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 1,2 that the primary event in phototransduction is a release of Ca 2+ inside the cell. More recent evidence 3–6 has cast doubt on the calcium hypothesis, and the observations of Yau and Nakatani 7 and Matthews et al. 6 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 3 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.