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. 2021 Jan;599(1):157-170.
doi: 10.1113/JP279917. Epub 2020 Oct 19.

A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction

Juan J Ferreira  1 , Germán Pequera  1 , Bradley S Launikonis  2 , Eduardo Ríos  3 , Gustavo Brum  1
Affiliations

A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction

Juan J Ferreira et al. J Physiol. 2021 Jan.

Abstract

Key points: Accumulation of inorganic phosphate (Pi ) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of Pi into SR are fully established. We showed that Pi promoted Ca2+ release at concentrations below 10 mm and decreased it at higher concentrations. This decrease correlated well with that of [Ca2+ ]SR . Pre-treatment of permeabilized myofibres with 2 mm Cl- channel blocker 9-anthracenecarboxylic acid (9AC) inhibited both effects of Pi . The biphasic dependence of Ca2+ release on [Pi ] is explained by a direct effect of Pi acting on the SR Ca2+ release channel, combined with the intra-SR precipitation of Ca2+ salts. The effects of 9AC demonstrate that Pi enters the SR via a Cl- pathway of an as-yet-undefined molecular nature.

Abstract: Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (Pi ). Pi diffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca2+ salts, thus contributing to the impairment of Ca2+ release. Information on the Pi entrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca2+ spark frequency is correlated with the Ca2+ load of the SR and can be used to monitor this variable. We studied the effects of Pi on Ca2+ sparks in permeabilized fibres of the frog. Relative event frequency (f/fref ) rose with increasing [Pi ], reaching 2.54 ± 1.6 at 5 mm, and then decreased monotonically, reaching 0.09 ± 0.03 at [Pi ] = 80 mm. Measurement of [Ca2+ ]SR confirmed a decrease correlated with spark frequency at high [Pi ]. A large [Ca2+ ]SR surge was observed upon Pi removal. Anion channels are a putative path for Pi into the SR. We tested the effect of the chloride channel blocker 9-anthracenecarboxylic acid (9AC) on Pi entrance. 9AC (400 µm) applied to the cytoplasm produced a non-significant increase in spark frequency and reduced the Pi effects on this parameter. Fibre treatment with 2 mm 9AC in the presence of high cytoplasmic Mg2+ suppressed the effects of Pi on [Ca2+ ]SR and spark frequency up to 55 mm [Pi ]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR, thus contributing to myogenic fatigue.

Keywords: 9-anthracenecarboxylic acid; chloride channel; fatigue; inorganic phosphate; sarcoplasmic reticulum; skeletal muscle.

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Conflict of interest statement

Competing Interests

None of the authors has any conflicts of interest to disclose.

Figures

Figure 1.
Figure 1.. High Pi reduces spark frequency.
A, representative xt images in a typical experiment. Reference images are shown before and after perfusing the fibre with solutions containing different Pi concentrations. Some sparks are identified by an arrow. B, summary of the same experiment, showing Ca2+ spark frequency at various [Pi]. Each bar represents average frequency in 10 consecutive images. The fibre was exposed to three Pi concentrations, intercalated with reference solution as indicated by the horizontal bars. Symbols represent averages in each solution. Error bars represent S.D.
Figure 2.
Figure 2.. Effect of Pi on Ca2+ spark frequency.
Spark frequency was measured at different [Pi] as described in Methods and normalized to the value in reference solution. Bars represent mean value, circles represent individual measurements. Note opposite effects of Pi at low or high concentrations. Other spark morphology parameters are listed in Table I. The effect is statistically significant for [Pi] 2, 5, 7.5, 30, 55 and 80 mM. Number of fibres in each concentration is indicated in table I. Error bars correspond to S.D.
Figure 3.
Figure 3.. The effect of Pi on [Ca2+]SR.
Mag-Indo-1 [Ca2+]SR measurements in two permeabilized fibres exposed to different [Pi]. At time 0 the fibres were perfused with a 100 nM Ca2+ reference glutamate solution. In A the reference solution was changed to a [Pi] = 20 mM and thereafter washed with a 50 mM EGTA solution with 0 Ca2+. In B a similar experiment as in A but [Pi] was 80 mM. Insets A and B show [Ca2+]SR time course after changing to the EGTA solution in an expanded time scale. C and D show two examples of [Ca2+]SR recorded in permeabilized muscle fibres exposed sequentially to glutamate, 80 mM SO42- and glutamate. Note the prominent Ca2+ surge upon returning to the glutamate reference solution. In all cases the measurements correspond to average signals recorded from the whole fibre.
Figure 4.
Figure 4.. Pi reduces free Ca2+ in the SR.
A. Fluorescence images in a Fluo-5N-loaded fibre. A permeabilized fibre was exposed to 50 mM Pi or 10 mM caffeine (horizontal bars). Pi and caffeine were removed by perfusion of reference glutamate solution. The typical pattern corresponding to SR is depicted in white on each image (see text). Arrows indicate the sequence of the solution changes. B. Average fluorescence intensity measured in sequentially obtained images is plotted as a function of time. Grey bars correspond to power of the fundamental Fourier component of the profiles represented on the images in A.
Figure 5.
Figure 5.. SR fluorescence as a function of [Pi] in the cytosol.
[Ca2+]SR-related fluorescence in Fluo-5N-loaded fibres, evaluated by subtraction of the fluorescence remaining in 10 mM caffeine, is plotted as a function of Pi. Number of fibres at each concentration tested is indicated in brackets, total N was 17 obtained from 9 animals. Bars correspond to mean values, error bars to ± S.D; circles represent individual measurements. Asterisks indicate significant differences (P<0.05).
Figure 6.
Figure 6.. Ca2+ spark frequency and [Ca2+]SR.
A, [Ca2+]SR-related fluorescence measured with Fluo-5N (a), and sparks recorded with Rhod-2 in the same fibre (b) . Fluorescence images and sparks were recorded alternately, first in K-glutamate solution (reference), then in 50 mM Pi, after washout of Pi and after perfusion of 10 mM caffeine. B, Fluorescence profile plotted from the same region of the fibre (rectangle in first image) in the four conditions: a, reference; b, 50 mM Pi; c, washout and d, 10 mM caffeine. C, normalized Fluo-5N fluorescence after subtraction of fluorescence in 10 mM caffeine (symbols) and normalized spark frequency (bars) averages obtained from similar experiments in four fibres. Error bars correspond to ± S.D. Significant differences from reference (P<0.05) are indicated by the stars for sparks and the asterisk for fluorescence.
Figure 7.
Figure 7.. Effect of chloride channel blocker 9AC on spark frequency.
Plot of mean relative spark frequency as a function of 9AC concentration. Bars correspond to mean values, symbols to individual measurements. Numbers in brackets correspond to the number, n, of fibres studied for the corresponding concentration. Reference value was obtained from n = 10. Error bars correspond to ±S.D. Differences are not statistically significant (One way ANOVA, P=0.442).
Figure 8.
Figure 8.. 9AC suppresses Pi potentiation in the low concentration range but reduces Pi effects at higher [Pi] concentrations.
Normalized mean spark frequency as a function of [Pi] (filled bars, same data as in figure 2) compared with data for the same [Pi] in the presence of 400 μM 9AC (hatched bars). Numbers in brackets correspond to n at each [Pi] for the fibres in 9AC; symbols correspond to individual measurements in 9AC. Asterisks indicate significant differences at the same [Pi] with or without 9AC, error bars correspond to ±S.D.
Figure 9.
Figure 9.. Simultaneous exposure to 9AC and high [Pi].
A, mean relative frequency of sparks vs. [Pi] in the absence of 9AC (filled bars, data from table I) and after incubation in 2 mM 9AC (hatched bars). B, Mean [Ca2+]SR-related fluorescence vs. [Pi] (filled bars, same data from figure 5) and after incubation with 2 mM 9AC (hatched bars). Symbols in A and B correspond to individual measurements; all differences in A and B at the same [Pi] are significant, error bars correspond to ±S.D. Number of fibres in each condition is indicated in brackets. C, normalized fluorescence vs. spark frequency in the same fibres. Solid line: best fit y= b*x + a. b=0.898±0.065, a=0.067±0.048 (value ± S.E. of fit). Standard error of estimate = 0.0676. Dotted lines 95% confidence interval. r=0.990. Error bars correspond to ± S.D.
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