Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase.
Single Ca2+-activated K+ channels of human erythrocytes were studied with the patch-clamp technique, to identify the mechanisms of their modulation by phosphorylation. In the cell-attached configuration, the openings of these channels were infrequent, as expected by the low cell Ca2+ content. After patch excision, the activity increased to levels determined by the Ca2+ concentration (0.5-10 microM) in the bath solution, then the channel activity ran down within a few minutes, to reach values of open probability lower than 0.10. The perfusion of the patch with MgATP increased the channel activity, with delayed and variable effects. Furthermore, the application of a mixture of cAMP (1 mM), MgATP (1 mM) and theophylline (1 mM) to the cytoplasmic side of excised patches led to dramatic enhancement of channel activity, which appeared within 20-120 s and decayed in tens of seconds after wash-out. The activation of the channel by the mixture was reversibly blocked by PKI5-24, a peptide inhibitor specific to cAMP-dependent protein kinase (PKA). The level of activation promoted by cAMP and ATP was dependent on the Ca2+ concentration in the bathing solution. These results provide direct evidence that an endogenous PKA modulates the calcium sensitivity of Ca2+-activated K+ channels of human erythrocytes.
Pellegrino
M
M
Dipartimento di Fisiologia e Biochimica "G. Moruzzi", Universita' di Pisa, Via S. Zeno 31, I-56127 Pisa, Italy. marpell@dfb.unipi.it
Pellegrini
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000328:Adult; D002118:Calcium / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology / Q000378:metabolism*; D005260:Female; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D008297:Male; D008875:Middle Aged; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D013806:Theophylline / Q000494:pharmacology; D015854:Up-Regulation
D010446:Peptide Fragments; D015221:Potassium Channels; D047428:Protein Kinase Inhibitors; C518603:protein kinase inhibitor peptide (5-24); D000255:Adenosine Triphosphate; D013806:Theophylline; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium