Protein kinase A-regulated Cl- channel in ML-1 human hematopoietic myeloblasts.
Using the inside-out patch clamp technique, we identified a Cl- channel in patches from the membrane of cultured human hematopoietic myeloblastic leukemia ML-1 cells. The Cl- channel was not seen at negative membrane potentials in excised patches until the membrane potential was depolarized to greater than +40 mV. The channel was also activated by addition of cAMP-dependent protein kinase (PKA) catalytic subunit at physiological membrane potential (-40 mV). Biophysical studies of the Cl- channel revealed that the current-voltage (I-V) relationship of the Cl- channel was outwardly rectifying in symmetrical 142 mM Cl- solutions. Single channel conductances were 48 pS for the outward current measured at +60 mV and 27 pS for the inward current at -60 mV. The open time constant of the channel was dependent on the membrane potential and was significantly prolonged at positive membrane potentials. Channels activated by cAMP-dependent protein kinase spent a significantly longer time in the open state compared to those channels activated by depolarization pulses. Pharmacological properties of the Cl- channel were also studied. Two anion transport inhibitors, anthracene-9-carboxylic acid (9-AC) and 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS) caused a flickering block of the channel. Half-inhibitory concentrations (IC50) for 9-AC and DIDS were 174 +/- 20 and 70 +/- 16 microM, respectively. Blockade of the Cl- channel by 9-AC or DIDS was completely reversible. Our findings suggest that outwardly rectifying Cl- channels (ORCC) are present in human hematopoietic myeloblasts. The function of ORCC may be involved in hormone-regulated cell growth, cell volume regulation and immune responses.
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Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45435.
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D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000873:Anthracenes / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002454:Cell Differentiation; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D006412:Hematopoietic Stem Cells / Q000502:physiology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D015470:Leukemia, Myeloid, Acute / Q000473:pathology; D008564:Membrane Potentials; D009363:Neoplasm Proteins / Q000502:physiology; D014411:Neoplastic Stem Cells / Q000502:physiology; D018408:Patch-Clamp Techniques; D014407:Tumor Cells, Cultured
D000873:Anthracenes; D018118:Chloride Channels; D009363:Neoplasm Proteins; C010030:9-anthroic acid; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid