Functionality of aquaporin-2 missense mutants in recessive nephrogenic diabetes insipidus

Aquaporin-2 (AQP2) missense mutants in recessive nephrogenic diabetes insip idus (NDI) are all retained in the endoplasmic reticulum (ER), but some cou ld function as water channels. No conclusions could be drawn about the wate r permeability (P-f) of others, because there was no method. for quantifyin g AQP2 expression in the plasma membrane. We recently developed such a meth od, which has allowed us to study the functionality of these AQP2 mutants. Immunoblot analysis of membranes of injected oocytes revealed that all muta nts (AQP2-G64R, AQP2-N68S, AQP2 T126M, AQP2-A147T, AQP2-R187C, AQP2-S216P) are expressed as unglycosylated and high-mannose glycosylated AQP2. The lev el of the high-mannose form of AQP2-A 147T in the plasma membranes was low, indicating that this mutation has a less severe effect on proper folding. Analysis of Pf values and plasma membrane expression levels reveals that AQ P2-N68S, AQP2-R187C and AQP2-S216P are non-functional, AQP2-A 147T is as fu nctional as wt-AQP2, while AQP2-T126M and AQP2-G64R retain 20% of the perme ability of wt-AQP2. Since G64 is highly conserved between AQPs and expected to form essential interactions with other amino acids within AQP1, the res idual functionality of AQP2-C64R is surprising. Our data furthermore indica te that an eventual therapy with chemical chaperones that restores the rout ing of AQP2 mutants to the apical membrane of collecting ducts cells might relieve NDI in patients encoding AQP2-A147T, and to a lesser extent AQP2-T1 26M and AQP2-G64R, but not in patients encoding AQP2-N68S, AQP2-R187C or AQ P2-S216P.