Measurement of the intracellular free calcium concentration in salamander rods

Measurement of the free calcium concentration within a photo-receptor outer segment has been considered an important aim since the proposal by Hagins and Yoshikami that the primary event in phototransduction is a release of Ca (2+) inside the cell. More recent evidence has cast doubt on the calcium hypothesis, and the observations of Yau and Nakatani and Matthews et al. suggest that the internal Ca (2+) concentration ([Ca (2+)]i), may decrease after a flash of light. In the present study we have measured [Ca (2+)]i directly by using a new method for incorporating the Ca-sensitive photoprotein aequorin into an isolated rod. We report that the light response is accompanied by a decrease in [Ca (2+)]i, caused by the closure of light-sensitive channels which are the main route for Ca (2+) entry into the outer segment. Of the Ca (2+) entering through light-sensitive channels, about 95% is sequestered by a rapid and reversible buffering mechanism. Calcium is removed from the cell by an electrogenic pump in which 3 Na (+) ions are exchanged for each Ca (2+); the pump is highly active and the free Ca (2+) in the cell declines with a time constant of ~0.5 s after a flash of light.