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© The Rockefeller University Press, 0022-1295/1999//81/ $5.00
Journal of General Physiology, Volume 113, Number 1, 1999

Article

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

Xinghua Lu*, Alan Fein{ddagger}, Maurice B. Feinstein*, and Flavia A. O'Rourke*

From the * Department of Pharmacology and {ddagger} Department of Physiology, The University of Connecticut Health Center, Farmington, Connecticut 06030

To study the role of the inositol 1,3,4,5-trisphosphate–binding protein GAP1IP4BP in store-operated Ca2+ entry, we established a human erythroleukemia (HEL) cell line in which the expression of GAP1IP4BP 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). GAP1IP4BP 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 GAP1IP4BP 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 GAP1IP4BP, 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.

Key Words: GAP1IP4BP • inositol 1,3,4,5-tetrakisphosphate • store-operated calcium entry • calcium-activated potassium channel

Address correspondence to Flavia A. O'Rourke, Department of Pharmacology, The University of Connecticut Health Center, Farmington, CT 06030. Fax: 860-679-3693; E-mail: orourke{at}nso1.uchc.edu

Abbreviations: AS-HEL, antisense-transfected HEL; CTX, charybdotoxin; HEL, human erythroleukemia; HPSS, HEPES-buffered physiological salt solution; ICRAC, calcium release activated calcium current; InsP3, inositol 1,4,5 trisphosphate; InsP4, inositol 1,3,4,5 tetrakisphosphate; IPTG, isopropylthio-β-D-galactoside; I–V, current–voltage; RSV, Rous Sarcoma virus; TEA, tetraethylammonium; TMRE, tetramethylrhodamine ethyl ester; V-HEL, vector-transfected HEL


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