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1.
Figure 5

Figure 5. WT (blue trace) and KO (red trace) fibres demonstrate virtually identical relative time courses of Ca2+ release. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

Average Ca2+ release time courses from designated voltage steps in WT and KO fibres. Inset in bottom two panels shows zoom in of the rising phase of the rate of Ca2+ release peak, demonstrating virtually identical time course of release.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
2.
Figure 7

Figure 7. Temporal comparison of charge movement and Ca2+ release in fibres exhibiting (WT fibres, n= 7) or lacking (KO fibres, n= 7) Qγ. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

Average Ca2+ release flux (top records each panel) and charge movement currents (bottom record each panel) are plotted in time during depolarizing steps to designated voltages. The green curve represents the difference charge current, or Qγ current. The initial dashed line represents the start of the rising phase of Ca2+ release, and the following dashed line represents the peak of release. Note the development of the Qγ current in time with release, and the similar peaks of release and the Qγ current.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
3.
Figure 1

Figure 1. Fluo-4 Ca2+ transients elicited by step depolarizations in a control voltage-clamped FDB fibre. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A, high speed (50 μs per line) confocal line scan x–t images synchronized with pulse protocol drawn above. Time scale is same as in B. The white box in the bottom panel (pulse to +20 mV) represents the region of interest drawn in the centre of the fibre that was averaged to calculate the change in fluorescence intensity. B, time course of change in fluo-4 fluorescence (ΔF) recorded from pulses above. A change in fluo-4 fluorescence was typically first detected at pulses to −30 mV, and saturated with pulses around +20 mV.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
4.
Figure 3

Figure 3. Calculation of voltage-dependent Ca2+ release flux from fluo-4 ΔF transients. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A, experimentally measured fluo-4 ΔF records from Fig. 1B, now expanded in time, with pulse protocol above and time scale at bottom of C. B, free Ca2+ waveforms derived from A and calculated using eqn (2) (see Methods). C, Ca2+ release flux derived from B, calculated using eqn (4) with 20 mm EGTA in the internal solution. EGTA is the major intracellular binding site for released Ca2+, and therefore the rate of Ca2+ binding to EGTA can be used as a first approximation of the rate of Ca2+ release. In B and C, for pulses to −40 and −30 mV, the derivative term from eqn (2) (see Methods) has been set to zero to improve signal to noise.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
5.
Figure 8

Figure 8. Potentiation of Ca2+ release in KO fibres by 4–CMC. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A, average Ca2+ release records of KO fibres (n= 6) before (red traces) and after addition of 100 μm 4–CMC (purple traces). There was a large potentiation of release at intermediate depolarizations that diminished with increasing depolarization. B, R vs. V plot of KO fibres before (red symbols) and after application of 4–CMC (purple symbols) demonstrates a leftward shift in the voltage dependence of release imparted by 4–CMC. C, normalized R vs. V comparing the differences in Ca2+ release isolated from 4–CMC application to KO fibres (KO+4–CMC – KO; dark grey line) with the difference in release between WT and KO fibres (WT – KO; green line). The release augmented by 4–CMC demonstrates a leftward shift, a peak, and then diminishes with increasing depolarization, while the difference between WT and KO fibres is sustained at maximal depolarizations and exhibits a similar voltage dependence to both the WT or KO release curves (dashed line).

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
6.
Figure 9

Figure 9. Potentiation of Ca2+ release with 4–CMC elicits a temporally delayed charge movement component in KO fibres. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A, average charge movement currents in same KO fibres from Fig. 8 before (red trace) and after (purple trace) the addition of 4–CMC, with resulting difference record (KO+4–CMC – KO, dark grey trace). The addition of 4–CMC to KO fibres isolated a temporally delayed ‘hump’ component similar to that seen in WT fibres. B, Q vs. V relationship of KO fibres before (red symbols) and after application of 4–CMC (purple symbols), and the resulting difference charge (dark grey symbols). 4–CMC led to additional charge moved at intermediate depolarizations that diminished in magnitude with increasing depolarization in KO fibres. C, normalized Q vs. V of difference charge isolated by application of 4–CMC to KO fibres (dark grey line) compared to difference charge between WT and KO fibres (green line). Similar to release, the charge augmented by 4–CMC in KO fibres exhibits a left-shifted activation, a peak, and then declines monotonically with increasing depolarization, in contrast to the sustained additional charge isolated between WT and KO fibres.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
7.
Figure 6

Figure 6. Correlation between Qγ and Ca2+ release in WT and KO fibres. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A and B, charge movement currents (left column traces) elicited by steps to −10 mV (top) and +20 mV (bottom) in representative WT and KO fibres demonstrating definite (green traces), slight (black traces), and no Qγ (orange traces). Ca2+ release records (middle column traces) from these same fibres at each voltage. Note the correlation between the presence or absence of Qγ and the peak of Ca2+ release. Right-most traces in B show normalized release records from +20 mV steps, showing that despite variable presence/absence of Qγ, the relative time course of Ca2+ release is virtually identical in WT and KO fibres. C, bar plot showing distribution of WT and KO fibres demonstrating definite, slight and no Qγ, and average peak release in these groups.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
8.
Figure 4

Figure 4. Peak rate of Ca2+ release is decreased at all voltages in S100A1 KO fibres. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A, average records of Ca2+ release flux from WT fibres (n= 9). B, average records of flux from KO fibres (n= 8), plotted on same scale as A to appreciate differences in release amplitude. C, peak release (R) plotted vs. voltage. Continuous lines through the symbols are best fits to a single Boltzmann function (eqn (5)) with parameters of 57 μm ms−1 for Rmax, 8.3 mV for k and −9.5 mV for Vhalf for average WT data, with corresponding values of 32 μm ms−1, 8.4 mV and −10.9 mV for the average KO fibres. The continuous red trace represents the fit to the KO average scaled up to the WT maximum to show virtually identical voltage dependence between WT and KO fibres. D, WT and KO R plotted vs. WT and KO charge movement from companion paper (Prosser et al. 2009). Traces are normalized to WT maximum R and Q to appreciate proportional differences.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
9.
Figure 2

Figure 2. Evaluation of resting fluorescence, evoked fluorescence transients, and charge movement currents during repetitive pulses in fibres exposed to an external solution with or without Cd2+ and Co2+. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A, F0 and ΔF plotted against time of dialysis with 50 μm fluo-4 in the patch pipette. The first four points represent pulses from −80 mV to +10 mV, the second four represent pulses to −60 mV, and the last four repeat pulses to +10 mV. As can be seen the resting fluorescence (F0) steadily increases with time, even with pulses to −60 mV where there is no detectable release, in the presence (F0-Cd2+, Co2+; filled circles), but not in the absence of Cd2+ and Co2+ (F0-Mg2+; open circles). The initial F0 value in both solutions was normalized to the initial value in the Cd2+/Co2+ containing solution to appreciate differences in F0 at the start of the experimental protocol. B, charge movement records from pulses in A show that charge movement currents are preserved with repeated pulsing and time in solution. C, ΔF records from pulses in AF-Cd2+, Co2+; filled squares) show stability of ΔF recordings, despite elevation in F0, with repetitive pulsing. Some slight rundown with time is evident after 25 min.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.
10.
Figure 10

Figure 10. Model for the effects of Ca2+ release on charge movement in WT and S100A1 KO fibres. From: Simultaneous recording of intramembrane charge movement components and calcium release in wild-type and S100A1−/− muscle fibres.

A, proposed effects of Ca2+ release on charge movement in WT fibres. Upon depolarization, charged residues in the α1s subunit of the DHPR (cylinders in DHPR cartoon) act as voltage sensors that transduce its movement (Qβ) through the II–III loop into conformational changes that are sensed by the RyR1 and initiate Ca2+ release (middle cartoon). In WT fibres, S100A1 binding to the cytoplasmic foot of RyR1 enhances channel open time and optimizes release. This optimized Ca2+ release results in an elevation of local Ca2+ in the vicinity of a putative Ca2+ binding site regulating the generation of Qγ by the DHPR (right cartoon). B, proposed effects of the absence of S100A1 on Ca2+ release and charge movement. In this scenario, the lower rate of Ca2+ release (middle cartoon) and the subsequently blunted local Ca2+ gradient, due to the lack of S100A1, simply does not reach the ‘optimal’ level required for the Ca2+-dependent step(s) needed for the generation of Qγ (right cartoon). Qγ may arise from the further movement of the same set of voltage sensors that generate Qβ, or from a parallel set of charges uncoupled to the activation pathway. The function of this additional component of charge movement remains to be determined.

Benjamin L Prosser, et al. J Physiol. 2009 September 15;587(Pt 18):4543-4559.

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