Reconstitution of water chan-nel function of aquaporins (AQP) 1 & 2 by expression in yeast secretory (SEC) vesicles

L. A. Courv, J. C. Mathai, J. L. Brpdskv, Peter C Agre, M. L. Zeideh

Research output: Contribution to journalArticle

Abstract

Five AQPs have been cloned in mammals, but only AQP1 from RBCs is available in sufficient quantities for reconstitution and biophysical studies. AQP2 mediates vasopressin-stimulated water flow across the apical membrane of kidney collecting duct cells. To develop a reconstitution system suitable for all AQPs and mutants, we utilized yeast sec mutants to overexpress AQP 1&2. The temperature sensitive mutant sc6-4 accumulates post-Oolgi plajma membrane targeted vesicles at the restrictive temperature (37 C). Both AQP genes were indutibly (pressed while the vesides were simultaneously accumulated at the 37 C. The vesicles were isolated, loaded with carboxyfluoroscein (CF), and osmotic water permeability (Pf) assayed by stopped-flow fluorescence quenching of entrapped CF. AQP 1&2 vesicles, but not control vesicles, contained abundant AQP proteins by immunoblotting. P, (cm/sec) for the vesicles were (mean ±SD): control P, = 0.0022 ±0.0004 (n=5); with AQP2, P, = 0.305 ±0.049 (n=4), a significant difference at p <0.001. With AQP1, P, values ranged from 0.016 to 0.185 depending oft the induction protocol. AQP1 was reconstituted as previously described (Zeidel, Ambudkar, Smith, Agre [1992] Biochemistry 31:7436-7440).Activation energies for water flow with AQPs were also much lower (-2 kcal/mole). We conclude: 1. Expression of AQP in sec vesicles reconstitutes water channel function. 2. Differences in P, values between AQP 1&2 vesicles likely relate to differences in protein expression. 3. This system will permit detailed analysis of native and mutant AQP.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number3
StatePublished - 1996
Externally publishedYes

Fingerprint

Aquaporin 1
Aquaporins
aquaporins
secretory granules
Secretory Vesicles
Yeast
Yeasts
yeasts
Water
water
mutants
Collecting Kidney Tubules
Membranes
water flow
Biochemistry
Temperature
Mammals
Vasopressins
Immunoblotting
Ducts

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Reconstitution of water chan-nel function of aquaporins (AQP) 1 & 2 by expression in yeast secretory (SEC) vesicles. / Courv, L. A.; Mathai, J. C.; Brpdskv, J. L.; Agre, Peter C; Zeideh, M. L.

In: FASEB Journal, Vol. 10, No. 3, 1996.

Research output: Contribution to journalArticle

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