Fig. 2. Localized depletion of the Ins(1,4,5)P₃-sensitive Ca²⁺ store depletes the entire store of Ca²⁺. At –70 mV, locally photolysed Ins(1,4,5)P₃ (, B) at a 10-µm-diameter region (photolysis site 1; bright spot in A, left-hand panel; see also patch electrode, left side) evoked Ca²⁺ transients (B). Results from photolysis site 1 are indicated by the red bars below the [Ca²⁺]_c traces in B. When repositioned to site 2 (A, right-hand panel), subsequent photolysis 90 seconds later produced reproducible [Ca²⁺]_c increases (B). Results from photolysis site 2 are indicated by the blue line below the [Ca²⁺]_c trace (B). In a Ca²⁺-free solution [containing EGTA (1 mM) and MgCl₂ (3 mM); unfilled line above the [Ca²⁺]_c trace], the [Ca²⁺]_c increase evoked by Ins(1,4,5)P₃ at photolysis site 2 (A) declined in amplitude as the store was depleted of Ca²⁺ (B). When the store content had been substantially reduced at photolysis site 2 (A) (as revealed by the smaller Ca²⁺ transients, B), Ins(1,4,5)P₃ was liberated by photolysis at site 1 (A). Again, as at photolysis site 2, the response was now almost abolished compared with that of the control. On restoring external Ca²⁺ (B, right-hand side), the Ca²⁺ increase evoked by Ins(1,4,5)P₃ at photolysis site 1 was restored towards control values. These results suggest that the SR is lumenally continuous and within it Ca²⁺ can diffuse freely throughout. [Ca²⁺]_c measurements (B) have been derived from fluorescence intensity changes occurring in a circle of diameter 5 µm in the center of the photolysis region. Thus, those results from photolysis site 1 are from a circle of diameter 5 µm positioned at site 1; results from photolysis site 2 are from a circle of diameter 5 µm at site 2. (C) Local photolysed Ins(1,4,5)P₃ () at photolysis site 1 (A, left-hand panel) increased [Ca²⁺]_c (C, right-hand panel), which was maximal at, and decreased with each 10 µm increment away from, the release site (C, right panel); region 1 is the photolysis site. [Ca²⁺]_c measurements were made at lines of width 1 pixel. The position of each measurement (regions 1-9) line is shown (C, left panel) at a width of two pixels to facilitate visualization. The second photolysis site lies between regions 8-9 – that is, 75 µm away from photolysis site 1.