Abstract
BackgroundPolycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells.MethodsWe investigated the subcellular PC2 localization by immunocytochemistry and confocal microscopy in primary cultures of human and rat proximal tubule cells after stimulating cytosolic Ca2+ signaling. Plasma membrane (PM) Ca2+ permeability was evaluated by Fura-2 manganese quenching using time-lapse fluorescence microscopy.ResultsWe demonstrated that PC2 exhibits a dynamic subcellular localization pattern. In unstimulated human or rat proximal tubule cells, PC2 exhibited a cytosolic/reticular distribution. Treatments with agents that in various ways affect the Ca2+ signaling machinery, those being ATP, bradykinin, ionomycin, CPA or thapsigargin, resulted in increased PC2 immunostaining in the PM. Exposing cells to the steroid hormone ouabain, known to trigger Ca2+ oscillations in kidney cells, caused increased PC2 in the PM and increased PM Ca2+ permeability. Intracellular Ca2+ buffering with BAPTA, inositol 1,4,5-trisphosphate receptor (InsP3R) inhibition with 2-aminoethoxydiphenyl borate (2-APB) or Ca2+/Calmodulin-dependent kinase inhibition with KN-93 completely abolished ouabain-stimulated PC2 translocation to the PM.ConclusionsThese novel findings demonstrate intracellular Ca2+-dependent PC2 trafficking in human and rat kidney cells, which may provide new insight into cyst formations in ADPKD.
Highlights
Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel
The amount of PC2 present in the endoplasmic reticulum (ER), where it functions as a Ca2+-activated intracellular Ca2+ release channel [3], is significantly larger than the amount of PC2 expressed in the plasma membrane (PM), where it functions as a non-selective cation channel [5,6]
To test the dynamic nature of PC2 localization, human proximal tubule (hPT) cells were challenged with the steroid ouabain, known to induce intracellular Ca2+ oscillations in rat kidney cells [14,15]
Summary
Polycystin-2 (PC2), encoded by the gene that is mutated in autosomal dominant polycystic kidney disease (ADPKD), functions as a calcium (Ca2+) permeable ion channel. Considerable controversy remains regarding the subcellular localization and signaling function of PC2 in kidney cells. ADPKD is caused by mutation in two associated proteins, polycystin-1 or −2 (PC1 and PC2), which are essential for the formation and maintenance of a proper structure of the renal tubule. These mutations in PC1 and PC2 are responsible for approximately 85% and 15% of all ADPKD cases, respectively [2]. The exact function and mechanism of PC2 activation in the cilium and/or other subcellular organelles remain largely unknown
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