treatment of serum deprivation with PD98059 or U0126 did not produce an additional increasing effect on [³H]STX binding, compared with either treatment alone. In cells subjected to either treatment, veratridine-induced maximum ²²Na⁺ influx was augmented by ~47%, with no change in the EC₅₀ value; Ptychodiscus brevis toxin-3 enhanced veratridine-induced ²²Na⁺ influx by 2-fold, as in nontreated cells. Serum deprivation, PD98059, or U0126 increased Na⁺ channel - but not ₁- subunit mRNA level by ~50% between 3 and 24 h; cycloheximide, an inhibitor of protein synthesis, increased -subunit mRNA level and nullified additional increasing effect of either treatment on -subunit mRNA level. Either treatment prolonged half-life of -subunit mRNA from 17.5 to ~26.3 h without altering -subunit gene transcription. Thus, constitutively phosphorylated/activated ERK destabilizes Na⁺ channel -subunit mRNA via translational event, which negatively regulates steady-state level of -subunit mRNA and cell surface expression of functional Na⁺ channels.