Measurement of the intracellular free calcium concentration in salamander rods

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 Yoshikami^1,2 that the primary event in phototransduction is a release of Ca²⁺ inside the cell. More recent evidence^3–6 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.