Mh. Levin et al., Diffusion in the endoplasmic reticulum of an aquaporin-2 mutant causing human nephrogenic diabetes insipidus, J BIOL CHEM, 276(24), 2001, pp. 21331-21336
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
.