Store-operated Ca²⁺ entry and coupling to Ca²⁺ pool depletion in thapsigargin-resistant cells

The release of Ca²⁺ from intracellular Ca²⁺ pumping pools and the entry of extracellular Ca²⁺ are tightly coupled events. The potent and specific intracellular Ca²⁺ pump inhibitor, thapsigargin, blocks Ca²⁺ accumulation and allows Ca²⁺ release from pools within mammalian cells, inducing major changes in endoplasmic reticulum function and cell growth. Recent studies characterized the pools of Ca²⁺ within permeabilized DC- 3F/TG2 cells (a thapsigargin-resistant variant form of the DC-3F Chinese hamster lung fibroblast line, able to grow in 2 μM thapsigargin), revealing highly thapsigargin-resistant intracellular Ca²⁺ pumping activity capable of accumulating Ca²⁺ within an inositol 1,4,5-trisphosphate-releasable Ca²⁺ pool (Waldron, R. T., Short, A. D., and Gill, D. L. (1995) J. Biol. Chem. 270, 11955-11961). Using intact fura-2-loaded thapsigargin-resistant DC-3F/TG2 cells, the present study investigated the role of this unusual Ca²⁺ pumping activity in maintaining cytosolic Ca²⁺, generating Ca²⁺ signals, and mediating Ca²⁺ entry. The thapsigargin-resistant Ca²⁺ pumping pool was capable of generating rapid cytosolic Ca²⁺ signals in response to the phospholipase C-coupled agonist, oleoyl lysophosphatidic acid. The resting level of cytosolic Ca²⁺ in DC-3F/TG2 cells was 2-fold elevated compared with control cells (the parent DC-3F line), and transient extracellular Ca²⁺ removal induced a large 'overshoot' in cytosolic Ca²⁺. The overshoot response was blocked by the Ca²⁺ influx inhibitor, SKF96365, and was kinetically identical to that induced in parent DC-3F cells after thapsigargin-induced Ca²⁺ pool emptying, indicating that the thapsigargin- resistant DC-3F/TG2 cells had 'constitutively' opened Ca²⁺ entry channels coupled to an emptied or partially emptied thapsigargin-sensitive Ca²⁺ pumping pool. Even though oleoyl lysophosphatidic acid-mediated Ca²⁺ release induced little Ca²⁺ entry, complete ionomycin-activated emptying of the thapsigargin-resistant Ca²⁺ pool in DC-3F/TG2 cells induced a large, sustained entry of Ca²⁺ that was also completely blocked by SKF96365. The results revealed that the thapsigargin-resistant Ca²⁺ pump does maintain physiological Ca²⁺ levels, is able to fill an agonist-responsive Ca²⁺ pool in DC-3F/TG2 cells, and is likely responsible for the ability of these cells to function and grow in the presence of thapsigargin. In addition, Ca²⁺ influx in the resistant DC-3F/TG2 cells reflects emptying of pools that accumulate Ca²⁺ by both thapsigargin-sensitive and -resistant Ca²⁺ pumps; since these pumps accumulate Ca²⁺ in distinct pools in parent DC-3F cells, it is possible that more than one pool is coupled to Ca²⁺ influx in the resistant DC-3F/TG2 cells.