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, and kT 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 the SR and for both surfaces of the SR 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 of SR. Values for the Ca2+-ATPase rich SR 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 the SR. 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 this SR 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 the SR lumen.
ASJC Scopus subject areas
- Cell Biology