Functional Analysis of Aquaporin-2 Mutants Associated with Nephrogenic Diabetes Insipidus by Yeast Expression

Title: Functional Analysis of Aquaporin-2 Mutants Associated with Nephrogenic Diabetes Insipidus by Yeast Expression
Authors: Shinbo, Itsuki; Fushimi, MD, Kiyohide; Kasahara, Michihiro; Yamauchi, MD, Atsushi; Sasaki, Sei; Marumo, MD, Fumiaki
Publisher: American Journal of Physiology
Date Published: November 01, 1999
Reference Number: 490
Mutations of aquaporin-2 (AQP2) vasopressin water channel cause nephrogenic diabetes insipidus (NDI). It has been suggested that impaired routing of AQP2 mutants to the plasma membrane causes the disease; however, no determinations have been made of mutation-induced alterations of AQP2 channel water permeability. To address this issue, a series of AQP2 mutants were expressed in yeast, and the osmotic water permeability (P(f)) of the isolated vesicles was measured. Wild-type and mutant AQP2 were expressed equally well in vesicles. P(f) of the vesicles containing wild-type AQP2 was 22 times greater than that of the control, which was sensitive to mercury and weakly dependent on the temperature. P(f) measurements and mercury inhibition examinations suggested that mutants L22V and P262L are fully functional, whereas mutants N68S, R187C, and S216P are partially functional. In contrast, mutants N123D, T125M, T126M, A147T, and C181W had very low water permeability. Our results suggest that the structure between the third and fifth hydrophilic loops is critical for the functional integrity of the AQP2 water channel and that disruption of AQP2 water permeability by mutations may cause NDI.
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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Mutations in aquaporin-2 (AQP2) genes produce mutant AQP2 proteins that are associated with nephrogenic diabetes insipidus (NDI). Normal AQP2s travel to the apical cell membrane of the principal cells of the kidney collecting duct and are inserted into it. There they act as channels through which water can flow out of the principal cells into the inner tissues of the kidney. This is a vital step in the process that allows the kidney to balance body water. AQP2s are predicted to have six coiled transmembrane structures connected by five loops that readily absorb water. (You can look at a diagram of AQP2 here.)

When researchers studied the function of mutant AQP2s in laboratory cell cultures (using a type of cell called Xenopus ousts), they found evidence to suggest that many of the mutants had structural faults that interfered with the AQP2s' ability to travel to the apical cell membrane, the location AQP2s' must be if they are to let water pass through them. Researchers thought that if the mutant AQP2s could not get to the membrane, they could not be in the proper position to act as channels through which water could pass.

However, until now, there has never been any research on these AQP2 mutants that directly measured their water channeling ability to see if the mutations affected their ability to channel water. This ability is measured in terms of the AQP2's water permeability (P(f)). Shinbo, et al., measured and analyzed the P(f) of ten AQP2 mutants. That is, they directly tested their ability to allow water to pass through them. To do this, the researchers expressed the mutants, not in Xenopus ousts, but in yeast.

Four of these mutants were found to be nonfunctional - they did not let any water through them. Three were partially functional - they let some water through, but not near normal levels. Two were fully functional - they let a similar amount of water through them as normal AQP2s do. The researchers noted that the mutations of AQP2s with diminished or no ability to channel water all occurred between the AQP2s' third and fifth loop. This finding confirms the importance of the structure between the third and fifth loop for AQP2s' ability to let water flow through them.

The researchers note that their findings warrant a reexamination of the notion that it is primarily the inability of mutant AQP2s to travel to the cell membrane that is responsible for symptoms of AQP2 related NDI.