Modulation of calcium channels in human erythroblasts by erythropoietin.
Erythropoietin (Epo) induces a dose-dependent increase in intracellular free Ca2+ ([Ca2+]i) in human erythroblasts, which is dependent on extracellular Ca2+ and blocked by high doses of nifedipine or Ni2+. In addition, pretreatment of human erythroblasts with mouse antihuman erythropoietin receptor antibody but not mouse immunopure IgG blocked the Epo-induced [Ca2+]i increase, indicating the specificity of the Ca2+ response to Epo stimulation. In this study, the erythropoietin-regulated calcium channel was identified by single channel recordings. Use of conventional whole cell patch-clamp failed to detect Epo-induced whole cell Ca2+ current. To minimize washout of cytosolic constituents, we next used nystatin perforated patch, but did not find any Epo-induced whole cell Ca2+ current. Using Ba2+ (30 mmol/L) as charge carrier in cell-attached patches, we detected single channels with unitary conductance of 3.2 pS, reversal potential of +72 mV, and whose unitary current (at +10 mV) increased monotonically with increasing Ba2+ concentrations. Channel open probability did not appreciably change over the voltage range (-50 to +30 mV) tested. Epo (2 U/mL) increased both mean open time (from 4.27 +/- 0.75 to 11.15 +/- 1.80 ms) and open probability (from 0.26 +/- 0.06 to 2.56 +/- 0.59%) of this Ba(2+)-permeable channel. Our data strongly support the conclusion that the Epo-induced [Ca2+]i increase in human erythroblasts is mediated via Ca2+ entry through a voltage-independent Ca2+ channel.
Cheung
J Y
JY
Department of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey, USA.
Zhang
X Q
XQ
Bokvist
K
K
Tillotson
D L
DL
Miller
B A
BA
D000818:Animals; D002118:Calcium / Q000097:blood*; D015220:Calcium Channels / Q000187:drug effects*; D004900:Erythroblasts / Q000187:drug effects* / Q000378:metabolism; D004921:Erythropoietin / Q000494:pharmacology*; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008564:Membrane Potentials; D051379:Mice; D009761:Nystatin / Q000494:pharmacology; D018408:Patch-Clamp Techniques; D017467:Receptors, Erythropoietin / Q000037:antagonists & inhibitors / Q000276:immunology
D015220:Calcium Channels; D007074:Immunoglobulin G; D007476:Ionophores; D017467:Receptors, Erythropoietin; D004921:Erythropoietin; D009761:Nystatin; D002118:Calcium