A, top, charge movement current trace elicited by an 80 ms step to −20 mV demonstrates ON/OFF conservation with minimal ionic interference. Shaded region marks the area of integration to establish Q_ON and Q_OFF, which are quantified in Fig. 2B. Bottom, at steeper depolarizing, 80 ms steps (such as to +10 mV, blue trace) significant ionic contamination leads to an overestimation of Q_OFF, as seen by the open triangles in Fig. 2B. For this purpose we used shorter, 25 ms steps (black trace) to minimize ionic contamination. Integrating these ON and OFF currents from shorter steps showed charge conservation, quantified with the open and filled circles in Fig. 2B. B, Q_ON and Q_OFFvs. voltage relationship in WT fibres. Filled circles are Q_ON established by integrating non-linear current traces during an 80 ms pulse. Open triangles are Q_OFF from the same 80 ms pulse; note overestimation of Q_OFF at steeper depolarizations, a clear indication of ionic contamination of the OFF records. Open circles are Q_OFF during a 25 ms pulse. Based on the above we averaged Q_ON and Q_OFF from a 25 ms pulse to establish ‘Q’ at steeper depolarizations. C, Q_ONvs. Q_OFF relationship demonstrates charge conservation when using above criteria, using Q_ON values from 80 ms pulses, and Q_OFF values from 80 ms pulses for steps to −80 through −10 mV and from 25 ms pulses for steps to 0 through +20 mV. The data were fitted with a linear function with a slope of 1.09 and R²= 0.986. D, sum response (black) to opposite polarity 20 mV voltage steps (red and grey traces) from a holding potential (HP) of −120 mV indicates negligible non-linearity of membrane properties over voltage range used to obtain leak templates with the P/4 leak subtraction protocol. E, non-linear capacitive current elicited by a test pulse of 80 mV amplitude (from −80 mV to 0 mV; top current trace) using P/4 method. Below is the amplitude of the P/4 leak template (Control, middle current trace) and full leak template (Control × 4; bottom current trace) for comparison.

A, KO data was well fitted by a single Boltzmann function with parameters of Q_max= 37.3 nC μF⁻¹, V_half=−25.8 mV and k= 12.6; n= 11 fibres. A single Boltzmann function did not accurately fit the WT data. B, based on the second, temporally delayed component of charge movement seen in WT, but not KO fibres, a double Boltzmann fit was applied to the WT data using fixed KO parameters for the first Boltzmann function and free values for the second component. This double Boltzmann function very accurately fitted the WT data. The second component, analogous to the theoretically derived difference component, had Boltzmann parameters of Q_max= 15.2 nC μF⁻¹, V_half=−13.0 mV and k= 4.8; n= 12 fibres. A curve with these parameters is simulated in the WT – KO (green) trace.

A, exposure of control fibres to 40 μm dantrolene for 3 min suppressed the action potential evoked Ca²⁺ transient by about 35%, measured with high speed confocal microscopy and Fluo4-AM Ca²⁺ indicator dye. This reduction in the Ca²⁺ transient recapitulates the suppression seen in KO fibres (Prosser et al. 2007). The inset shows the normalized peak values of the Ca²⁺ transient (reported as ΔF/F₀±s.e.m.) as a function of time of exposure to dantrolene. B, charge movement currents from a control fibre (left), before (blue trace) and after (brown trace) 3 min exposure to 40 μm dantrolene in the bath, and the resulting difference current (orange trace). As can be seen, the RyR inhibitor dantrolene suppressed a temporally delayed hump component at intermediate voltages, without affecting the initial component of charge movement currents, similar to the difference current between WT and KO fibres. On the right of the figure are charge movement currents elicited by the same voltage steps in 4 KO fibres exposed to the same concentration of dantrolene (red traces). There was no suppression of charge movement in any of these fibres. C, Q–V relationship of 6 control fibres before and after treatment with dantrolene. The steeply voltage-dependent difference component (Ctrl – Dantro; orange Q–V) was fitted to a single Boltzmann function to −10 mV and displayed parameters Q_max= 6.3 nC μF⁻¹, V_half=−21.5 mV and k= 3.0 mV. At higher voltages the difference component decreased monotonically with voltage and was therefore fitted to a linear function.

A, the average KO recording was subtracted from each individual WT current trace, and the resulting difference trace was integrated to establish Q during the step. The green trace represents the fit to the average difference Q–V, with Boltzmann parameters of Q_max= 12.1 nC μF⁻¹, V_half=−17.7 mV and k= 5, similar to those from the theoretically isolated difference component seen in Fig. 3B. The black trace is the slightly steeper curve generated from averaging the Boltzmann parameters of the individual fits to the difference Q–Vs. B, average difference (WT – KO) Q–V plotted in scale with WT and KO Q–V relationships from Fig. 3B. C, normalized Q–V relationships of fits from panel B.

A, charge movement currents recorded using an external solution lacking Cd²⁺ and Co²⁺ exhibit a temporally delayed component and similar current kinetics to fibres bathed in our standard control solution. Ca²⁺ was reduced to 0.5 mm and 10 mm Mg²⁺ was added to the external solution to block inward ionic components. B, fibres bathed in control external solution (open circles, dashed line) demonstrate a very similar Q vs. V relationship to fibres bathed in Cd²⁺ and Co²⁺ free external solution (filled circles, continuous line). C, charge movement currents recorded using an external solution supplemented with 0.3 mm La³⁺ exhibit rapid current kinetics and no clear temporally delayed ‘hump’ component. D, fibres bathed in our control external solution supplemented with La³⁺ demonstrate decreased maximal charge moved and a rightward shift in the voltage dependence of charge movement when compared to fibres bathed in our standard external solution. E, charge movement currents recorded in control and La³⁺-treated fibres in response to depolarizing pulses to −10 mV of increasing duration (2 ms increments) to evaluate charge movement conservation. F, Q_ON or Q_OFFvs. pulse duration for the complete set of pulses of corresponding traces illustrated in E. The normalized charge–pulse duration relationships for both Q_ON and Q_OFF are fitted to single exponential functions (continuous lines). The calculated time constant for charge development in the Cd²⁺/Co²⁺ exposed fibre was 13 ms, compared to 2.4 ms for the fibre exposed to La³⁺.

A and B, representative charge movement current traces elicited by 80 ms depolarizing steps from a holding potential of −80 mV. In all traces dotted line represents the zero baseline. C, zoom in of the ON current of superimposed WT and KO current traces demonstrates suppression of temporally delayed ‘hump’ component (Q_γ) in KO fibres. Typically WT fibres developed a noticeable hump around −20 mV, whereas no distinct second component was seen in KO fibres. Colour-coordinated hash marks on current traces represent integration times to establish ‘Q’ for WT and KO fibres, set to 20, 25, or 30 ms depending on the appearance of a temporally delayed component. A single hash mark implies that the same time to integration was used for both traces.

A, charge movement currents recorded in response to depolarizing pulses to −10 and +20 mV of increasing duration (2 ms increments) to evaluate charge movement conservation. B, Q_ON or Q_OFFvs. pulse duration for the complete set of pulses of corresponding traces illustrated in A. The normalized charge-pulse duration relationships for both Q_ON and Q_OFF are fitted to single exponential functions (continuous lines). The calculated Q_ON time constants were 12.7 ms for pulses to −10 mV, and 2.3 ms for pulses to +20 mV.

Note virtually identical charge movement recordings at −40 and −30 mV steps, followed by development of delayed, steeply voltage dependent hump component (Q_γ) suppressed in KO fibres. At steeper depolarizations this component shifts back earlier in time and saturates in area.

A, KO fibres dialysed, via the patch pipette, with 10 μm Alexa-488-S100A1 show restoration of Q–V relationship to WT levels. S100A1 protein concentration in KO fibres was estimated using confocal fluorescence microscopy and averaged 2.8 ± 0.21 μm at the time of recordings. B, zoom in of ON current traces from KO AVG and representative KO + S100A1 fibre show that kinetic properties of charge movement currents can be restored in KO fibres dialysed with S100A1, specifically the temporally delayed Q_γ component.

A, representative Western blots with anti-α₁s from lysates of WT and S100A1^−/− muscle show similar DHPR expression, with GAPDH used as a loading control. The bar graph is the result of triplicate experiments in WT and KO muscle, showing that the expression of the DHPR is not altered with the ablation of S100A1. Data are presented as a percentage of the average WT DHPR expression, normalized to the GAPDH signal. B, representative macroscopic Ca²⁺ current recordings of WT (blue) and KO (red) fibres. In these experiments Cd²⁺ and Co²⁺ were eliminated from the external solution and Ca²⁺ was increased to 2 mm. Vertical dashed lines illustrate the time used to measure peak current values to create the I–V plots. C, top, I–V relationship of average data from WT and KO fibres reveals no significant differences in L-type channel current density and voltage dependence. The data are fitted to a Boltzmann-Ohm function. At bottom is the activation profile of WT and KO fibres derived from parameters of a Boltzmann-Ohm function. These results suggest that suppressed charge movement in KO fibres is not linked to changes in L-type Ca²⁺ channel expression and voltage-dependent activation.

A, charge movement in WT fibres consisted of two components, the first eliciting a current with an initial rise to peak and simple decay, similar to Q_β (red), and the second, a temporally delayed, steeply voltage dependent component, similar to Q_γ (green). Q_γ was suppressed in fibres lacking S100A1, consistent with decreased RyR-mediated Ca²⁺ release. B, comparison of the Q–V relationships of WT (blue; total charge movement) and KO (red; Q_β) fibres, along with difference Q–V plot (WT−KO, green; Q_γ). Transient application of S100A1 restored Q_γ, suggesting that this suppression of charge movement is not due to developmental or compensatory effects of S100A1 KO (KO+S100A1, black). Inhibition of RyR with dantrolene also suppressed a temporally delayed, steeply voltage dependent component similar to Q_γ (Ctrl – Dantro, orange), suggesting this component may be a product of crosstalk between the voltage sensor of EC coupling, DHPR, and the mechanically coupled SR Ca²⁺ release channel, RyR. All curves are Boltzmann fits to average data.