Calcium buffering properties of sarcoplasmic reticulum and calcium-induced Ca(2+) release during the quasi-steady level of release in twitch fibers from frog skeletal muscle

Abstract

Experiments were performed to characterize the properties of the intrinsic Ca(2+) buffers in the sarcoplasmic reticulum (SR) of cut fibers from frog twitch muscle. The concentrations of total and free calcium ions within the SR ([Ca(T)](SR) and [Ca(2+)](SR)) were measured, respectively, with the EGTA/phenol red method and tetramethylmurexide (a low affinity Ca(2+) indicator). Results indicate SR Ca(2+) buffering was consistent with a single cooperative-binding component or a combination of a cooperative-binding component and a linear binding component accounting for 20% or less of the bound Ca(2+). Under the assumption of a single cooperative-binding component, the most likely resting values of [Ca(2+)](SR) and [Ca(T)](SR) are 0.67 and 17.1 mM, respectively, and the dissociation constant, Hill coefficient, and concentration of the Ca-binding sites are 0.78 mM, 3.0, and 44 mM, respectively. This information can be used to calculate a variable proportional to the Ca(2+) permeability of the SR, namely d[Ca(T)](SR)/dt ÷ [Ca(2+)](SR) (denoted release permeability), in experiments in which only [Ca(T)](SR) or [Ca(2+)](SR) is measured. In response to a voltage-clamp step to -20 mV at 15°C, the release permeability reaches an early peak followed by a rapid decline to a quasi-steady level that lasts ~50 ms, followed by a slower decline during which the release permeability decreases by at least threefold. During the quasi-steady level of release, the release amplitude is 3.3-fold greater than expected from voltage activation alone, a result consistent with the recruitment by Ca-induced Ca(2+) release of 2.3 SR Ca(2+) release channels neighboring each channel activated by its associated voltage sensor. Release permeability at -60 mV increases as [Ca(T)](SR) decreases from its resting physiological level to ~0.1 of this level. This result argues against a release termination mechanism proposed in mammalian muscle fibers in which a luminal sensor of [Ca(2+)](SR) inhibits release when [Ca(T)](SR) declines to a low level.

Figure 1.

Phenol red and TMX absorbance signals and cooperative Ca²⁺–calsequestrin binding reaction. (A) The top trace shows voltage measured in the V1 end pool. The top pair of traces shows the ΔA_PR and ΔA_TMX signals determined from absorbance signals measured at the same time as the voltage trace. This same ΔA_TMX signal (without a scale bar) is shown with its associated [Ca²⁺]_SR signal in the next pair of traces (from top to bottom). The [Ca²⁺]_SR signal was determined from the ΔA_TMX signal using Eq. 7 with [Ca²⁺]_SR,R = 0.6656 mM, the ratio [Ca²⁺]_SR,R/K_app = 0.256 (the [Ca²⁺]_SR,R and K_app values are those for case 1 in Table 1). The next trace shows the ΔpH signal determined from the ΔA_PR signal. In the bottom pair of traces, the [Ca²⁺]_SR signal is the same one shown above, and the [Ca_T]_SR signal was determined from the ΔpH signal. (See text for details). (B) The black dots plot values of [Ca_T]_SR versus [Ca²⁺]_SR from the signals in A. The blue line plots [Ca²⁺]_SR, and the red curve plots the sum of [Ca²⁺]_SR and [CaTMX]. The [CaTMX] component of this sum was determined from the ΔA_TMX signal using Eq. 6. (C) The black dots plot the estimated concentration of Ca in the SR bound to intrinsic buffers, [CaIB]; they are given by [Ca_T]_SR − [Ca²⁺]_SR − [CaTMX]. The red curve is a least-squares best fit of a cooperative binding function (Eqs. 9 and 10) to the [CaIB] data. A description of the fit and the best-fit parameters is given in the text. Fiber reference, 724011; time after saponin treatment, 77 min; time after adding dye, 37 min; sarcomere length, 3.9 µm; fiber diameter, 84 µm; holding current, −50 nA; 17.0°C.

Figure 2.

Method 2 for assessing Ca binding to intrinsic buffers in the SR. Results in this figure are from the same multi-pulse stimulation shown in Fig. S3, which has the same format as Fig. 1. (A) The top trace is the voltage signal measured in the V1 end pool. The stimulation protocol started with a 400-ms pulse to −60 mV, followed by three 200-ms pulses to −45 mV, and then a 1,000-ms pulse to −20 mV. The durations of the returns to the holding potential of −90 mV between pulses was 1,000 ms, except for the 600-ms period between the final pulse to −45 mV and the pulse to −20 mV. In the top set of traces, the blue trace is the same [Ca²⁺]_SR signal shown in Fig. S3, and the green trace is the [CaTMX] signal. The green trace mostly covers the blue trace because they have approximately the same amplitude. The [Ca_T]_SR signal is very similar to the [Ca_T]_SR,1 signal in Fig. S3, except that the final slope and value were constrained to be the same as those for the [Ca_T]_SR,model signal (see text and Section 4 of the supplemental text for how it is determined). The [Ca_T]_SR,model signal is the least-squares best-fit estimate of [Ca_T]_SR determined from the [Ca²⁺]_SR signal using the cooperative model of Ca binding to intrinsic buffers given by Eqs. 9–11 in Materials and methods. The best-fit values of the Ca-binding parameters for the [Ca_T]_SR,model signal are those for case 1 for fiber reference 211021 given in columns 2–4 of Table 1. (B) The curves show Ca-binding curves (f_CB vs. [Ca²⁺]_SR,R) corresponding to different sets of Ca-binding parameters, with the values for each of the axes normalized by the corresponding resting value. The different sets of parameters are given by the four cases in Table 1.

Figure 3.

Release permeability significantly decreases with decreasing [Ca²⁺]_SR. (A) The top trace is voltage measured in the V1 end pool in response to an 800-ms pulse to −20 mV. The next trace (from top to bottom) is the associated phenol red absorbance change (ΔA_PR). In the pair of traces below the ΔA_PR signal, the black [Ca_T]_SR trace was determined from the ΔpH signal (not depicted). The blue [Ca_T]_SR trace was determined from the ΔpH signal corrected for proton loss to give a constant final level. In the next pair of traces, the [Ca²⁺]_SR signal (black trace) was derived from the [Ca_T]_SR signal with Eq. 13 using the brute-force method described in the text using the average values for K_CB, n, and [CaIB]_max for case 1 in Table 1. The blue trace in this pair is the same blue [Ca_T]_SR signal in the previous pair, normalized to have the same height as the [Ca²⁺]_SR trace. The next trace shows the rate of SR Ca²⁺ release (−d[Ca_T]_SR/dt) plotted on an expanded time scale. The next trace shows the DCRR determined from the d[Ca_T]_SR/dt and [Ca_T]_SR signals with Eq. 4 in Materials and methods. The last trace is the release permeability signal determined from the d[Ca_T]_SR/dt and [Ca²⁺]_SR signals with Eq. 15. (B) The voltage trace at the top is a portion of the same voltage trace shown in A plotted on an expanded time scale. In the four pairs of traces, the black trace is the same release permeability signal shown in A (i.e., that corresponding to the case 1 set of parameters in Table 1), and each of the red traces is a release permeability signal determined with a different set of assumptions. All release permeability traces were normalized to have the same peak heights. For the top two pairs of traces, the red traces were obtained with the average values for K_CB, n, and [CaIB]_max for cases 2 and 3 in Table 1, as indicated. For the third and fourth pairs of traces, the red signals are those obtained with the linear binding parameter f_LB = 0.1 and 0.2, respectively, as indicated. The peak values of the release permeability signal for cases 1, 2, and 3 and for f_LB = 0.1 and 0.2 were 0.556, 0.860, 0.219, 0.518, and 0.554 ms⁻¹, respectively. See text for details concerning the traces and the various markers and line segments. Fiber reference, 908991; time after saponin treatment, 74 min; time after adding dye, 63 min; sarcomere length, 3.5 µm; fiber diameter, 106 µm; holding current, −18 nA; 14.5°C; concentration of phenol red at optical site, 1.20 mM; estimated resting pH, 6.82; C_app, 0.0172 µF.

Figure 4.

DCRR versus [Ca_T]_SR and release permeability versus [Ca²⁺]_SR. (A) DCRR versus [Ca_T]_SR obtained from the DCRR and [Ca_T]_SR versus time signals in Fig. 3 A. (B) Release permeability versus [Ca²⁺]_SR also determined from the corresponding signals in Fig. 3 A. Details for the experiment in A and B are given in the legend of Fig. 3. Details for the experiment in C and D are given at the end of this legend. (C) The red circles plot versus [Ca_T]_SR the values at the peaks of DCRR signals in response to voltage-clamp steps to −45 mV. Each point in this case is from a different stimulation done 5 min apart. The variation of [Ca_T]_SR in this case was achieved by varying the resting SR Ca load, [Ca_T]_SR,R. Each black circle plots the steady level of DCRR reached during a 300-ms voltage-clamp step to −60 mV measured 400 ms before the pulse to −45 mV. (D) Release permeability versus [Ca²⁺]_SR obtained from the data in C. Values for [Ca²⁺]_SR on the abscissa were obtained by converting the values of [Ca_T]_SR in A with Eq. 13 using the average values for K_CB, n, and [CaIB]_max for case 1 in Table 1. Each release permeability value was obtained by multiplying the corresponding DCRR value in C by the ratio [Ca_T]_SR/[Ca²⁺]_SR for that point. The fiber reference for the experiment in C and D is 510971; details for this experiment are given in the legends of Fig. 2 in Pape and Carrier (1998) and Fig. 5 of Pape et al. (2002). The internal and external solutions were the same as those given in Materials and methods for the two points with the highest concentrations of Ca. After these two points were obtained, the internal solution in the end pools was exchanged for the same Cs internal solution except that there was no Ca present and all of the other points were obtained with this Ca-free internal solution (see Pape and Carrier, 1998).