Protein kinase C activators induce membrane retrieval of type IINa+-phosphate cotransporters expressed in Xenopus oocytes

1. The rate of inorganic phosphate (P-i) reabsorption in the mammalian kidn ey is determined by the amount of type II sodium-coupled inorganic phosphat e (Na+-P-i) cotransport protein present in the brush border membrane. Under physiological conditions, parathyroid hormone (PTH) leads to an inhibition of Na+-P-i cotransport activity, most probably mediated by the protein kin ase A (PKA) and/or C (PKC) pathways. 2. In this study, PKC-induced inhibition of type II Na+-P-i cotransport act ivity was characterized in Xenopus laevis oocytes using electrophysiologica l and immunodetection techniques. Transport function was quantified in term s of P-i-activated current. 3. Oocytes expressing the type IIa rat renal, type IIb flounder renal or ty pe IIb mouse intestinal Na+-P-i cotransporters lost > 50% of P-i-activated transport function when exposed to the PKC activators DOG (1,2-dioctanoyl-s n-glycerol) or PMA (phorbol 12-myristate 13-acetate). DOG-induced inhibitio n was partially reduced with the PKC: inhibitors staurosporine and bisindol ylmaleimide I. Oocytes exposed to the inactive phorbol ester 4 alpha-PDD (4 alpha-phorbol 12,13-didecanoate) showed no significant loss of cotransport er function. 4. Oocytes expressing the rat renal Na+-SO42- cotransporter alone, or coexp ressing this with the type IIa rat renal Na+-P-i cotransporter, showed no d ownregulation of SO42--activated cotransport activity by DOG. 5. Steady-state and presteady-state voltage-dependent kinetics of type II N a+-P-i cotransporter function were unaffected by DOG 6. DOG induced a decrease in membrane capacitance which indicated a reducti on in membrane area, thereby providing evidence for PKC-mediated endocytosi s. 7. Immunocytochemical studies showed a redistribution of type II Na+-P-i co transporters from the oolemma to the submembrane region after DOG: treatmen t. Surface biotinylation confirmed a DOG-induced internalization of the tra nsport protein. 8. These findings document a specific retrieval of exogenous type II Na+-P- i cotransporters induced by activation of a PKC pathway in the Xenopus oocy te.