Regulation of free cytosolic Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. I. Efficiency of transport and interactions between cations.
Regulation of free cytosolic Ca2+ concentration in the rod outer segments (ROS) isolated from bovine retinas was examined with the fluorescent Ca(2+)-indicating dye fluo-3. In situ calibration of cytosolic fluo-3 was done in the presence of the Ca2+ ionophore A23187 and yielded a dissociation constant of 500 nM for the Ca(2+)-fluo-3 complex. Ca2+ influx in Ca(2+)-depleted ROS was completely abolished when internal Na+ was removed suggesting that Ca2+ influx exclusively occurred via Na-Ca-K exchange. The most striking observation was that Na-Ca-K exchange could mediate a rapid increase in cytosolic free Ca2+ over the most of the usable indicating range of fluo-3 (from 10 nM to 2 microM), even when exposed to free external Ca2+ concentrations as low as 10 nM. From a comparison between changes in free Ca2+ and changes in total Ca2+, we conclude that physiologically occurring changes in cytosolic free Ca2+ are mediated by exchange fluxes less than 1% of the maximal Na-Ca-K exchange flux. The Na-Ca-K exchanger could mediate both K(+)-dependent and K(+)-independent Ca2+ influx; Li+ caused a complete inhibition of K(+)-independent Ca2+ influx, but had no effect on K(+)-dependent Ca2+ influx. We examined the complex interactions of alkali cations with Ca2+ influx and discuss the results in terms of a three-site model for the Na-Ca-K exchanger (Schnetkamp, P. P. M. and Szerencsei, R. T. (1991) J. Biol. Chem. 266, 189-197). Ca2+ competed with one Mg2+ ion or two Na+ ions for binding to a common site. High K+ concentration greatly diminished the ability of Na+ and Mg2+ to compete with Ca2+ for this common site on the exchanger protein. As a result, high internal K+ induced a conformation of the exchange protein that kinetically favoured Ca2+ extrusion.
Schnetkamp
P P
PP
Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Li
X B
XB
Basu
D K
DK
Szerencsei
R T
RT
D000814:Aniline Compounds; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D003600:Cytosol / Q000378:metabolism*; D005456:Fluorescent Dyes; D007474:Ion Exchange; D007700:Kinetics; D011188:Potassium / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D014966:Xanthenes
D000814:Aniline Compounds; D005456:Fluorescent Dyes; D014966:Xanthenes; C059715:Fluo-3; D012964:Sodium; D011188:Potassium; D002118:Calcium