Membrane insertion of betaine/GABA transporter during hypertonic stress correlates with nuclear accumulation of TonEBP

Abstract

MDCK cells stably transfected with betaine/GABA transporter tagged with EGFP (EGFP–BGT) were used to study plasma membrane insertion of EGFP–BGT. Adaptive response to hypertonicity requires nuclear migration of TonEBP. Confocal microscopy showed that after 6 h hypertonicity, the nuclear/cytoplasmic ratio of TonEBP fluorescence was increased to 2.4 compared to 1.4 in isotonic controls ( P<0.001). The ratio in hypertonic cells was reduced by the proteasome inhibitor MG-132 in a dose-dependent way. Inhibition was 50% at 3 μM. After 6 h, hypertonicity expressed EGFP–BGT was localized in the plasma membrane, but there was no change in total EGFP–BGT abundance compared to isotonic controls. In contrast, EGFP–BGT remained mostly intracellular when 3 μM MG-132 was included in the hypertonic medium. The transport function of EGFP–BGT was studied as Na +-dependent uptake of [ 3H]GABA. This was not changed by MG-132 in isotonic controls, but MG-132 produced dose-dependent inhibition of hypertonic upregulation of Na +/GABA cotransport. Inhibition was 80% at 3 μM MG-132. Transport likely reflects membrane insertion of EGFP–BGT and there was a positive correlation ( P<0.05) between Na +/GABA cotransport and the N/C ratio of TonEBP. Results are consistent with a role for TonEBP-mediated transcription in synthesis of additional proteins required for membrane insertion of EGFP–BGT protein.