Spatiotemporal patterning of IP3-mediated Ca2+ signals in Xenopus oocytes by Ca2+-binding proteins

Abstract

Ca(2+)-binding proteins (CaBPs) are expressed in a highly specific manner across many different cell types, yet the physiological basis underlying their selective distribution patterns remains unclear. We used confocal line-scan microscopy together with photo-release of IP(3) in Xenopus oocytes to investigate the actions of mobile cytosolic CaBPs on the spatiotemporal properties of IP(3)-evoked Ca(2+) signals. Parvalbumin (PV), a CaBP with slow Ca(2+)-binding kinetics, shortened the duration of IP(3)-evoked Ca(2+) signals and 'balkanized' global responses into discrete localized events (puffs). In contrast, calretinin (CR), a presumed fast buffer, prolonged Ca(2+) responses and promoted 'globalization' of spatially uniform Ca(2+) signals at high [IP(3)]. Oocytes loaded with CR or PV showed Ca(2+) puffs following photolysis flashes that were subthreshold in controls, and the spatiotemporal properties of these localized events were differentially modulated by PV and CR. In comparison to results we previously obtained with exogenous Ca(2+) buffers, PV closely mimicked the actions of the slow buffer EGTA, whereas CR showed important differences from the fast buffer BAPTA. Most notably, puffs were never observed after loading BAPTA, and this exogenous buffer did not show the marked sensitization of IP(3) action evident with CR. The ability of Ca(2+) buffers and CaBPs with differing kinetics to fine-tune both global and local intracellular Ca(2+) signals is likely to have significant physiological implications.

Figure 1. Parvalbumin (PV) and calretinin (CR) modulate IP₃-evoked Ca²⁺ signals

Confocal line-scan images from two oocytes illustrate Ca²⁺ signals evoked by photoreleased IP₃ in the presence of increasing [PV] or [CR]. Data are representative of similar findings in 37 oocytes (PV, n = 18; CR, n = 19). Identical photolysis flashes (normalized durations of 1.4 in A and 1.7 in B) were delivered at the arrows. Traces below each image show fluorescence profiles averaged over 21 pixels (1.4 μm) regions. A, top panel shows control response prior to loading buffer, and subsequent panels illustrate responses after sequentially loading the same oocyte with PV to the final intracellular concentrations stated. B, similar records from a different oocyte showing the effects of increasing concentrations of CR.

Figure 2. Parvalbumin and calretinin differentially modulate the decay kinetics of IP₃-evoked Ca²⁺ transients

A, buffer actions at varying [IP₃]. Representative fluorescence profiles show superimposed Ca²⁺ transients evoked by increasing photorelease of IP₃ in the absence of exogenous CaBP (top panels) and after loading increasing concentrations of PV (left) or CR (right). Traces correspond to different photolysis flash durations, indicated in normalized units. B, Ca²⁺ transients are shortened by PV but prolonged by CR. Families of curves illustrating Ca²⁺ transients evoked by a fixed photolysis flash in the presence of the indicated concentrations of PV (left) and CR (right) (normalized flash durations were 1.4 and 1.7, for PV and CR, respectively). Responses are scaled to same peak height to facilitate comparison of kinetics.

Figure 3. CaBPs modulate the concentration–response relationship of IP₃-evoked Ca²⁺ signals

A and B, mean peak amplitude (ΔF/F_o) of Ca²⁺ signals as a function of normalized photolysis flash duration, plotted for various intracellular concentrations of PV (A: n = 16 oocytes) and CR (B: n = 18 oocytes). Curves were fitted using the Hill equation. C–E, parameters derived from Hill fits to the concentration–response relationships. In each panel, ○ represent values derived at different concentrations of PV, and ▪ show corresponding values for CR. Horizontal lines represent control values (i.e. before loading CaBP). C, V_max (maximal fluorescence signal at infinite [IP₃]) as a function of [CaBP]. D, Hill coefficients (n_H) as functions of [CaBP]. E, EC₅₀ as a function of [CaBP].

Figure 4. Biphasic Ca²⁺ liberation through IP₃Rs, and selective reduction of the slow component of Ca²⁺ release by parvalbumin

A, representative fluorescence signals evoked by photoreleased IP₃ in control conditions (top), and in the presence of 100 μm PV (middle) or 100 μm CR (bottom). B, rates of Ca²⁺ flux into the cytosol derived from the records in A, assuming that cytosolic Ca²⁺ clearance follows a first order process with a time constant of about 1 s (see text for further details). Traces were smoothed using 15 point adjacent averaging. The vertical calibration bars correspond to a rate of increase in fluorescence (d(ΔF/F_o)/dt). Control traces (in A and B) are from the same oocyte as the CR traces. The fluorescence trace for the PV-paired control (not shown) was closely similar to that of the CR-paired control.

Figure 5. Parvalbumin ‘balkanizes’ Ca²⁺ signals into discrete, autonomous units, whereas calretinin promotes spatially uniform global signals

A, line-scan images and fluorescence profiles (averaged over 4 μm regions) showing responses to photolysis flashes (red arrows) of increasing duration (indicated in normalized units) before injecting buffer. B, corresponding records in a different oocyte after loading 100 μm PV (records in this oocyte before loading PV were similar to those in A). Two representative fluorescence profiles are illustrated from each image, recorded at different puff sites (black arrows). C, corresponding records after loading with 100 μm CR, from the same oocyte as in A.

Figure 6. IP₃-evoked Ca²⁺ puffs terminated more rapidly in the presence of parvalbumin

Averaged line-scan images (n = 18 events for each) and their corresponding fluorescence profiles (averaged over 0.6 μm regions) of Ca²⁺ puffs evoked by low photo-release of IP₃ in the absence of added buffer (left panel) and in the presence of 50 μm PV (centre panel) or 50 μm CR (right panel). Images were acquired at a scan rate of 2.6 ms line⁻¹ using the indicator dye Fluo-4-dextran (low affinity version). Red curves represent single (for PV oocyte) or double (for control and CR-containing oocyte) exponential fits to the decay phase of puffs.

Figure 7. Comparison of actions of synthetic Ca²⁺ buffers and CaBPs on IP₃-evoked Ca²⁺ signals

Graphs summarize data obtained using PV (light blue) and CR (green), together with data taken from Dargan & Parker (2003) obtained previously using EGTA (red) and BAPTA (dark blue). In order to facilitate comparison, buffer concentrations are expressed as the equivalent concentration of Ca²⁺ binding sites (see text for further explanation). Further, all fluorescence data are scaled relative to the maximum of the peak signal obtained at high [IP₃] before loading buffer. Control measurements (before loading buffer) are indicated in black. Different batches of control oocytes were used for each buffer, but the inset plot in A shows that normalized control data from these four groups matched closely. For clarity, control data points are shown only in the lower panels of A and B, and the fitted control Hill curves are replicated in the other panels. A, plots show the peak amplitude of Ca²⁺ signals as a function of normalized photolysis flash duration for three different concentrations of slow buffers (EGTA and PV). B, corresponding concentration–response relationships for the ‘fast’ buffers BAPTA and CR. C, data derived from the Hill curves in A and B showing changes in apparent cooperativity of IP₃ action, V_max and EC₅₀ as a result of increasing concentrations of buffers. Horizontal black lines mark control values in the absence of added buffer. Hill coefficients varied between about 4.5 and 10.5 among the different batches of control oocytes, and the cooperativity is therefore expressed as a percentage of that in each respective control group. Similarly, values of V_max are scaled as a percentage of each control group.