Quantifying Photoreceptor Calcium Dynamics

To quantify how photoreceptor Ca²⁺ concentration changes during exposure to light, both the K_d of fluo-3 and the maximum and minimum fluorescence that can be elicited from the rod outer segment in high and low Ca²⁺ (F_max and F_min) are required (Minta et al., 1989 (link)). The K_d of the Ca²⁺ indicator was determined with a haemocytometer (100 μm path length) containing 100 μM fluo-3 free acid in pseudo-intracellular solutions of buffered Ca²⁺ (World Precision Instruments, Sarasota, FL), whose precise free Ca²⁺ concentrations were verified using a Ca²⁺ electrode (Calcium Ionophore I−cocktail A; Fluka, Ronkonkoma, NY). The sigmoidal variation of the fluorescence signal (F) with free Ca²⁺ concentration (Fig. 1, inset) could be fitted by the Michaelis-Menten equation for Ca²⁺ binding:
which yielded a value for K_d of 400 nM.
F_max and F_min were determined in situ using the four-laser pulse protocol described above while exposing the photoreceptor outer segment to solutions of low and high Ca²⁺ concentration containing 25 μM ionomycin or 40 mg/ml saponin. The low Ca²⁺_i solution that was used to determine F_min consisted of 111 mM NaCl, 2.5 mM KCl, 2.05 mM MgCl₂, 3 mM HEPES, and 2 mM EGTA. The high Ca²⁺ solution that was used to determine F_max contained 76.6 mM CaCl₂, 2.5 mM KCl, and 3 mM HEPES. In both cases, the pH was titrated to 7.7 with NaOH. Values of F_min and F_max measured in this way were used in Eq. 1 to determine the free Ca²⁺ concentration in the same dark-adapted rod before and after exposure to saturating laser illumination. The value of F_min determined in situ was a greater fraction of F_max than was obtained from fluo-3 in the cuvette (see Fig. 1, inset); this discrepancy may reflect nonspecific binding of the dye within the rod outer segment (see discussion).