CALCIUM HOMEOSTASIS IN THE OUTER SEGMENTS OF RETINAL RODS FROM THE TIGER SALAMANDER

1. The processes regulating intracellular calcium in the outer segments of salamander rods have been investigated. The main preparation used was the isolated rod loaded with the Ca2+-sensitive photoprotein aequorin, from which outer segment membrane current and free [Ca2+]i could be recorded simultaneously. Two other preparations were also used: outer segment membrane current was recorded from intact, isolated rods using a suction pipette, and from detached outer segments using a whole-cell pipette. 2. Measurements of free intracellular [Ca2+] in Ringer solution were obtained from two aequorin-loaded rods. Mean [Ca2+]i in darkness was 0.41-mu-M, and after a bright flash [Ca2+]i fell to below detectable levels (< 0.3-mu-M). No release of intracellular Ca2+ by a bright flash of light could be detected (< 0.2-mu-M). 3. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused an increase in the size of the light-sensitive current and a rise in [Ca2+]i, but application of IBMX either when the light-sensitive channels had been closed by a bright light or in the absence of external Ca2+ caused no detectable rise in [Ca2+]i. It is concluded that IBMX increases [Ca2+]i by opening light-sensitive channels, and does not release Ca2+ from stores within the outer segment. 4. Removal of external Na+ caused a rise in [Ca2+]i to around 2-mu-M and completely suppressed the light-sensitive current. 5. The Na+-Ca2+, K+ exchange current in aequorin-loaded rods was activated in first-order manner by internal free calcium, with a mean Michaelis constant, K(Ca), of 1.6-mu-M. 6. The K(Ca) of the Na+-Ca2+, K+ exchange was increased by elevating internal [Na+]. 7. The Michaelis relation between [Ca2+]i and the activity of the Na+-Ca2+, K+ exchange was used to calculate the change in [Ca2+]i occurring during the response to a bright light. In aequorin-loaded rods in Ringer solution the mean change in free [Ca2+]i after a bright flash was 0.34-mu-M. In these rods 10 % of the dark current was carried by Ca2+. 8. Most of the calcium entering the outer segment was taken up rapidly and reversibly by buffer systems. The time constant of equilibration between free and rapidly bound Ca2+ was less than 20 ms. No slow component of calcium uptake was detected. 9. Two components of calcium buffering could be distinguished in the outer segments of aequorin-loaded rods. A buffer of low capacity and high affinity (Michaelis constant K(buff) almost-equal-to 0.7-mu-M) was important at free [Ca2+]i below 1-mu-M. At high free [Ca2+]i calcium buffering was dominated by a low-affinity buffer of large capacity. 10. The average ratio of free-to-bound Ca2+ at low [Ca2+]i was 1 : 74, that is to say one Ca2+ ion remains free out of every seventy-five entering the outer segment. At high [Ca2+]i the ratio fell to 1 : 16. 11. High- and low-affinity components of internal Ca2+ buffering were also observed in intact rods, but the mean capacity of the high-affinity buffer, 241-mu-M, was an order of magnitude larger than in aequorin-loaded rods. The difference is attributed to loss of this component of the buffer during the process of internal perfusion used to load aequorin. 12. In detached outer segments internally perfused by a whole-cell pipette the capacity of the high-affinity calcium buffer declined with a time constant of around 15 min. It is concluded that this component of buffering is associated with a large, diffusible molecule. 13. During internal perfusion the affinity for Ca2+ of the Na+-Ca2+, K+ exchange declined relative to that of the high-affinity buffer, probably because the affinity of the Na+-Ca2+, K+ exchange is controlled by a diffusible cytoplasmic modulator.