Representative electrophysiological data obtained from a single whole-cell patch clamped acinar cell held under voltage clamp at a membrane potential of −30 mV in the presence of 50 μm EGTA. The upper horizontal line shows the zero current level in this figure (and in others). The record was obtained immediately after breaking through to the whole-cell configuration and shows the stable nature of the spike responses, which are of similar frequency and amplitude throughout the time of recording. The period of time used in the analysis of spike parameters was always 150 s after breakthrough to whole cell and was itself 150 s long. The period of analysis, for this cell, is indicated below the figure.

EGTA, ▪ and continuous line; BAPTA, • and dashed line. Spike frequency was measured independent of spike size. The minimum spike size measured was 10 pA. The graphs show divergent effects of the different buffers. Increasing concentrations of EGTA lead to an increase in spike frequency, whereas increasing concentrations of BAPTA lead to a decrease in spike frequency.

The typical high-frequency, low-amplitude spikes seen with EGTA are in contrast with the large-amplitude, low-frequency spikes seen in the presence of BAPTA. The long horizontal line indicates the zero current level. The short filled bar above the zero current line indicates the part of the record shown expanded below. The membrane potential was voltage clamped at −30 mV.

A-C, expanded traces correspond to the region of the record indicated by the short filled bar above the zero current line. At 200 μm EGTA a higher frequency of smaller events and the appearance of steps in the spike activation phase were observed (note the different amplitude scale). D, frequency-amplitude histograms drawn up from this dataset. At 50 μm EGTA there is a clustering of high amplitude events which contrasts with the spread of amplitudes seen at the higher Ca²⁺ buffer concentration.

A, a single clear step is seen in the Cl_(Ca) current activation phase. B, the series of fluorescence images (upper panel) show the Ca²⁺ signal at the time points indicated (a-g). The signal looks similar to that in control (Fig. 7) in terms of amplitude but has an additional slow spreading component. This is seen more clearly in the subtracted images (lower panel) where the change in Ca²⁺ response moves across the secretory pole in a right to left direction. In all of the data showing steps in the current activation phase, the analysed images were consistent with multiple components to the secretory pole response. A scale bar of 10 μm is shown on the black and white image of the cell.

The current is of large amplitude and the Ca²⁺ response shows a Ca²⁺ change very similar to that in the control (Fig. 7). This is despite the large increase in buffering power of the cytosol and indicates an increase in the amount of Ca²⁺ released. The scale bar on the black and white image of the cell represents 10 μm.

EGTA, ▪ and continuous line; BAPTA, • and dashed line. These graphs and those of Figs 3 and 4, show the mean ±s.e.m. spike amplitudes obtained from at least 3 separate cells at each buffer concentration. In the presence of EGTA increasing buffer led to a steady decrease in spike amplitude. In contrast a complex relationship of initial decline followed by variable amplitudes was observed in the presence of increasing BAPTA concentrations.

EGTA, ▪ and continuous line; BAPTA, • and dashed line. In both cases EGTA and BAPTA led to a decrease in the average rate of rise of the Cl_(Ca) current signal.

Examples of normalised current spikes in the presence of 50 μm BAPTA and EGTA are shown in the figure. The activation phase is monotonic and relatively slow (≈1 s to reach peak). The figure also shows the Cl_(Ca) current response recorded in response to the rapid photolytic release of Ca²⁺ from DM-nitrophen. This experiment was conducted after the partial replacement of the extracellular monovalent cations with NMDG and the cells were voltage clamped at a membrane potential of −17 mV (the equilibrium potential for the remaining cations). For this reason the current amplitude was slightly smaller and therefore we have scaled the responses to their maxima. The figure shows the much more rapid initial activation phase observed in response to photolytic release of Ca²⁺ compared with the responses to Ins(2,4,5)P₃.

A, time course of the whole-cell current response. B, ΔF/F_o digital images at the points a-e in A. The response originates at a single spot and spreads rapidly through the secretory pole over the time course of the activation phase of the spike. It appears as if the current inactivates much more rapidly than the decay in the bulk cytosolic Ca²⁺. In support of this the time to reach half peak amplitude (t_½) of the Ca²⁺ signal was 1.2 ± 0.24 s (mean ±s.e.m., n = 3), and of the current was 0.53 ± 0.07 s (mean ±s.e.m., n = 3, means significantly different, P < 0.05, Student's one-tailed t test). Scale bar, 10 μm.

The whole-cell current records are shown in A and simultaneously recorded fluorescence images at the time points indicated (a-f) are shown in B. A scale bar representing 10 μm is shown on the black and white image of the cell. Any given series of images were limited in length by the memory limitations of the computer and image capture was triggered by the experimenter. In this example the first images and Cl_(Ca) currents show evidence of spike activity. The ‘control’ image is therefore shown in b, where there is neither current nor fluorescence evidence for a response. Images c and d show a very small Ca²⁺ response in the same region of the cell (our spatial resolution for the centre of the Ca²⁺ response is 200 nm). In image a, an apparently smaller Ca²⁺ response in a different region of the cell gives rise to a larger Cl_(Ca) current spike. Image e was taken at the peak of a larger Cl_(Ca) current spike and shows a diffuse Ca²⁺ signal in a region distinct from the Ca²⁺ release sites of the previous images. Image f shows the Ca²⁺ response during a larger Cl_(Ca) current response. The differences in amplitude of the Ca²⁺ signal may well be due to Ca²⁺ release events occurring in and out of the plane of focus. We therefore make no conclusions about the size of the Ca²⁺ release events. It is clear, however, that individual Cl_(Ca) current spikes arise from spatially separate Ca²⁺ release events.