Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C.
Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.
Dipartimento di Fisiologia e Biochimica, G. Moruzzi, Università di Pisa, Via S. Zeno 31, 56127, Pisa, Italy.
Barbara
B
Del Carlo
Monica
M
Pellegrini
Mario
M
Pellegrino
D000328:Adult; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008875:Middle Aged; D024681:Potassium Channels, Calcium-Activated / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology*; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D024681:Potassium Channels, Calcium-Activated; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate