Measurement of surface potential and surface charge densities of sarcoplasmic reticulum membranes

Summary

The binding of the anionic fluorescent probe 1-anilino-8-naphthalene-sulfonate (ANS⁻) was used to estimate the surface potential of fragmented sarcoplasmic reticulum (SR) derived from rabbit skeletal muscle. The method is based on the observation that ANS⁻ is an obligatory anion whose equilibrium constant for binding membranes is proportional to the electrostatic function of membrane surface potential, exp(eΨ₀/kT, where Ψ₀ is the membrane surface potential,e is the electronic charge, andkT has its usual meaning. The potential measured is characteristic of the ANS⁻ bindings of phosphatidylcholine head groups and is about one-third as large as the average surface potential predicted by the Gouy-Chapman theory. At physiological ionic strength the surface potentials, measured by ANS⁻, referred to as the aqueous phase bathing the surface, were in the range −10 to −15 mV. This was observed for the outside and inside surfaces of the Ca²⁺-ATPase-rich fraction of theSR and for both surfaces of theSR fraction rich in acidic Ca²⁺ binding proteins. The inside and outside surfaces were differentiated on the basis of ANS⁻ binding kinetics observed in stopped-flow rapid mixing experiments. A mechanism by which changes in Ca²⁺ concentration could give rise to an electrostatic potential across the membrane and possibly result in changes in Ca²⁺ permeability.

The dependence of the surface potential on the monovalent ion concentration in the medium was used together with the Gouy-Chapman theory to determine the lower limits for the surface charge density for the inside and outside surfaces of the two types ofSR. Values for the Ca²⁺-ATPase richSR fraction were between 2.9×10³ and 3.8×10³ esu/cm², (0.96×10⁻⁶ and 1.26×10⁻⁶ C/cm²) with no appreciable transmembrane asymmetry. A small amount of asymmetry was observed in the values for the inside and outside surfaces of the fraction rich in acidic binding proteins which were ca. 6.6×10³ and ca. 2.2×10³ esu/cm² (2.2×10⁻⁶ and 0.73×10⁻⁶ C/cm). The values could be accounted for by the known composition of negatively-charged phospholipids in theSR. The acidic Ca²⁺ binding proteins were shown to make at most a small contribution to the surface charge, indicating that their charge must be located at least several tens of Å from the membrane surface. The experiments gave evidence for a Donnan effect on the K⁺ distribution in the fraction rich in acidic binding proteins. This could be accounted for by the known concentration of acidic binding proteins in thisSR fraction.

The equilibrium constant for ANS⁻ was shown to be more sensitive to changes in the divalent cation concentration than to changes in the monovalent cation concentration, as predicted by the Gouy-Chapman theory. Use of these findings together with the stopped-flow rapid mixing techniques constitutes a method for rapid and continuous monitoring of changes in ion concentrations in theSR lumen.