Characterization of D150E and G196D aquaporin-2 mutations responsible for nephrogenic diabetes insipidus: importance of a mild phenotype

Abstract

Aquaporin-2 (AQP2) is a water channel responsible for the final water reabsorption in renal collecting ducts. Alterations in AQP2 function induce nephrogenic diabetes insipidus (NDI), a condition characterized by severe polyuria and polydipsia. Three patients affected with severe NDI, who were compound heterozygous for the AQP2 mutations D150E and G196D, are presented here along with a mildly affected D150E homozygous patient from another family. Using Xenopus oocytes as an expression system, these two mutations (G196D and D150E) were compared with the wild-type protein (AQP2-wt) for functional activity (water flux analysis), protein maturation, and plasma membrane targeting. AQP2-wt induces a major increase in water permeability (P(f) = 47.4 +/- 12.2 x 10(-4) cm/s) whereas D150E displays intermediate P(f) values (P(f) = 12.5 +/- 3.0 x 10(-4) cm/s) and G196D presents no specific water flux, similar to controls (P(f) = 2.1 +/- 0.8 x 10(-4) cm/s and 2.2 +/- 0.7 x 10(-4) cm/s, respectively). Western blot and immunocytochemical evaluations show protein targeting that parallels activity levels with AQP2-wt adequately targeted to the plasma membrane, partial targeting for D150E, and complete sequestration of G196D within intracellular compartments. When coinjecting AQP2-wt with mutants, no (AQP2-wt + D150E) or partial (AQP2-wt + G196D) reduction of water flux were observed compared with AQP2-wt alone, whereas complete loss of function was found when both mutants were coinjected. These results essentially recapitulate the clinical profiles of the family members, showing a typical dominant negative effect when G196D is coinjected with either AQP2-wt or D150E but not between AQP2-wt and D150E mutant.

New autosomal recessive mutations in aquaporin‐2 causing nephrogenic diabetes insipidus through deficient targeting display normal expression in Xenopus oocytes

“…In comparison to AQP2‐wt, when considering functionality (equivalent), plasma membrane targeting (equivalent) and protein abundance (20–40%), we find that K228E and V24A are the two most efficiently expressed AQP2 mutations evidenced so far in oocytes. Previous data, including ours (Guyon et al 2009 a ), globally reports overall efficiencies of about 1/10 or less (Marr et al 2002 a ; Robben et al 2006). With such functional characteristics in oocytes, V24A and even more K228E could basically be considered as functional variants of AQP2 with reduced expression levels, which may not satisfactory explain the NDI phenotype observed in compound mutation individuals.…”

“…4), which is in agreement with other reports using the same systems (Tamarappoo & Verkman, 1998; Yamauchi et al 1999; Marr et al 2001, 2002 b ). Even though oocytes and mammalian cells usually generate similar plasma membrane targeting results for AQP2 mutants (Guyon et al 2009 a ), the diverging targeting results found with K228E and V24A underline existing differences between both expression systems and stress the need to confirm key features such as targeting using a more appropriate cell model such as mIMCD‐3 cells. Thus, even though both K228E and V24A are functional proteins, the basic defect most probably resides in their profound inability to adequately reach the plasma membrane, worsened by a low protein synthesis level (20–40% in comparison to AQP2‐wt, in oocytes) and a possible lack of maturation.…”

“…Since most AQP2‐related NDI are found to be recessive hereditary traits (Robben et al 2006), heterozygotes bearing one wild‐type form of the protein are usually non‐symptomatic as two defective alleles are required to induce the disease. Even though altered forms of the AQP2 protein were often found to be less efficiently synthesized, as shown in studies performed using Xenopus oocytes (Marr et al 2002 a ; Guyon et al 2009 b ), the lack of function usually originates from their inadequate synthesis and routing (Deen et al 1995; Lin et al 2002; Marr et al 2002 a ). The mutated protein, believed to be misfolded, is usually retained in intracellular stores (endoplasmic reticulum, ER) and eventually routed for degradation (class II mutations) (Robben et al 2006).…”

X-ray structure of human aquaporin 2 and its implications for nephrogenic diabetes insipidus and trafficking

“…Within the cytoplasmic loop D, the D150E mutant has been identified in patients suffering from recessive NDI (34,35). Our X-ray structure reveals that Asp150 mediates an interaction between loop D and the proximal end of the C-terminal tail, with Asp150 connecting to Pro225 via hydrogen bonds mediated by Arg152 (Fig.…”

Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

“…Since both mutations are well described in literature [45], correlations of protein energy/stability and experimental observations can be developed.…”