Translocon closure to calcium leak in vascular smooth muscle cells

Abstract

The translocon is an endoplasmic reticulum (ER) membrane protein complex that mediates translocation of nascent polypeptide into the ER. Recent studies have suggested it also acts as a calcium leak channel of the ER (Flourakis et al 2006 FASEB J, 20, 1215–7). Our objective was to investigate the relevance of this hypothesis to proliferating vascular smooth muscle cells. Intracellular calcium and protein synthesis were measured using fura‐2 and 35S‐methionine respectively. Chemical inhibitors of protein synthesis (puromycin, emetine and anisomycin) all act via the translocon and were confirmed as strong inhibitors of protein synthesis in smooth muscle cells after 1 hr of incubation. Puromycin (which holds the translocon open) evoked a transient increase in calcium that was inhibited by pre‐incubation with thapsigargin. Emetine and anisomycin (which close the translocon) inhibited calcium‐release evoked by puromycin, but not the calcium‐release evoked by extracellular ATP or ionomycin. Thapsigargin‐evoked calcium release was slightly reduced by emetine but unaffected by anisomycin. The data suggest the translocon is not a physiological calcium leak channel in vascular smooth muscle cells but that it can enable calcium‐release when acted on by puromycin.The research is supported by the Wellcome Trust and The Egyptian Ministry of Higher Education.