Modulation of Xenopus laevis Ca-activated Cl currents by protein kinase C and protein phosphatases: implications for studies of anesthetic mechanisms.
Ca-activated Cl currents (I(Cl(Ca))) are used frequently as reporters in functional studies of anesthetic effects on G protein-coupled receptors using Xenopus laevis oocytes. However, because anesthetics affect protein kinase C (PKC), they could indirectly affect I(Cl(Ca)) if this current is regulated by phosphorylation. We therefore studied the effect of modulation of either PKC or protein phosphatases PP1alpha and PP2A on I(Cl(Ca)) stimulated either by lysophosphatidate (LPA) signaling or by microinjection of Ca. X. laevis oocytes were studied under voltage clamp. Rat PP1alpha and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP(3)), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl(2) were directly injected into the oocyte. Responses to agonists (LPA 10(-6) M, IP(3) 10(-4) M, CaCl(2) 0.5 M) were measured at a holding potential of -70 mV in the presence or absence of the PP inhibitors cantharidin or OA. PP1 alpha and PP2A inhibited I(Cl(Ca)) from 7.6 +/- 0.9 microC to 2.5 +/- 0.9 microC and 3.2 +/- 1.4 microC, respectively. PP inhibition enhanced I(Cl(Ca)) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 alpha or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl(2)-induced I(Cl(Ca)). Our data indicate that the Xenopus I(Cl(Ca)) is modulated by phosphorylation. This may complicate design and interpretation of studies of G protein-coupled receptors using this model.
Department of Anesthesiology and Intensive Care, University Hospital, Muenster, Germany.
Klaus
K
Hahnenkamp
Marcel E
ME
Durieux
Hugo
H
van Aken
Sascha
S
Berning
Thomas J
TJ
Heyse
Christian W
CW
Hönemann
Bettina
B
Linck
D000465:Algorithms; D000777:Anesthetics / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects* / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D015544:Inositol 1,4,5-Trisphosphate / Q000502:physiology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008564:Membrane Potentials / Q000187:drug effects; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D010957:Plasmids / Q000235:genetics; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000502:physiology*; D018160:Receptors, Cytoplasmic and Nuclear / Q000187:drug effects / Q000502:physiology; D014982:Xenopus laevis
D000777:Anesthetics; D015220:Calcium Channels; D018118:Chloride Channels; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D053496:Inositol 1,4,5-Trisphosphate Receptors; D018160:Receptors, Cytoplasmic and Nuclear; D015544:Inositol 1,4,5-Trisphosphate; D011493:Protein Kinase C; D010749:Phosphoprotein Phosphatases; D002118:Calcium