Spontaneous autocrine release of protons activates ASIC-mediated currents in HEK293 cells.
When examining HEK293 cells by whole-cell patch-clamp electrophysiology we found spontaneous currents, present in almost all cells. These currents were carried by Na(+) ions, were inhibited by amiloride and by cells exposure to acidic (pH 6.3) extracellular solutions. These properties (ion carrier, amiloride-sensitivity, and inactivation by constant lowering of extracellular pH) were similar to the properties of proton-activated currents measured from the same cells. Spontaneous currents required intracellular ATP, were completely inhibited by intracellular Ca(2+) buffering with BAPTA and were suppressed by intracellular administration of vesicular H(+)ATPase inhibitor bafilomycin. ATP-induced Ca(2+) influx through P2X receptors in HEK293 cells stably transfected with P2X(2), P2X(2/3) or P2X(4) purinoreceptor subunits transiently potentiated amplitude and frequency of spontaneous currents; this effect was antagonized by bafilomycin. We concluded that spontaneous currents represent activation of acid-sensitive ion channels (ASICs) by autocrine vesicular release of protons from HEK cells.
Lalo
Ulyana
U
Faculty of Life Sciences, The University of Manchester, Manchester, UK.
Pankratov
Yuri
Y
North
R Alan
RA
Verkhratsky
Alexei
A
D062565:Acid Sensing Ion Channels; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000584:Amiloride / Q000494:pharmacology; D019898:Autocrine Communication / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005089:Exocytosis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011522:Protons; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D058848:Receptors, Purinergic P2X4; D022142:Secretory Vesicles / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D015222:Sodium Channels / Q000378:metabolism*; D014162:Transfection
D062565:Acid Sensing Ion Channels; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C546401:P2RX2 protein, human; C546407:P2RX3 protein, human; C548226:P2RX4 protein, human; D011522:Protons; D018048:Receptors, Purinergic P2; D058476:Receptors, Purinergic P2X2; D058477:Receptors, Purinergic P2X3; D058848:Receptors, Purinergic P2X4; D015222:Sodium Channels; D004533:Egtazic Acid; D000584:Amiloride; D000255:Adenosine Triphosphate; D012964:Sodium; D006180:Proton-Translocating ATPases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium