Anion channels modulate store-operated calcium influx in human microglia.
Recent work from this laboratory has demonstrated that purinergic-mediated depolarization of human microglia inhibited a store-operated pathway for entry of Ca2+. We have used Fura-2 spectrofluorometry to investigate the effects on store-operated Ca2+ influx induced by replacement of NaCl with Na-gluconate in extracellular solutions. Three separate procedures were used to activate store-operated channels. Platelet activating factor (PAF) was used to generate a sustained influx of Ca2+ in standard physiological saline solution (PSS). The magnitude of this response was depressed by 70% after replacement of PSS with low Cl- PSS. A second procedure used ATP, initially applied in Ca2+-free PSS solution to deplete intracellular stores. The subsequent perfusion of PSS solution containing Ca2+ resulted in a large and sustained entry of Ca2+, which was inhibited by 75% with low Cl- PSS. The SERCA inhibitor cyclopiazonic acid (CPA) was used to directly deplete stores in zero-Ca2+ PSS. Following the introduction of PSS containing Ca2+, a maintained stores-operated influx of Ca2+ was evident which was inhibited by 77% in the presence of the low Cl- PSS. Ca2+ influx was linearly reduced with cell depolarization in elevated K+ (7.5 to 35 mM) suggesting that changes in external Cl- were manifest as altered electrical driving force for Ca2+ entry. However, 50 mM external KCl effectively eliminated divalent entry which may indicate inactivation of this pathway with high magnitudes of depolarization. Patch clamp studies showed low Cl-PSS to cause depolarizing shifts in both holding currents and reversal potentials of currents activated with voltage ramps. The results demonstrate that Cl- channels play an important role in regulating store-operated entry of Ca2+ in human microglia.
McLarnon
J G
JG
Department of Pharmacology and Therapeutics, Faculty of Medicine, The University of British Columbia, Vancouver, British Columbia, Canada. Mclarnon@interchange.ubc.ca
Helm
J
J
Goghari
V
V
Franciosi
S
S
Choi
H B
HB
Nagai
A
A
Kim
S U
SU
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000838:Anions / Q000378:metabolism; D002021:Buffers; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors; D002478:Cells, Cultured; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005942:Gluconates / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007473:Ion Channels / Q000502:physiology*; D017628:Microglia / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010972:Platelet Activating Factor / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D012965:Sodium Chloride / Q000494:pharmacology
D000838:Anions; D002021:Buffers; D004791:Enzyme Inhibitors; D005942:Gluconates; D007211:Indoles; D007473:Ion Channels; D010972:Platelet Activating Factor; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; C030691:gluconic acid; D011188:Potassium; D002118:Calcium; C000543:cyclopiazonic acid