Antisense Knock Out of the Inositol 1,3,4,5-Tetrakisphosphate Receptor GAP1IP4BP in the Human Erythroleukemia Cell Line Leads to the Appearance of Intermediate Conductance K(Ca) Channels that Hyperpolarize the Membrane and Enhance Calcium Influx

To study the role of the inositol 1,3,4,5-trisphosphate–binding protein GAP1^IP4BP in store-operated Ca²⁺ entry, we established a human erythroleukemia (HEL) cell line in which the expression of GAP1^IP4BP was substantially reduced by transfection with a vector containing antisense DNA under control of a Rous Sarcoma virus promoter and the Escherichia coli LacI repressor (AS-HEL cells). Control cells were transfected with vector lacking antisense DNA (V-HEL cells). GAP1^IP4BP protein, which is a member of the GTPase-activating protein (GAP1) family, was reduced by 85% in AS-HEL cells and was further reduced by 96% by treatment with isopropylthio-β-d- galactoside to relieve LacI repression. The loss of GAP1^IP4BP was associated with both a membrane hyperpolarization and a substantially increased Ca²⁺ entry induced by thrombin or thapsigargin. The activation of intermediate conductance Ca²⁺-activated K⁺ channels in AS-HEL cells (not seen in V-HEL cells) was responsible for the membrane hyperpolarization and the enhanced Ca²⁺ entry, and both were blocked by charybdotoxin. Stimulated V-HEL cells did not hyperpolarize and basal Ca²⁺ influx was unaffected by charybdotoxin. In V-HEL cells hyperpolarized by removal of extracellular K⁺, the thapsigargin-stimulated Ca²⁺ influx was increased. Expression of mRNA for the human Ca²⁺-activated intermediate conductance channel KCa4 was equivalent in both AS-HEL and V-HEL cells, suggesting that the specific appearance of calcium-activated potassium current (I_K(Ca)) in AS-HEL cells was possibly due to modulation of preexisting channels. Our results demonstrate that GAP1^IP4BP, likely working through a signaling pathway dependent on a small GTP-binding protein, can regulate the function of K(Ca) channels that produce a hyperpolarizing current that substantially enhances the magnitude and time course of Ca²⁺ entry subsequent to the release of internal Ca²⁺ stores.