Reduced osmotic water permeability of KR/AQP1 chimeras after CALI. FRT cells were transfected with YFP alone or together with indicated KR/AQP1 chimeras. (A, top) AQP1 immunoblot after SDS/PAGE of cells expressing indicated KR/AQP1 chimeras. Also shown are AQP1 (in kidney lysate) and FRT cells expressing a GFP-AQP1 chimera. Gels that are apart were run separately. (A, bottom) Time course of YFP fluorescence in cells expressing AQP1 (without KR) after rapid reduction in osmolality of the extracellular solution from 300 to 150 mOsm. (B) Osmotic water permeability as in A (bottom) for FRT cells expressing YFP and KR in cytoplasm, KR targeted to the plasma membrane (KRmem), and indicated KR/AQP1 chimeras. (C) Summary of relative osmotic water permeabilities (expressed as reciprocal exponential time constant τ−1) deduced from experiments in B (n = 5–8 cells on three cover glasses, error bars indicate SE; *, P < 0.01). (D) Dependence of AQP1 water permeability inactivation on KR bleaching for KR-AQP1 short. (D, top) Osmotic water permeability in cells exposed to CALI for the indicated times. (D, bottom) Relative water permeability versus CALI bleach time (n = 5–8 cells on three cover glasses, error bars indicate SE; *, P < 0.01). (D, inset) Percentage inhibition of water permeability versus percentage of KR bleach (reduction in fluorescence).
