Membrane potentials associated with Ca-induced K conductance in human red blood cells: Studies with a fluorescent oxonol dye, WW 781

Abstract

A divalent anionic dye, bis-[3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-pentamethine oxonol (WW 781) is a rapidly responding fluorescent indicator of KCl diffusion potentials induced in human red blood cells with valinomycin, gramicidin, and with the Ca ionophore A 23187 in the presence of external Ca. WW 781 has a sensitivity of 0.13% delta F/mV, a detection limit of 10 mV, a response time of less than 1 sec, and exhibits a decrease in fluorescence intensity upon hyperpolarization without detectable shifts in absorption or emission peaks. This dye does not perturb the normal resting potential, and unlike the slow permeant cyanine dyes, does not inhibit Ca-induced K conductance in human red blood cells. However, WW 781 does stimulate Ca-induced unidirectional Rb efflux. With Ca plus A 23187, the initial rapid change in dye fluorescence is sensitive to [Ca]o and to [A 23187], is reversible with excess EGTA, and is inhibited by quinine, oligomycin, and by trifluoperazine. A biphasic dependence of hyperpolarization on Ko is evident at pH 6, where the ionic selectivity of activation is K, Rb greater than Cs greater than Na and that of conductance is K, Rb greater than Cs. Conditions were defined which permitted continuous monitoring of Em for at least 10 min, and the time dependence of the Ca-induced potentials was characterized. Since the properties of the Ca-induced changes in dye fluorescence correlate well with the known characteristics of Ca-induced K permeability, we conclude that WW 781 is a useful indicator of changes in Em, provided that sufficient controls are employed to separate direct effects of Ca on dye fluorescence from the effects of Em on fluorescence.

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

“…The membrane potential-sensitive oxonol dyes DiBAC4(3) (Br'duner et al, 1984) and WW78 1 (Freedman and Novak, 1983) were added to 0.2 pM (final concentration) from stock solutions in dimethyl sulfoxide (0.1% final concentration). For fluorescence measurements using DiBAC4(3), the emission wavelength was 520 nm with the excitation wavelength set at 490 nm.…”

Relationship between the shape and the membrane potential of human red blood cells

“…The resultant increase in intra-cellular Ca 2", or Ca,, induces a selective increase in K + conductance, resulting in changes in E~ dependent on Ko. Increases in K ~ permeability attributed to Ca 2+ were first described by Gardos (1959), and the associated changes in Em dependent on Ca 2+ have recently been characterized by the fluorescent potentiometric indicator technique (Freedman & Novak, 1983). While previous studies indicate that increased Cat is echinocytogenic at about 200 /./,M (Sarkadi et al, 1976), and that increased K + conductance is induced at/xmolar or sub-/xmolar levels of Car (Blum & Hoffman, 1972), we sought to compare the Ca 2+ activation curves for the change in Em and for echinocytosis under equivalent conditions, and also to determine to what extent the Ca2+-activation curve for the shape change might be affected by Em in cells treated with A23187.…”

Membrane sidedness and the interaction of H+ and K+ on Ca2(+)-activated K+ transport in human red blood cells.

“…It was also observed (12,14) that the inhibitory properties of H' were antagonized by Kt. Clearly, in order to sort out the sided effects of H', as well as K+, it was necessary to inhibit pH equilibration across the cell membrane.…”