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Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes.
Forster IC, Traebert M, Jankowski M, Stange G, Biber J, Murer H.
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1. The rate of inorganic phosphate (Pi) reabsorption in the mammalian kidney is determined by the amount of type II sodium-coupled inorganic phosphate (Na+-Pi) cotransport protein present in the brush border membrane. Under physiological conditions, parathyroid hormone (PTH) leads to an inhibition of Na+-Pi cotransport activity, most probably mediated by the protein kinase A (PKA) and/or C (PKC) pathways. 2. In this study, PKC-induced inhibition of type II Na+-Pi cotransport activity was characterized in Xenopus laevis oocytes using electrophysiological and immunodetection techniques. Transport function was quantified in terms of Pi-activated current. 3. Oocytes expressing the type IIa rat renal, type IIb flounder renal or type IIb mouse intestinal Na+-Pi cotransporters lost > 50 % of Pi-activated transport function when exposed to the PKC activators DOG (1,2-dioctanoyl-sn-glycerol) or PMA (phorbol 12-myristate 13-acetate). DOG-induced inhibition was partially reduced with the PKC inhibitors staurosporine and bisindolylmaleimide I. Oocytes exposed to the inactive phorbol ester 4alpha-PDD (4alpha-phorbol 12,13-didecanoate) showed no significant loss of cotransporter function. 4. Oocytes expressing the rat renal Na+-SO42- cotransporter alone, or coexpressing this with the type IIa rat renal Na+-Pi cotransporter, showed no downregulation of SO42--activated cotransport activity by DOG. 5. Steady-state and presteady-state voltage-dependent kinetics of type II Na+-Pi cotransporter function were unaffected by DOG. 6. DOG induced a decrease in membrane capacitance which indicated a reduction in membrane area, thereby providing evidence for PKC-mediated endocytosis. 7. Immunocytochemical studies showed a redistribution of type II Na+-Pi cotransporters from the oolemma to the submembrane region after DOG treatment. Surface biotinylation confirmed a DOG-induced internalization of the transport protein. 8. These findings document a specific retrieval of exogenous type II Na+-Pi cotransporters induced by activation of a PKC pathway in the Xenopus oocyte.
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