Protein kinase C activators induce membrane retrieval of type II Na+-phosphate cotransporters expressed in Xenopus oocytes.
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.
Forster
I C
IC
Institute of Physiology, University of Zurich, Zurich, Switzerland. forster@physiol.unizh.ch
Traebert
M
M
Jankowski
M
M
Stange
G
G
Biber
J
J
Murer
H
H
D000818:Animals; D002352:Carrier Proteins / Q000187:drug effects / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism / Q000502:physiology; D004075:Diglycerides / Q000494:pharmacology; D004553:Electric Conductivity; D004789:Enzyme Activation / Q000502:physiology; D005260:Female; D005432:Flounder; D006706:Homeostasis / Q000502:physiology; D007700:Kinetics; D051379:Mice; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D010710:Phosphates / Q000494:pharmacology; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters; D014982:Xenopus laevis
D002352:Carrier Proteins; D004075:Diglycerides; D010710:Phosphates; D020033:Protein Isoforms; C493555:Slc34a1 protein, mouse; C493556:Slc34a1 protein, rat; C493558:Slc34a2 protein, mouse; C493559:Slc34a2 protein, rat; D050608:Sodium-Phosphate Cotransporter Proteins; D050610:Sodium-Phosphate Cotransporter Proteins, Type II; D050613:Sodium-Phosphate Cotransporter Proteins, Type IIa; D050614:Sodium-Phosphate Cotransporter Proteins, Type IIb; D027981:Symporters; C044295:1,2-dioctanoylglycerol; D011493:Protein Kinase C