Calcium translocation and storage of isolated intact cattle rod outer segments in darkness

Bovine rod outer segments (rods), isolated with an intact plasma membrane and a stable calcium exchange and storage capacity, contain 2-3 mol endogenous calcium/mol rhodopsin. By means of 45Ca accumulation experiments and concomitant 40Ca analysis, the calcium metabolism of these organelles has been studied with the following results: 1. The majority of endogenous calcium is localized within disks. 2. In the presence of the ionophore A23187 the intradiskal binding sites can be titrated with external calcium. 3. The Scatchard plot of calcium binding of rods indicates the presence of a single set of intradiskal binding sites with a maximal capacity of 8-9 mol calcium/mol rhodopsin and an affinity constant of 55 microM to calcium. 4. Without A23187 more than 99% of the rod calcium appears in a bound state in equilibrium with a free calcium concentration of 15-25 microM. 5. External calcium exchanges with endogenous calcium in a fast (t 1/2 = 12 s) process with a uniform rate constant, whereas net calcium transport is very slow (t 1/2 greater than 2 h). 6. Intact rods contain a calcium translocation system, presumably located in the plasma membrane, which performs Ca-Ca exchange with a high unidirectional flux of 2 . 10(6) calcium ions/rod per s. 7. This translocation system can be saturated by external calcium (Km = 0.5 -1 microM) and has a low Q10 (1.08). Both the calcium translocation system and the calcium binding system appear to depend on the structural integrity of the stacked disks and are very sensitive to the experimental conditions. The relevance of these findings is discussed in relation to the proposed role of calcium ions as the intracellular transmitter in vertebrate rod photoreceptor cells.