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ANESTHETIC PHARMACOLOGY

Modulation of Xenopus laevis Ca-Activated Cl Currents by Protein Kinase C and Protein Phosphatases: Implications for Studies of Anesthetic Mechanisms

Klaus Hahnenkamp, MD^*, Marcel E. Durieux, MD PhD^*, Hugo van Aken, MD PhD^*, Sascha Berning^*, Thomas J. Heyse^*, Christian W. Hönemann, MD^*, and Bettina Linck, MD PhD

*Department of Anesthesiology and Intensive Care, and Institute of Pharmacology and Toxicology, University Hospital, Muenster, Germany

Address correspondence to Marcel E. Durieux, MD, PhD, Department of Anesthesiology, University of Virginia, PO Box 800710, Charlottesville, VA 22908–0710. Address email to durieux{at}virginia.edu

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 PP1 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 PP1 and PP2A were overexpressed in oocytes. PP, inositoltrisphosphate (IP₃), the PP inhibitor okadaic acid (OA), the PKC inhibitor chelerythrine, or CaCl₂ were directly injected into the oocyte. Responses to agonists (LPA 10^–6 M, IP₃ 10^–4 M, CaCl₂ 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 and PP2A inhibited I_Cl(Ca) from 7.6 ± 0.9 µC to 2.5 ± 0.9 µC and 3.2 ± 1.4 µC, respectively. PP inhibition enhanced I_Cl(Ca) in control oocytes and reversed the inhibitory effect in oocytes expressing PP1 or PP2A. PKC inhibition by chelerythrine enhanced both LPA- and CaCl₂-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.

IMPLICATIONS: The Xenopus I_Cl(Ca), commonly used as a reporter current in studies of anesthetic effects on G protein-coupled signaling, is modulated by phosphorylation. Anesthetic effects on channel phosphorylation state can therefore be misinterpreted as effects on receptor signaling.