Antisense knock out of the inositol 1,3,4,5-tetrakisphosphate receptor GAP1(IP4BP) in the human erythroleukemia cell line leads to the appearance of intermediate conductance K(Ca) channels that hyperpolarize the membrane andenhance calcium influx

To study the role of the inositol I,3,4,5-trisphosphate-binding protein GAP 1(IP4BP) in store-operated Ca2+ entry, we established a human erythroleukem ia (HEL) cell line in which the expression of GAP1(IP4BP) was substantially reduced by transfection with a vector containing antisense DNA under contr ol of a Rous Sarcoma virus promoter and the Escherichia coli LacI repressor (AS-HEL cells). Control cells were transfected with vector lacking antisen se DNA (V-HEL cells). GAP1(IP4BP) protein, which is a member of the GTPase- activating protein (GAP1) family, was reduced by 85% in AS-MEL cells and wa s further reduced by 96% by treatment with isopropylthio-beta-D-galactoside to relieve LacI repression. The loss of GAP1(IP4BP) was associated with bo th a membrane hyperpolarization and a substantially increased Ca2+ entry in duced by thrombin or thapsigargin. The activation of intermediate conductan ce Ca2+-activated K+ channels in AS-MEL 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 hy perpolarize and basal Ca2+ influx was unaffected by charybdotoxin. In V-MEL cells hyperpolarized by removal of extracellular K+, the thapsigargin-stim ulated Ca2+ influx was increased. Expression of mRNA for the human Ca2+-act ivated intermediate conductance channel KCa4 was equivalent in both AS-MEL and V-HEL cells, suggesting that the specific appearance of calcium-activat ed potassium current (I-K(Ca)) in AS-MEL cells was possibly due to modulati on of preexisting channels. Our results demonstrate that GAP1(IP4BP), likel y working through a signaling pathway dependent on a small GTP-binding prot ein, can regulate the function of K(Ca) channels that produce a hyperpolari zing current that substantially enhances the magnitude and time course of C a2+ entry subsequent to the release of internal Ca2+ stores.