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Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons.

ABSTRACT: Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-proline-alanine (NPA) motifs, the central constriction, and an aromatic/arginine (ar/R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar/R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individual or joint replacement of His-180 and Arg-195 by alanine and valine residues, respectively (AQP1-H180A, AQP1-R195V, and AQP1-H180A/R195V), did not affect water permeability. The double mutant AQP1-H180A/R195V allowed urea to pass. In line with the predicted solute discrimination by size, replacement of both Phe-56 and His-180 (AQP1-F56A/H180A) enlarged the maximal diameter of the ar/R constriction 3-fold and enabled glycerol and urea to pass. We further show that ammonia passes through all four AQP1 mutants, as determined (i) by growth complementation of yeast deletion strains with ammonia, (ii) by ammonia uptake from the external solution into oocytes, and (iii) by direct recordings of ammonia induced proton currents in oocytes. Unexpectedly, removal of the positive charge in the ar/R constriction in AQP1-R195V and AQP1-H180A/R195V appeared to allow the passage of protons through AQP1. The data indicate that the ar/R constriction is a major checkpoint for solute permeability, and that the exquisite electrostatic proton barrier in AQPs comprises both the NPA constriction as well as the ar/R constriction.

SUBMITTER: Beitz E

PROVIDER: S-EPMC1326162 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons.

Beitz Eric E Wu Binghua B Holm Lars M LM Schultz Joachim E JE Zeuthen Thomas T

Proceedings of the National Academy of Sciences of the United States of America 20060103 2

Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-proline-alanine (NPA) motifs, the central constriction, and an aromatic/arginine (ar/R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar/R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individu ...[more]

PMID: 16407156

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Project description:Twenty four hour ammonia profiles and correlates of drug effect were examined in a phase 2 comparison of sodium phenylbutyrate (NaPBA) and glycerol phenylbutyrate (GPB or HPN-100), an investigational drug being developed for urea cycle disorders (UCDs).Protocol HPN-100-005 involved open label fixed-sequence switch-over from the prescribed NaPBA dose to a PBA-equimolar GPB dose with controlled diet. After 7 days on NaPBA or GPB, subjects underwent 24-hour blood sampling for ammonia and drug metabolite levels as well as measurement of 24-hour urinary phenyacetylglutamine (PAGN). Adverse events (AEs), safety labs and triplicate ECGs were monitored.Eleven subjects (9 OTC, 1 ASS, 1 ASL) enrolled and completed the switch-over from NaPBA (mean dose=12.4 g/d or 322 mg/kg/d; range=198-476 mg/kg/d) to GPB (mean dose=10.8 mL or 0.284 mL/kg/d or 313 mg/kg/d; range=192-449 mg/kg/d). Possibly-related AEs were reported in 2 subjects on NaPBA and 4 subjects on GPB. All were mild, except for one moderate AE of vomiting on GPB related to an intercurrent illness. No clinically significant laboratory or ECG changes were observed. Ammonia was lowest after overnight fast, peaked postprandially in the afternoon to early evening and varied widely over 24h with occasional values >100 ?mol/L without symptoms. Ammonia values were ~25% lower on GPB vs. NaPBA (p?0.1 for ITT and p<0.05 for per protocol population). The upper 95% confidence interval for the difference between ammonia on GPB vs. NaPBA in the ITT population (95% CI 0.575, 1.061; p=0.102) was less than the predefined non-inferiority margin of 1.25 and less than 1.0 in the pre-defined per-protocol population (95% CI 0.516, 0.958; p<0.05). No statistically significant differences were observed in plasma phenylacetic acid and PAGN exposure during dosing with GPB vs. NaPBA, and the percentage of orally administered PBA excreted as PAGN (66% for GPB vs. 69% for NaPBA) was very similar. GPB and NaPBA dose correlated best with urinary-PAGN.These findings suggest that GPB is at least equivalent to NaPBA in terms of ammonia control, has potential utility in pediatric UCD patients and that U-PAGN is a clinically useful biomarker for dose selection and monitoring.

Project description:ObjectiveTo evaluate glycerol phenylbutyrate (GPB) in the treatment of pediatric patients with urea cycle disorders (UCDs).Study designUCD patients (n=26) ages 2months through 17years were treated with GPB and sodium phenylbutyrate (NaPBA) in two short-term, open-label crossover studies, which compared 24-hour ammonia exposure (AUC0-24) and glutamine levels during equivalent steady-state dosing of GPB and sodium phenylbutyrate (NaPBA). These 26 patients plus an additional 23 patients also received GPB in one of three 12-month, open label extension studies, which assessed long-term ammonia control, hyperammonemic (HA) crises, amino acid levels, and patient growth.ResultsMean ammonia exposure on GPB was non-inferior to NaPBA in each of the individual crossover studies. In the pooled analyses, it was significantly lower on GPB vs. NaPBA (mean [SD] AUC0-24: 627 [302] vs. 872 [516] μmol/L; p=0.008) with significantly fewer abnormal values (15% on GPB vs. 35% on NaPBA; p=0.02). Mean ammonia levels remained within the normal range during 12months of GPB dosing and, when compared with the 12months preceding enrollment, a smaller percentage of patients (24.5% vs. 42.9%) experienced fewer (17 vs. 38) HA crises. Glutamine levels tended to be lower with GPB than with NaPBA during short-term dosing (mean [SD]: 660.8 [164.4] vs. 710.0 [158.7] μmol/L; p=0.114) and mean glutamine and branched chain amino acid levels, as well as other essential amino acids, remained within the normal range during 12months of GPB dosing. Mean height and weight Z-scores were within normal range at baseline and did not change significantly during 12months of GPB treatment.ConclusionsDosing with GPB was associated with 24-hour ammonia exposure that was non-inferior to that during dosing with NaPBA in individual studies and significantly lower in the pooled analysis. Long-term GPB dosing was associated with normal levels of glutamine and essential amino acids, including branched chain amino acids, age-appropriate growth and fewer HA crises as compared with the 12month period preceding enrollment.

Dataset Information

Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons.

Publications

Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons.

Similar Datasets

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