Buffer kinetics shape the spatiotemporal patterns of IP3-evoked Ca2+ signals

Abstract

Ca2+ liberation through inositol 1,4,5-trisphosphate receptors (IP3Rs) plays a universal role in cell regulation, and specificity of cell signalling is achieved through the spatiotemporal patterning of Ca2+ signals. IP3Rs display Ca2+-induced Ca2+ release (CICR), but are grouped in clusters so that regenerative Ca2+ signals may remain localized to individual clusters, or propagate globally between clusters by successive cycles of Ca2+ diffusion and CICR. We used confocal microscopy and photoreleased IP3 in Xenopus oocytes to study how these properties are modulated by mobile cytosolic Ca2+ buffers. EGTA (a buffer with slow 'on-rate') speeded Ca2+ signals and 'balkanized' Ca2+ waves by dissociating them into local signals. In contrast, BAPTA (a fast buffer with similar affinity) slowed Ca2+ responses and promoted 'globalization' of spatially uniform Ca2+ signals. These actions are likely to arise through differential effects on Ca2+ feedback within and between IP3R clusters, because Ca2+ signals evoked by influx through voltage-gated channels were little affected. We propose that cell-specific expression of Ca2+-binding proteins with distinct kinetics may shape the time course and spatial distribution of IP3-evoked Ca2+ signals for specific physiological roles.

Figure 2. Buffer actions at varying [IP₃]

Representative fluorescence profiles (from different oocytes to those in Fig. 1) show superimposed Ca²⁺ transients evoked by increasing photorelease of IP₃ in the absence of exogenous buffer (upper panels) and after loading increasing concentrations of EGTA (A) or BAPTA (B). Traces correspond to different photolysis flash strengths, indicated in normalized units.

Figure 1. Modulation of IP₃-evoked Ca²⁺ signals by EGTA and BAPTA

Confocal line-scan images illustrate Ca²⁺ signals evoked by photoreleased IP₃ in the presence of increasing concentrations of buffer. In each image, distance is depicted vertically, time runs from left to right and increasing Oregon Green 488 BAPTA 1 (OG-1) fluorescence ratio (ΔF/F₀: proportional to [Ca²⁺]_free) is represented by ‘warmer’ colours as denoted by the colour bar. Identical photolysis flashes (normalized intensities of 1.5 in A and 1.9 in B) were delivered at the arrows. Traces below each image show fluorescence profiles averaged over 100 pixel (6.6 μm) regions. A, upper panel shows response before loading buffer, and subsequent panels illustrate responses after sequentially loading the same oocyte with EGTA to the final intracellular concentrations stated. B, similar records from a different oocyte showing the effects of increasing concentrations of BAPTA.

Figure 3. Buffers both facilitate and depress IP₃-evoked Ca²⁺ signals

A and B, mean peak amplitude (ΔF/F₀) of Ca²⁺ signals as a function of photolysis flash strength, plotted for various intracellular concentrations of EGTA (A: n = 21 oocytes) and BAPTA (B: n = 24). Curves were fitted using the Hill equation. C and D, changes in peak fluorescence signal as a function of increasing [buffer] plotted as a percentage of that in the same oocyte before loading buffer. Data are pooled for weak (black symbols) and strong (red) photolysis flashes (respective normalized flash strengths 0.6–0.8 and 1.9–2.6) in the presence of EGTA (C, n = 14) and BAPTA (D, n = 17).

Figure 4. Ca²⁺ buffers reduce the apparent cooperativity of IP₃ action

A, Hill coefficients, derived from the curves fitted to data in Fig. 3A and B, are plotted as functions of [EGTA] (open circles) and [BAPTA] (filled squares). B, plot shows V_max (maximal fluorescence signal at infinite [IP₃] derived from Hill-fits) as functions of [EGTA] (open circles) and [BAPTA] (filled squares). Horizontal lines on each graph represent control values (i.e. no exogenous buffer).

Figure 5. Ca²⁺ transients are speeded by EGTA but prolonged by BAPTA

A, families of curves illustrate Ca²⁺ transients evoked by a fixed photolysis flash (normalized strength 2.5) in the presence of the indicated concentrations of EGTA (left) and BAPTA (right). Responses are scaled to same peak height to facilitate comparison. B, Ca²⁺ transients evoked by various photolysis flashes of various strengths (normalized strengths indicated) before loading buffer (left); in the same oocyte after loading 135 μm EGTA (middle); and in a different oocyte loaded with 135 μm BAPTA (right). Red curves illustrate fitting of single or double exponentials to derive fast (τ_fast) and slow (τ_slow) decay time constants.

Figure 6. EGTA and BAPTA differentially affect the fast and slow decay components of IP₃-evoked Ca²⁺ transients

A and C, graphs show, respectively, the time constants for the fast (τ_fast) and slow (τ_slow) decay components as a function of photolysis flash strength, in the presence of various concentrations of EGTA. B and D, similar data, with corresponding concentrations of BAPTA. Because the decay of Ca²⁺ transients without added buffer was mono-exponential, control time constants are repeated in the upper and lower panels (black symbols joined by lines). E, plot shows the relative magnitude of the fast decay component (τ_fast: measured as shown in Fig. 5B) at various concentrations of EGTA (red symbols) and BAPTA (black symbols). The amplitudes of the fast and slow components (a_fast and a_slow, respectively) were derived at the time of the peak signal from bi-exponential fits, and the graph shows the percentage contribution of a_fast to the total signal (i.e. a_fast/(a_fast+a_slow) × 100). Open and filled symbols represent signals evoked, respectively, by weak and strong photolysis flashes.

Figure 7. The decay of IP₃-evoked Ca²⁺ transients is largely determined by the kinetics of Ca²⁺ release, not sequestration

A, line-scan image shows a Ca²⁺ transient evoked by a 300 ms duration depolarization to +30 mV to activate expressed N-type channels, followed 10 s later by a UV flash to photorelease IP₃. The lower trace shows the fluorescence profile averaged across a 6.6 μm region of the scan line, and superimposed traces on the right compare the voltage-gated signal (black) with the IP₃-evoked signal (red) after normalizing to the same peak height. B, corresponding image and traces recorded under identical conditions in the same oocyte after loading 270 μm EGTA. C, similar records in a different oocyte loaded with 270 μm BAPTA. Control records in this oocyte before loading BAPTA were similar to one in A.

Figure 8. EGTA abolishes the ‘tail’ of IP₃-evoked Ca²⁺ liberation that normally prolongs Ca²⁺ transients

A, fluorescence signals evoked by Ca²⁺ entry through voltage-gated channels (bar) and by photoreleased IP₃ (arrow), before (upper) and after (lower) loading 270 μm EGTA, derived from images similar to Fig. 7A and B. B, rates of Ca²⁺ flux into the cytosol derived from the records in A, as described in the text. Traces were smoothed using 15 point adjacent averaging. Calibration bar corresponds to a rate of increase in fluorescence (d(ΔF/F₀)/dt) of 3 s⁻¹. Inset graphs show the same records with expanded vertical axes to better illustrate the abolition of the persistent ‘tail’ of Ca²⁺ liberation by EGTA. C and D, similar data from a different oocyte, illustrating the lack of action of BAPTA (270 μm) on the tail of Ca²⁺ liberation.

Figure 9. ²⁺ signals into discrete, autonomous units, whereas BAPTA 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 strength (indicated in normalized units) before injecting buffer. B, corresponding records in the same oocyte after loading 135 μm EGTA. Two representative fluorescence profiles are illustrated from each image, recorded at different puff sites (arrowed). C, corresponding records in a different oocyte loaded with 135 μm BAPTA. Records in this oocyte before loading BAPTA were similar to those in A.

Figure 10. Cartoon illustrating the distribution of Ca²⁺ ions around IP₃R clusters, and how communication between IP₃Rs may be affected by mobile buffers with differing binding kinetics

A, without added buffer, Ca²⁺ ions liberated through open IP₃ at a cluster diffuse a few micrometres and, depending on [IP₃], may or may not trigger CICR at adjacent clusters. B, EGTA acts too slowly to disrupt Ca²⁺ diffusion and CICR within clusters, but binds Ca²⁺ ions as they diffuse between clusters and rapidly ‘shuttles’ them away, so as to disrupt cluster–cluster interactions. C, BAPTA binds Ca²⁺ ions sufficiently quickly to disrupt Ca²⁺ communication between IP₃Rs within individual clusters, whilst promoting Ca²⁺ communication between clusters by acting as a rapid Ca²⁺ shuttle.