On the nature of ion leaks in energy-transducing membranes.

Diffusion is the implicit null hypothesis for ion transport across biological membranes. A proper model of ionic diffusion across the permeability barrier is needed to distinguish among leaks, channels and carriers and to determine whether changes in flux reflect changes in permeability (regulation) or merely changes in the driving force. These issues arise in all biomembranes, but they are particularly confounding in energy-transducing membranes on account of their characteristically high electrical gradients. This paper examines the nature of the barrier to ion leaks, using the classical Eyring rate theory. We introduce new practical procedures for estimating permeability coefficients from ion flux data. We also reach some general conclusions regarding ion leaks across energy-transducing membranes. (1) The dependence of ion flux on the electrical membrane potential is invariably non-linear (non-ohmic). (2) Non-ohmic behavior does not imply variable permeability. (3) Ohmic behavior is exceptional and its occurrence should alert us to the possibility of an underlying carrier or channel. (4) Leak pathways are very likely localized to protein-lipid interfaces and will exhibit quasi-specific properties such as saturation and competition. (5) The inherent non-ohmicity of leaks and the requirement for efficient energy transduction impose constraints upon the magnitude of allowable Gibbs free-energy changes in biological systems. (6) Nature adapts to these constraints by devising mechanisms for step-wise splitting of the partial reactions of energy transduction.

Drug distribution within human milk phases.

Phase distribution and protein binding of drugs in human milk have been measured. The analytical method is reproducible, rapid, and requires only small sample volumes. Five drugs were studied: diazepam, phenobarbital, warfarin, phenytoin, and disopyramide. Experiments were carried out at 37 degrees C on milk samples with variable fat and protein contents. Results for the distribution of drugs between the skimmed-milk phase and fat-rich phase are presented, as well as the results of the dialysis of drugs in skimmed milk. It is shown that, among the physicochemical properties of a drug, the lipid solubility seems to be the most important property for predicting variations in drug concentrations in milk. The potential significance of the findings with respect to in vivo distribution of drugs into human milk is discussed.

Active uptake of glutamate in vesicles of Halobacterium salinarium.

Uptake of glutamate into vesicles of Halobacterium salinarium has been studied during respiration and in the nonrespiring state. Uptake requires respiration or a minimum gradient in NaCl, which is consistent with an Na+ symport mechanism for uptake, as proposed for H. halobium. By replacing KCl or NaCl by choline chloride, it has been possible to distinguish between the effects of gradients and/or absolute concentration effects of NaCl and KCl. Uptake depends on the concentration of KCl on the inside, but not on a gradient in KCl. This points to a role for K+ as a regulator of uptake rate, but not of total uptake. The uptake of glutamate is not inhibited by a number of acids with similar chemical groups. Inhibition is, however, caused by D-glutamate. This indicates a specific transport site for glutamate. Parallel results are obtained for binding of glutamate to a Triton extract of the vesicle membrane. The variation in binding and uptake properties with the salt concentration is discussed with reference to transport kinetics.

Amiloride inhibition of Na+-entry into corneal endothelium.

Amiloride in 10(-3) M concentration inhibits incompletely the short circuit current and active potential difference across the bovine corneal endothelium in vitro. The drug effect is reversible and unilateral, e.g. the drug is effective only from the aqueous side. The amiloride effect is compared to the effect of ouabain, nystatin and vasopressin on the same electrical parameters. The effect of these drugs support a model for active Na+ transport across the corneal endothelium with two separate pathways for Na+ transport - one for extrusion and one for reentry.

ATP synthesis by Ca2+ + Mg2+-ATPase in detergent solution at constant Ca2+ levels.

ATP has been synthesized by the purified Ca2+ + Mg2+-dependent ATPase from sarcoplasmic reticulum (SR) solubilized in nonionic detergent dodecyloctaoxyethylenglycol-monoether in a solution containing inorganic phosphate and glycerol by changing pH upon addition of ADP. The Ca2+ concentration is kept constant during the experiment. Optimum synthesis is found at CaCl2 = 0.6 mM and the delta pH = 2.9 +/- 0.2. The enzyme has been digested by trypsin for 1 and 20 min, and it is found that synthesis of ATP is correlated with the Ca2+-uptake into SR. The data indicate that the enzyme alone is responsible for active transport of Ca2+ in SR. The driving force for the ATP synthesis of the process may be due to various ion-protein interactions. H+ cannot substitute for Ca2+ in the synthesis of ATP but acts probably through a modification of the Ca2+ binding sites. The data give support that the integrity of the enzyme molecule between its hydrolytic site and the Ca2+-binding sites is essential for the overall Ca2+ transport.

Ionic transport across corneal endothelium.

The active potential difference across bovine corneal endothelium was measured in vitro at different temperatures, pH, osmolality and salt compositions. The measurements were made using either identical or different solutions on each side of the membrane. The experimental results are consistent with a model in which sodium is actively transported into the intercellular cleft. We propose that Na+ re-enters the cell electroneutrally by coupled co-transport with carbonate, derived from bicarbonate in the solution.