title: 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 and enhance calcium influx.
abstract: 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-beta-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 (IK(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.
authors.affiliation: Department of Pharmacology, The University of Connecticut Health Center, Farmington, Connecticut 06030, USA.
authors.forename: X
authors.initials: X
authors.lastname: Lu
authors.forename: A
authors.initials: A
authors.lastname: Fein
authors.forename: M B
authors.initials: MB
authors.lastname: Feinstein
authors.forename: F A
authors.initials: FA
authors.lastname: O'Rourke
mesh_terms: D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D016373:DNA, Antisense / Q000096:biosynthesis / Q000235:genetics*; D004558:Electric Stimulation; D004594:Electrophysiology; D016257:Fura-2; D006801:Humans; D004915:Leukemia, Erythroblastic, Acute / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D015221:Potassium Channels / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D018160:Receptors, Cytoplasmic and Nuclear / Q000096:biosynthesis* / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
chemical_list: D016373:DNA, Antisense; D015221:Potassium Channels; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; C061107:inositol-1,3,4,5-tetrakisphosphate receptor; D002118:Calcium; D016257:Fura-2