Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells

In human embryonic kidney (HEK) cells stably transfected with green fluores cent protein targeted to the endoplasmic reticulum (ER), elevation of intra cellular Ca2+ ([Ca2+](i)) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented ci rcular and branched rearrangements of the endoplasmic reticulum, but did no t involve obvious swelling or pathological fragmentation. Activation of pro tein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the eff ects of ionomycin on ER structure without affecting the elevation of [Ca2+] (i). These results suggest that protein kinase C activation alters cytoplas mic or ER components underlying the effects of high [Ca2+](i) on ER structu re. Treatment of HEK cells with PMA also reduced the size of the thapsigarg in-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin . Thus, protein kinase C activation has multiple actions on the calcium sto rage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitativ e Ca2+ entry, and (3) protection of the endoplasmic reticulum against the e ffects of high [Ca2+](i). (C) 2000 Harcourt Publishers Ltd.