Elevated cAMP increases aquaporin-3 plasma membrane diffusion.
Regulated urine concentration takes place in the renal collecting duct upon arginine vasopressin (AVP) stimulation, where subapical vesicles containing aquaporin-2 (AQP2) are inserted into the apical membrane instantly increasing water reabsorption and urine concentration. The reabsorped water exits via basolateral AQP3 and AQP4. Upon long-term stimulation with AVP or during thirst, expression levels of both AQP2 and AQP3 are increased; however, there is so far no evidence for short-term AVP regulation of AQP3 or AQP4. To facilitate the increase in transepithelial water transport, AQP3 may be short-term regulated via changes in protein-protein interactions, incorporation into lipid rafts, and/or changes in steady-state turnover, which could result in changes in the diffusion behavior of AQP3. Thus we measured AQP3 diffusion coefficients upon stimulation with the AVP mimic forskolin to reveal if AQP3 could be short-term regulated by AVP. k-Space image correlation spectroscopy (kICS) analysis of time-lapse image sequences of basolateral enhanced green fluorescent protein-tagged AQP3 (AQP3-EGFP) revealed that the forskolin-mediated elevation of cAMP increased the diffusion coefficient by 58% from 0.0147 ± 0.0082 μm(2)/s (control) to 0.0232 ± 0.0085 μm(2)/s (forskolin, P < 0.05). Quantum dot-conjugated antibody labeling also revealed a significant increase in AQP3 diffusion upon forskolin treatment by 44% [0.0104 ± 0.0040 μm(2)/s (control) vs. 0.0150 ± 0.0016 μm(2)/s (forskolin, P < 0.05)]. Immunoelectron microscopy showed no obvious difference in AQP3-EGFP expression levels or localization in the plasma membrane upon forskolin stimulation. Thus AQP3-EGFP diffusion is altered upon increased cAMP, which may correspond to basolateral adaptations in response to the increased apical water readsorption.
Institute of Molecular Biology and Genetics and Interdisciplinary Nanoscience Center, Aarhus University, Aarhus, Denmark; and.
Saw
S
Marlar
Eva C
EC
Arnspang
Jennifer S
JS
Koffman
Else-Merete
EM
Løcke
Birgitte M
BM
Christensen
Lene N
LN
Nejsum
D000818:Animals; D051400:Aquaporin 3 / Q000235:genetics / Q000378:metabolism*; D001127:Arginine Vasopressin / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism*; D005576:Colforsin / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism*; D004285:Dogs; D004848:Epithelium / Q000378:metabolism; D055459:Facilitated Diffusion; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000378:metabolism; D061985:Madin Darby Canine Kidney Cells; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D014867:Water / Q000378:metabolism*
D016271:Proto-Oncogene Proteins c-myc; C485184:enhanced green fluorescent protein; D014867:Water; D001127:Arginine Vasopressin; D049452:Green Fluorescent Proteins; D051400:Aquaporin 3; D005576:Colforsin; D000242:Cyclic AMP
AQP3; AVP; MDCK; aquaporin; image correlation spectroscopy; kICS; kidney; single particle tracking; urine; vasopressin