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

Mutations in the aquaporin-2 (AQP2) water channel cause the hereditary rena l disease nephrogenic diabetes insipidus (NDI), The missense mutation PIQP2 -T126M causes human recessive NDI by retention at the endoplasmic reticulum (ER) of renal epithelial cells, To determine whether the ER retention of A QP2-T126M is due to relative immobilization in the ER, we measured by fluor escence recovery after photobleaching the intramembrane mobility of green f luorescent protein (GFP) chimeras containing human wild-type and mutant AQP 2, In transfected LLC-PK1 renal epithelial cells, GFP-labeled AQP2-T126M wa s localized to the ER, and wild-type AQP2 to endosomes and the plasma membr ane; both were localized to the ER after brefeldin A treatment. Photobleach ing with image detection indicated that the GFP-AQP2 chimeras were freely m obile throughout the ER, Quantitative spot photobleaching revealed a diffus ion-dependent irreversible process whose recovery depended on spot size and was abolished by paraformaldehyde fixation, In addition, a novel slow reve rsible fluorescence recovery (t(1/2) similar to2 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; dif fusion 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 (di OC(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 wa s not significantly affected by up-regulation of molecular chaperones, cAMP activation, or actin filament disruption. ATP depletion by S-deoxyglucose and azide resulted in comparable slowing/immobilization of wild-type and T1 26M AQP2, These results indicate that the ER retention of AQP2-T126M does n ot result from restricted or slowed mobility and suggest that the majority of AQP2-T126M is not aggregated or bound to slowly moving membrane proteins .