Dynamic, spatially nonuniform calcium regulation in frog rods exposed to light

1. In intact rods of the bullfrog, Rana Catesbeiana, that were loaded with Fura-2 by incubation, we made high-resolution measurements of Na:Ca,K exchange currents and measured cytosolic free calcium concentrations during exposure to steps of illumination. The calcium dynamics we observed are indicative of unmanipulated rods because Fura-2 had little effect on calcium buffering within the outer segment. 2. In the dark, the total concentration of calcium within the outer segment, determined by integrating the exchange current, was near 50 microM. The free calcium concentration in darkness was 200-400 nM, indicating that > of = 99% of the internal calcium was bound to buffer molecules or equivalently sequestered. 3. During saturating illumination, the concentration of free calcium near the membrane (assayed by the exchange current) fell more rapidly than the space-averaged free calcium concentration (measured with Fura-2), but both had time courses that were best described by a sum of three exponential terms. The time constants were the same for each assay, but the weighting factors were different. 4. The relationship between the exchange current and space-averaged calcium concentration is consistent with significant concentration gradients within the outer segment resulting from high buffering power, diffusional restrictions, and the fact that all net gain and loss of calcium occurs at the membrane. The data further indicate that effective buffering, and hence calcium mobility, is not uniform within the outer segment. 5. Calcium kinetics were independent of the calcium concentration, indicating that the dominant buffers effectively have a low affinity for calcium (KD >> [Ca2+]dark free). 6. The dynamics of calcium changes and of exchange currents evoked by saturating and nonsaturating illumination are completely predictable from changes in the circulating current. Calcium and current are related by a linear transformation, indicating that calcium fluxes within the outer segment are passive and that buffers equilibrate rapidly. 7. Although calcium concentrations change slowly with respect to changes in the circulating current, both measured and calculated calcium dynamics are well correlated with changes in light adaptation. Responses to test flashes depended weakly on the detailed time course of the adapting stimulus but strongly on the free cytosolic calcium concentration at the time the test flash was delivered.