Protein Kinase A Anchoring Proteins Are Required for Vasopressin-mediated Translocation of Aquaporin-2 into Cell Membranes of Renal Principal Cells

Enno Klussmann,Kenan Maric,Burkhard Wiesner,Michael Beyermann,Walter Rosenthal

doi:10.1074/jbc.274.8.4934

Abstract

The antidiuretic hormone arginine-vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells by inducing a cAMP-dependent translocation of water channels (aquaporin-2, AQP-2) from intracellular vesicles into the apical cell membranes. In subcellular fractions from primary cultured rat inner medullary collecting duct (IMCD) cells, enriched for intracellular AQP-2-bearing vesicles, catalytic protein kinase A (PKA) subunits and several protein kinase A anchoring proteins (AKAPs) were detected. In nonstimulated IMCD cells the majority of AQP-2 staining was detected intracellularly but became mainly localized within the cell membrane after stimulation with AVP or forskolin. Quantitative analysis revealed that preincubation of the cells with the synthetic peptide S-Ht31, which prevents the binding between AKAPs and regulatory subunits of PKA, strongly inhibited AQP-2 translocation in response to forskolin. Preincubation of the cells with the PKA inhibitor H89 prior to forskolin stimulation abolished AQP-2 translocation. In contrast to H89, S-Ht31 did not affect the catalytic activity of PKA. These data demonstrate that not only the activity of PKA, but also its tethering to subcellular compartments, are prerequisites for cAMP-dependent AQP-2 translocation.

Highlights

The antidiuretic action of arginine-vasopressin (AVP)1 is mediated by renal collecting duct principal cells
Disruption of the Binding between protein kinase A (PKA) and AKAPs or Inhibition of PKA Prevents AQP-2 Translocation to the Cell Membranes of Rat inner medullary collecting duct (IMCD) Cells—Rat IMCD cells were cultured for 6 days as described [20]
Prior to stimulation with AVP (1 ␮M, 30 min) or forskolin (100 ␮M, 15 min), they were incubated with the membrane-permeable peptide S-Ht31 (100 ␮M, 30 min), which inhibits the binding of PKA to AKAPs, or with the corresponding control peptide S-Ht31-P (100 ␮M, 30 min)

Summary

Introduction

The antidiuretic action of arginine-vasopressin (AVP)1 is mediated by renal collecting duct principal cells. Disruption of the Binding between PKA and AKAPs or Inhibition of PKA Prevents AQP-2 Translocation to the Cell Membranes of Rat IMCD Cells—Rat IMCD cells were cultured for 6 days as described [20]. Prior to stimulation with AVP (1 ␮M, 30 min) or forskolin (100 ␮M, 15 min), they were incubated with the membrane-permeable peptide S-Ht31 (100 ␮M, 30 min), which inhibits the binding of PKA to AKAPs, or with the corresponding (inactive) control peptide S-Ht31-P (100 ␮M, 30 min).

Results

Conclusion