It was found that the absorbance and fluorescence of green fluorescent protein (GFP) mutants are strongly pH dependent in aqueous solutions and intracellular compartments in living cells. pH titrations of purified recombinant GFP mutants indicated >10-fold reversible changes in absorbance and fluorescence with pKa values of 6.0 (GFP-F64L/S65T), 5.9 (S65T), 6.1 (Y66H), and 4.8 (T203I) with apparent Hill coefficients of 0.7 for Y66H and approximately 1 for the other proteins. For GFP-S65T in aqueous solution in the pH range 5-8, the fluorescence spectral shape, lifetime (2.8 ns), and circular dichroic spectra were pH independent, and fluorescence responded reversibly to a pH change in <1 ms. At lower pH, the fluorescence response was slowed and not completely reversed. These findings suggest that GFP pH sensitivity involves simple protonation events at a pH of >5, but both protonation and conformational changes at lower pH. To evaluate GFP as an intracellular pH indicator, CHO and LLC-PK1 cells were transfected with cDNAs that targeted GFP-F64L/S65T to cytoplasm, mitochondria, Golgi, and endoplasmic reticulum. Calibration procedures were developed to determine the pH dependence of intracellular GFP fluorescence utilizing ionophore combinations (nigericin and CCCP) or digitonin. The pH sensitivity of GFP-F64L/S65T in cytoplasm and organelles was similar to that of purified GFP-F64L/S65T in saline. NH4Cl pulse experiments indicated that intracellular GFP fluorescence responds very rapidly to a pH change. Applications of intracellular GFP were demonstrated, including cytoplasmic and organellar pH measurement, pH regulation, and response of mitochondrial pH to protonophores. The results establish the application of GFP as a targetable, noninvasive indicator of intracellular pH.
Neuromyelitis optica is an inflammatory demyelinating disorder of the CNS. The discovery of circulating IgG1 antibodies against the astrocyte water channel protein aquaporin 4 (AQP4) and the evidence that AQP4-IgG is involved in the development of neuromyelitis optica revolutionised our understanding of the disease. However, important unanswered questions remain—for example, we do not know the cause of AQP4-IgG-negative disease, how astrocyte damage causes demyelination, the role of T cells, why peripheral AQP4-expressing organs are undamaged, and how circulating AQP4-IgG enters neuromyelitis optica lesions. New drug candidates have emerged, such as aquaporumab (non-pathogenic antibody blocker of AQP4-IgG binding), sivelestat (neutrophil elastase inhibitor), and eculizumab (complement inhibitor). Despite rapid progress, randomised clinical trials to test new drugs will be challenging because of the small number of individuals with the disorder.
Aquaporin-4, the major water-selective channel in astroglia throughout the central nervous system, facilitates water movement into and out of the brain. Here, we identify a novel role for aquaporin-4 in astroglial cell migration, as occurs during glial scar formation. Astroglia cultured from the neocortex of aquaporin-4-null mice had similar morphology, proliferation and adhesion, but markedly impaired migration determined by Transwell migration efficiency (18±2 vs 58±4% of cells migrated towards 10% serum in 8 hours; P<0.001) and wound healing rate (4.6 vs 7.0 μm/hour speed of wound edge; P<0.001) compared with wild-type mice. Transwell migration was similarly impaired (25±4% migrated cells) in wild-type astroglia after ∼90% reduction in aquaporin-4 protein expression by RNA inhibition. Aquaporin-4 was polarized to the leading edge of the plasma membrane in migrating wild-type astroglia, where rapid shape changes were seen by video microscopy. Astroglial cell migration was enhanced by a small extracellular osmotic gradient, suggesting that aquaporin-4 facilitates water influx across the leading edge of a migrating cell. In an in vivo model of reactive gliosis and astroglial cell migration produced by cortical stab injury, glial scar formation was remarkably impaired in aquaporin-4-null mice, with reduced migration of reactive astroglia towards the site of injury. Our findings provide evidence for the involvement of aquaporin-4 in astroglial cell migration, which occurs during glial scar formation in brain injury, stroke, tumor and focal abscess.
Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (ΔF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the ΔF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote ΔF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of ΔF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.
To investigate the role of aquaporin-1 (AQP1) water channels in proximal tubule function, in vitro proximal tubule microperfusion and in vivo micropuncture measurements were done on AQP1 knockout mice. The knockout mice were generated by targeted gene disruption and found previously to be unable to concentrate their urine in response to water deprivation. Unanesthetized knockout mice consumed 2.8-fold more f luid than wild-type mice and had lower urine osmolality (505 ؎ 40 vs. 1081 ؎ 68 milliosmolar). An important function of the kidney proximal tubule is the near-isosmolar reabsorption of a significant fraction of fluid that is filtered by the glomerulus. The proximal tubule also reabsorbs nearly all of the filtered glucose, amino acids, and bicarbonate. The apical and basolateral plasma membranes of proximal tubule cells contain water channel protein aquaporin-1 (AQP1), which is thought to provide an important water-selective pathway for transcellular fluid transport (1-3). However, there is conflicting evidence that significant paracellular water transport occurs (4), and it has been suggested that other water channels (AQP7, ref. 5) and transporters (glucose transporter GLUT1, refs. 6, 7; sodium-glucose cotransporter SGLT1, ref. 8) might contribute to transcellular water movement. It is generally believed, but without direct evidence, that high proximal tubule water permeability is important to permit the efficient coupling of solute and water transport to accomplish near-isosmolar fluid absorption.The AQP1 water channel is a water-selective transporter (9, 10) that is found in membranes as tetramers (11) in which each functionally independent monomer (12) contains six transmembrane, tilted helical domains surrounding a putative aqueous pore (13-15). In kidney, AQP1 is strongly expressed in apical and basolateral plasma membranes of epithelial cells in proximal tubule and thin descending limb of Henle and in endothelial cells of descending vasa recta (1-3, 16, 17). Recently, a transgenic AQP1 knockout mouse was generated by targeted gene disruption and shown to manifest a severe defect in urinary concentrating ability (18). When given access to water, the mice appeared grossly normal except for mild growth retardation compared with wild-type mice. When deprived of water, the mice were unable to concentrate their urine and conserve fluid, resulting in marked dehydration and serum hyperosmolality in 1-2 days.The purpose of this study was to define the role of AQP1 in proximal tubule water transport and fluid reabsorption. Isolated tubule microperfusion was used to measure transepithelial osmotic water permeability and fluid absorption under defined in vitro conditions. Free-flow micropuncture was used to determine the in vivo consequences of decreased proximal tubule water permeability. A remarkable decrease in proximal tubule water permeability and fluid reabsorption was found in the AQP1 knockout mice. The results have important implications regarding the mechanisms of proximal tubule fluid reabsorpt...
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