Phasic characteristic of elementary Ca(2+) release sites underlies quantal responses to IP(3)

Abstract

Ca(2+) liberation by inositol 1,4,5-trisphosphate (IP(3)) is 'quantal', in that low [IP(3)] causes only partial Ca(2+) release, but further increasing [IP(3)] evokes more release. This characteristic allows cells to generate graded Ca(2+) signals, but is unexpected, given the regenerative nature of Ca(2+)-induced Ca(2+) release through IP(3) receptors. Two models have been proposed to resolve this paradox: (i) all-or-none Ca(2+) release from heterogeneous stores that empty at varying [IP(3)]; and (ii) phasic liberation from homogeneously sensitive stores. To discriminate between these hypotheses, we imaged subcellular Ca(2+) puffs evoked by IP(3) in Xenopus oocytes where release sites were functionally uncoupled using EGTA. Puffs were little changed by 300 microM intracellular EGTA, but sites operated autonomously and did not propagate waves. Photoreleased IP(3) generated flurries of puffs-different to the prolonged Ca(2+) elevation following waves in control cells-and individual sites responded repeatedly to successive increments of [IP(3)]. These data support the second hypothesis while refuting the first, and suggest that local Ca(2+) signals exhibit rapid adaptation, different to the slower inhibition following global Ca(2+) waves.

Fig. 1. Functional uncoupling of IP₃-sensitive Ca²⁺ release sites following intracellular injection of EGTA. Panels show linescan images of Ca²⁺-dependent fluorescence, with distance along the scan line depicted vertically and time running from left to right. Increasing fluorescence ratio (ΔF/F) (increasing free [Ca²⁺]) is denoted by increasingly ‘warm’ colors (as indicated by the color bar). (A) Records showing responses to photolysis flashes of varying duration (indicated in ms) delivered at the times indicated by vertical white lines in the images. (B) Comparable responses evoked in the same oocyte after injecting EGTA to a final intracellular concentration of 300 µM, together with 150 µM Ca²⁺. Results are representative of findings in 17 oocytes (three donor frogs). (C) Images show averages of puffs in a single cell before injecting EGTA, and after progressively loading EGTA to final intracellular concentrations of 300 and 900 µM. Each frame is an average of eight events, aligned to their peaks in space and time. Traces show the time course of fluorescence signals measured from three pixel (0.4 µm) regions centered on the averaged puff images.

Fig. 2. Responses to repeated photolysis flashes before and after loading with EGTA. (A) Linescan image shows calcium signals in a control oocyte in response to two identical photolysis flashes (50 ms duration), delivered when marked by the arrows. The first flash evoked calcium waves originating at two sites (‘V’ patterns), which propagated across all but the lower region of the scan line. The second flash evoked little additional calcium release in regions that responded to the first flash, but triggered a wave in the region that failed to respond. The traces show fluorescence profiles (averaged over 22 pixels) measured at the two sites (a and b) indicated on the image. (B) Responses in a different oocyte, loaded with 300 µM EGTA plus 150 µM Ca²⁺. The cell was stimulated with four repeated photolysis flashes of decreasing durations, as indicated in ms. The trace shows fluorescence monitored from a 20 µm region near the center of the image.

Fig. 3. Ca²⁺ release sites exhibit phasic responses following step increases in [IP₃]. (A) Linescan images illustrate puffs evoked by photolysis flashes of varying durations delivered at the times indicated by the arrows. Histograms show the corresponding frequencies of puff occurrence as a function of time after the flashes. Data were obtained from a fixed scan line in a single oocyte, and numbers of events were measured in 250 ms time bins by counting all puffs occurring along the scan line in response to eight, four and five repeated trials in a, b and c, respectively. Similar distributions were observed in two additional oocytes. (B) Latency distributions measured in another oocyte, plotting separately the occurrence of the first (green) and subsequent (red) puffs at each release site following flashes of varying duration (indicated in ms).

Fig. 4. Estimation of decay of [IP₃] following photorelease, using paired-pulse facilitation. (A) Linescan images show Ca²⁺ responses evoked by paired photolysis flashes, delivered when marked by the arrows at varying inter-pulse intervals (indicated in s). All photolysis flashes were of a fixed duration (20 ms), chosen so that a single flash evoked only a very slight response. Periods of 90 s were allowed between each trial. To conserve space, records are omitted during the time between flashes at longer intervals (15 and 30 s). (B) Measurements from images like those in (A), obtained in two oocytes, showing the decay of facilitation of the second response as a function of interval between the flashes. Data were obtained by integrating the Ca²⁺ signal along the entire scan line, and are plotted as the peak fluorescence ratio change evoked by the second flash minus that evoked by the first flash in that trial.

Fig. 5. Incremental responses of Ca²⁺ release sites in response to paired photolysis flashes. (A and B) Linescan image and latency histogram illustrating puffs evoked by a single photolysis flash (40 ms duration). (C and D) Corresponding records showing responses to paired flashes at an interval of 1 s. Data were obtained from the same scan line as in (A), and with an identical first flash: the duration of the second flash was 20 ms. Latency distributions were derived from seven trials in (B) and nine trials in (D). (E) Instances when given sites exhibited puffs in response to only the first or second flash (a and b) or to both flashes (c). Traces show fluorescence ratios measured from five pixel (0.65 µm) regions of the scan line, centered on release sites. Results are representative of trials at 65 linescan locations in 12 oocytes.

Fig. 6. The probability of occurrence and amplitude of puffs in response to the second flash are independent of whether a particular site generated a puff in response to the first flash. (A) Percentage of times that sites failed to show puffs in response to a second flash, as a function of whether or not each site gave a puff to the first flash. (B) Amplitudes of puffs generated by a second flash, as a function of whether or not that site responded to the first flash. Data in (A) and (B) are from nine repeated trials (paired flashes) at a single scan line encompassing 16 discrete release sites. Error bars indicate ± 1 SEM. Paired flashes were delivered at a 1 s interval, with respective durations of 30 and 20 ms. Puffs were evoked by the first flash in 38% (54/144) of instances. (C) Scatter plot showing measurements of the amplitude (F/F_o) of second puffs as a function of the size of the first puff during individual trials at given sites. Data are included only from instances when puffs were evoked by both flashes, and are normalized as a percentage of the mean puff size averaged over repeated trials at each site.

Fig. 7. Incremental responses of puff sites at short intervals. (A) Linescan images and puff latency distributions showing responses to a single flash of 30 ms duration (a), and to paired flashes with durations of 30 and 20 ms delivered at an interval of 400 ms (b). Flashes were delivered when marked by the arrows. Latency distributions were derived from three trials in each instance, in a single oocyte. (B) Examples of second puffs evoked at short latencies following initial events, shown on expanded time and distance scales. Traces on the right show fluorescence measurements from 0.5 µm regions at the sites marked by the red arrows.