Eeyarestatin Compounds Selectively Enhance Sec61-Mediated Ca²⁺ Leakage from the Endoplasmic Reticulum

Eeyarestatin 1 (ES1) inhibits p97-dependent protein degradation, Sec61-dependent protein translocation into the endoplasmic reticulum (ER), and vesicular transport within the endomembrane system. Here, we show that ES1 impairs Ca²⁺ homeostasis by enhancing the Ca²⁺ leakage from mammalian ER. A comparison of various ES1 analogs suggested that the 5-nitrofuran (5-NF) ring of ES1 is crucial for this effect. Accordingly, the analog ES24, which conserves the 5-NF domain of ES1, selectively inhibited protein translocation into the ER, displayed the highest potency on ER Ca²⁺ leakage of ES1 analogs studied and induced Ca²⁺-dependent cell death. Using small interfering RNA-mediated knockdown of Sec61α, we identified Sec61 complexes as the targets that mediate the gain of Ca²⁺ leakage induced by ES1 and ES24. By interacting with the lateral gate of Sec61α, ES1 and ES24 likely capture Sec61 complexes in a Ca²⁺-permeable, open state, in which Sec61 complexes allow Ca²⁺ leakage but are translocation incompetent.