Measurement of the intracellular free calcium concentration in salamander rods

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Abstract
Measurement of the free calcium concentration within a photo-receptor outer segment has been considered an important aim since the proposal by Hagins and Yoshikami1,2 that the primary event in phototransduction is a release of Ca2+ inside the cell. More recent evidence3–6 has cast doubt on the calcium hypothesis, and the observations of Yau and Nakatani7 and Matthews et al.6 suggest that the internal Ca2+ concentration ([Ca2+]i) may decrease after a flash of light. In the present study we have measured [Ca2+]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 [Ca2+]i, caused by the closure of light-sensitive channels which are the main route for Ca2+ entry into the outer segment. Of the Ca2+ 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 pump3 in which 3 Na+ ions are exchanged for each Ca2+; the pump is highly active and the free Ca2+ in the cell declines with a time constant of ∼0.5 s after a flash of light.