The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum.

The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca2+ transport. The absorbance and fluorescence responses of 3,3'-diethyl-2,2'-indodicarbocyanine and oxonol VI during ATP-dependent Ca2+ transport are influenced by the effect of accumulated Ca2+ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca2+ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca2+, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulum in vivo and in vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca2+ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca2+ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca2+ translocation. The potential generated during Ca2+ transport is rapidly dissipated by passive ion fluxes across the membrane.