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  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 language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology