Lipid domain structure correlated with membrane protein function in placental microvillus vesicles

Membrane fluidity properties of placental microvillus membrane vesicles (MVV) were determined from fluorescence anisotropy (r), dynamic depolarization, and lifetime heterogeneity studies of diphenylhexatriene (DPH), trimethylamino-DPH (TMA-DPH), and cis- and trans-parinaric acids (c-PnA and t-PnA). Plots of r against temperature for DPH and TMA-DPH in MVV had slope discontinuities at 26 degrees C (Tc, transition temperature); however, analysis of r in terms of probe rotational rate (R), limiting anisotropy (r infinity), and lifetime (tau) revealed that DPH reported a phase transition because of changes in r infinity, whereas the phase transition observed by TMA-DPH occurred primarily because of changes in R. Heterogeneity analysis using phase and modulation lifetimes at three frequencies showed that DPH and TMA-DPH lifetimes were homogeneous in MVV. Both long (greater than 25 ns) and short (less than 6 ns) lifetime components were detected for c-PnA and t-PnA in MVV, corresponding to the probes in solid and fluid lipid phases. The fractional amplitude of the long lifetimes (solid phase) decreased from 0.86 to 0.12 with increasing temperature (5-55 degrees C) as the membrane passed through the phase transition, with 50% of the change occurring at 27 degrees C (c-PnA) or 33 degrees C (t-PnA). The activation energies for alkaline phosphatase, aminopeptidase M, and sodium-proton antiporter activities all showed discontinuities in the temperature range 27-31 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic transport model for cellular regulation of pH and solute concentration in the renal proximal tubule.

An open circuit kinetic model was developed to calculate the time course of proximal tubule cell pH, solute concentrations, and volume in response to induced perturbations in luminal or peritubular fluid composition. Solute fluxes were calculated from electrokinetic equations containing terms for known carrier saturabilities, allosteric dependences, and ion coupling ratios. Apical and basolateral membrane potentials were determined iteratively from the requirements of cell electroneutrality and equal opposing transcellular and paracellular currents. The model converged to membrane potentials accurate to 0.05% in one to four iterations. Model variables included cell concentrations of Na, K, HCO3, glucose, pH (uniform CO2), volume, and apical and basolateral membrane potentials. The basic model contained passive apical membrane transport of Na/H, Na/glucose, H and K, basolateral transport of Na/3HCO3, K, H, and glucose, and paracellular transport of Na, K, Cl, and HCO3; apical H and basolateral 3Na/2K-ATPases were present. Apical Na/H and basolateral K transport were regulated allosterically by pH. Apical Na/H transport, basolateral Na/3HCO3 transport, and the 3Na/2K-ATPase were saturable. Model parameters were chosen from data in the rat proximal tubule. Model predictions for the magnitude and time course of cell pH, Na, and membrane potential in response to rapid changes in apical and peritubular Na and HCO3 were in excellent agreement with experiment. In addition, the model requires that there exist an apical H-ATPase, basolateral Na/3HCO3 transport saturable with HCO3, and electroneutral basolateral K transport.     

Identification of protein kinase A phosphorylation sites on NBD1 and R domains of CFTR using electrospray mass spectrometry with selective phosphate ion monitoring.

HPLC-electrospray mass spectrometry was used to identify the phosphorylated sites on a bacterially expressed cystic fibrosis transmembrane conductance regulator (CFTR) fragment containing the first nucleotide binding domain (NBD1) and the regulatory domain (R). Tryptic digests of NBD1-R (CFTR residues 404-830) were analyzed after protein kinase A (PKA) treatment for all possible peptides and phosphopeptides (a total of 118 species) containing Ser residues within "high-probability" PKA consensus sequences: R-R/K-X-S/T, R-X-X-S/T, and R-X-S/T. Three criteria were used to assign phosphorylated sites: (1) an 80-Da increase in the predicted average molecular weight of the tryptic peptides; (2) co-elution with the PO3- ion induced by stepped energy collision; and (3) the relative elution positions of the phosphorylated and unmodified peptides. Ser residues within the eight dibasic sites in the NBD1 and R domains (positions 422, 660, 700, 712, 737, 768, 795, and 813) were phosphorylated, a pattern similar to that observed for full-length CFTR. The serine at position 753, which in CFTR is phosphorylated in vivo, was not phosphorylated. The remaining potential PKA sites, Ser489, Ser519, Ser557, Ser670, and Thr788, were not phosphorylated. The "low-probability" PKA sites (those not containing an Arg residue) were not phosphorylated. The results suggest that isolated domains of CFTR developed useful models for investigating the biochemical and structural effects of phosphorylation within CFTR. The mass spectrometry approach in this study should prove useful for defining phosphorylation sites of CFTR in vitro and in vivo.     

Single photon radioluminescence. I. Theory and spectroscopic properties.

The excitation of a fluorescent molecule by a beta-decay electron (radioluminescence) depends upon the electron energy, the distance between radioactive 'donor' and fluorescent 'acceptor', and the excitation characteristics and solvent environment of the fluorophore. The theory for calculation of single photon radioluminescence (SPR) signals is developed here; in the accompanying paper, measurement methods and biological applications are presented. To calculate the three-dimensional spatial profile for electron energy deposition in an aqueous environment, a Monte Carlo calculation was performed incorporating theories of electron energy distributions, energy loss due to interactions with matter, and deflections in electron motion due to collisions. For low energy beta emitters, 50% of energy deposition occurs within 0.63 micron (3H, 18.5 keV), 22 microns (14C, 156 keV), 25 microns (35S, 167 keV), and 260 microns (36Cl, 712 keV) of the radioisotope. In close proximity to the beta emitter (100 nm, 3H; 10 microns, 14C) the probability for fluorophore excitation is approximately proportional to the inverse square of the distance between the beta emitter and fluorophore. To investigate the other factors that determine the probability for fluorophore excitation, SPR measurements were carried out in solutions containing 3H and a series of fluorophores in different solvents. In water, the probability of fluorescence excitation was nearly proportional to the integrated absorbance over a > 1,000-fold variation in absorbances. The probability of fluorescence excitation was enhanced up to 2,600-fold when the fluorophore was in a "scintillant" aromatic or hydrocarbon solvent. SPR emission spectra were similar to fluorescence emission spectra obtained with photon excitation. The single photon signal due to Bremsstrahlung increased with wavelength in agreement with theory. The distance dependence for the SPR signal predicted by the model was in good agreement with measurements in which a 14C donor was separated by known thicknesses of water from a fluorescently-coated coverglass. Quantitative predictions for radioluminescence signal as a function of donor-acceptor distance were developed for specific radioisotope-fluorophore geometries in biological samples.     

Quantitative fluorescence measurement of chloride transport mechanisms in phospholipid vesicles

A quantitative fluorescence assay has been developed to measure Cl flux across liposomal membranes for use in chloride transporter reconstitution studies. A Cl-sensitive fluorophore [6-methoxy-N-(3-sulfopropyl)quinolinium; SPQ] was entrapped into phospholipid/cholesterol liposomes formed by bath sonication, high-pressure extrusion, and detergent dialysis. Liposomes containing entrapped SPQ were separated from external SPQ by passage down a Sephadex G25 column. There was less than 10% leakage of SPQ from liposomes in 8 h at 4 degrees C and in 2 h at 23 degrees C. Cl influx (JCl in millimolar per second or nanomoles per second per centimeter squared) was determined from the time course of SPQ fluorescence, measured by cuvette or stopped-flow fluorometry, in response to inward Cl gradients. In 90% phosphatidylcholine (10% cholesterol liposomes at 23 degrees C, JCl in response to a 50 mM inward Cl gradient was 0.06 +/- 0.01 mM.s-1 (SD, n = 3) in the absence and 0.27 +/- 0.02 mM.s-1 in the presence of a K/valinomycin voltage clamp (0 mV), showing that the basal Cl "leak" is conductive; JCl increased (1.7 +/- 0.1)-fold in the presence of a 60-mV inside-positive diffusion potential. Accuracy of chloride influx rates determined by the SPQ method was confirmed by measurement of 36Cl uptake. In liposomes voltage-clamped to 0 mV, JCl was linear with external [Cl] (0-100 mM), independent of pH gradients, and strongly dependent on temperature (activation energy 18 +/- 1 kcal/mol, 12-42 degrees C) as predicted for channel-independent Cl diffusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Functional water channels are present in clathrin-coated vesicles from bovine kidney but not from brain

Targeting of water channels in renal epithelia may involve trafficking of clathrin-coated vesicles. We have isolated and measured the osmotic water permeability (Pf) of purified clathrin-coated vesicles from bovine kidney cortex and inner medulla, and bovine brain, a tissue not expected to contain "water channels." Brain-coated vesicles had a diameter of 80 nm in negatively stained preparations. Pf was measured by a stopped-flow light scattering technique. In brain-coated vesicles, water transport was functionally homogeneous with a low Pf of 0.0016 +/- 0.0001 cm/s (seven preparations, 23 degrees C). Pf was independent of osmotic gradient size (25-300 mOsm), not inhibited by mercurials, and not altered by removal of the clathrin coat. The activation energy (Ea) for Pf was high (11 +/- 1 kcal/mol less than 34 degrees C, 17 +/- 2 kcal/mol greater than 34 degrees C). Therefore, water channels are absent from brain-coated vesicles. In contrast, there were two functional populations of vesicles in coated vesicle preparations from both kidney cortex and medulla. One population of vesicles had low water permeability and no water channels, whereas a second population had high Pf (0.02 cm/s, 21 degrees C) that was inhibited by HgCl2, and low Ea (2-3 kcal/mol). The fraction of vesicles with high Pf was 52 +/- 3% (S.D., n = 3, cortical vesicles) and 26 +/- 3% (medullary vesicles). These results provide evidence that functional water channels are not present in clathrin-coated vesicles from the brain, whereas they are found in a population of coated vesicles from kidney cortex and medulla, tissues in which water channels are recycled between the plasma membrane, and an intracellular compartment.     

Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.     

Pre-steady-state analysis of the turn-on and turn-off of water permeability in the kidney collecting tubule

Summary Water transport across the mammalian collecting tubule is regulated by vasopressin-dependent water channel insertion into and retrieval from the cell apical membrane. The time course of osmotic water permeability (P _f ) following addition and removal of vasopressin (VP) and 8-Br-cAMP was measured continuously by quantitative fluorescence microscopy using an impermeant fluorophore perfused in the lumen. Cortical collecting tubules were subjected to a 120 mOsm bath-to-lumen osmotic gradient at 37°C with 10–15 nl/min lumen perfusion and 10–20 ml/min bath exchange rate. With addition of VP (250 U/ml), there was a 23±3 sec (sem,n=16) lag in whichP _f did not change, followed by a rise inP _f (initial rate 1.4±0.2×10^–4 cm/sec²) to a maximum of 265±10×10^–4 cm/sec. With addition of 8-Br-cAMP (0.01–1mm) there was an 11±2 sec lag. For [8-Br-cAMP]=0.01, 0.1 and 1mm, the initial rate ofP _f increase following the lag was (units 10^–4 cm/sec²): 1.1±0.1, 1.2±0.1 and 1.7±0.3. MaximumP _f was (units 10^–4 cm/sec): 64±4, 199±9 and 285±11. With removal of VP,P _f decreased to baseline (12×10^–4 cm/sec) with aT _1/2 of 18 min; removal of 0.1 and 1mm 8-Br-cAMP gaveT _1/2 of 4 and 8.5 min. These results demonstrate (i) a brief lag in theP _f response, longer for stimulation by VP than by 8-Br-cAMP, representing the transient build-up of biochemical intermediates proximal to the water channel insertion step, (ii) similar initialdP _f /dt (water channel insertion) over a wide range of [8-Br-cAMP] and steady-stateP _f values, and (iii) more rapidP _f decrease with removal of 8-Br-cAMP than with VP. These pre-steady-state results define the detailed kinetics of the turn-on and turn-off of tubuleP _f and provide kinetic evidence that the rate-limiting step for turn-on ofP _f is not the step at which VP regulates steady-stateP _f . If water channel insertion is assumed to be the rate-limiting step in the turn-on ofP _f , these results raise the possibility that water channels must be activated following insertion into the apical membrane.