Figure 9. Force responses in a fibre stimulated by two APs at different intervals.

Force responses in a rat EDL skinned fibre loaded to maximal capacity and stimulated in the presence of TBQ and 280 μM BAPTA by a single AP (S) or a pair of APs 10 ms, 80 ms or 440 ms apart (labelled D10, D80 and D440). Force responses are expressed relative to the maximum Ca²⁺-activated force and are shown superimposed, aligned with the first (or only) AP positioned at 0 ms. The vertical ticks indicate the timing of the APs. The various stimuli were applied at least 15 s apart (in the order D440, S, D10, D80). The fibre was first stimulated with the pair of APs spaced 440 ms apart (dashed line); the force response to the first AP of this pair elicited a slightly smaller force response (≈1 % of maximum) than did a single AP applied subsequently (≈2 % of maximum force, thin line S), but the peak force reached after the second AP of the pair was substantially higher than that reached with a pair of APs 10 ms apart (D10). When a pair of APs spaced 80 ms apart was applied, the peak force reached was even higher (D80). The total amount of Ca²⁺ released by the single or paired stimuli (calculated from the peak of the response) was 203 μM (S), 288 μM (D10), 311 μM (D80) and 301 μM (D440), though the latter two estimates are considerable underestimates owing to the evident loss of Ca²⁺ from the fibre (and binding to Ca²⁺-Mg²⁺ sites on TnC - see Methods) occurring over the interval between the first and second AP. If two APs applied in rapid succession (e.g. 10 ms apart) had released twice the amount of Ca²⁺ released by a single AP (i.e. 406 μM) it would have produced a force response between ≈85 and 95 % of maximum. The force responses in the figure can be reasonably accounted for by a model in which (1) Ca²⁺ is lost from the fibre with an exponential time constant of ≈600 ms, and (2) the amount of Ca²⁺ released by the second AP in a pair is reduced to ≈38 % of that released by the first for very short intervals (< 10 ms) and recovers with a time constant of ≈120 ms. Thus, if Δt is the time interval (in ms) between the two APs, the released Ca²⁺ (Ca_T) present in the fibre at the peak of the response to the second AP is calculated as Ca_T = 203 μM (e^-Δt/600 + 0.38 + 0.62(1 - e^-Δt/120)), which gives values of 287, 316 and 298 μM for the D10, D80 and D440 cases, respectively.