Diffusion in the endoplasmic reticulum of an aquaporin-2 mutant causing human nephrogenic diabetes insipidus

Abstract

Mutations in the aquaporin-2 (AQP2) water channel cause the hereditary renal disease nephrogenic diabetes insipidus (NDI). The missense mutation AQP2-T126M causes human recessive NDI by retention at the endoplasmic reticulum (ER) of renal epithelial cells. To determine whether the ER retention of AQP2-T126M is due to relative immobilization in the ER, we measured by fluorescence recovery after photobleaching the intramembrane mobility of green fluorescent protein (GFP) chimeras containing human wild-type and mutant AQP2. In transfected LLC-PK1 renal epithelial cells, GFP-labeled AQP2-T126M was localized to the ER, and wild-type AQP2 to endosomes and the plasma membrane; both were localized to the ER after brefeldin A treatment. Photobleaching with image detection indicated that the GFP-AQP2 chimeras were freely mobile throughout the ER. Quantitative spot photobleaching revealed a diffusion-dependent irreversible process whose recovery depended on spot size and was abolished by paraformaldehyde fixation. In addition, a novel slow reversible fluorescence recovery (t(12) approximately 2 s) was characterized whose recovery was independent of spot size and not affected by fixation. AQP2 translational diffusion in the ER was not slowed by the T126M mutation; diffusion coefficients were (in cm(2)/s x 10(-)10) 2.6 +/- 0.5 (wild-type) and 3.0 +/- 0.4 (T126M). Much faster diffusion was found for a lipid probe (diOC(4)(3), 2.7 x 10(-)8 cm(2)/s) in the ER membrane and for unconjugated GFP in the aqueous ER lumen (6 x 10(-)8 cm(2)/s). ER diffusion of GFP-T126M was not significantly affected by up-regulation of molecular chaperones, cAMP activation, or actin filament disruption. ATP depletion by 2-deoxyglucose and azide resulted in comparable slowing/immobilization of wild-type and T126M AQP2. These results indicate that the ER retention of AQP2-T126M does not result from restricted or slowed mobility and suggest that the majority of AQP2-T126M is not aggregated or bound to slowly moving membrane proteins.