Fluo 4

Tyrode solution contained (in mM) 140 NaCl, 5 KCl, 2.5 CaCl₂, 2 MgCl₂, and 10 HEPES. The standard extracellular solution for voltage-clamp experiments contained (in mM) 140 TEA-methane-sulfonate, 2.5 CaCl₂, 2 MgCl₂, 1 4-aminopyridine, 10 HEPES, and 0.002 tetrodotoxin. For the experiments described in Figs. 1 and 2, the extracellular solution also contained 0.33% DMSO. The standard pipette solution contained (in mM) 120 K-glutamate, 5 Na₂-ATP, 5 Na₂-phosphocreatine, 5.5 MgCl₂, 5 glucose, and 5 HEPES. For measurements of rhod-2 Ca²⁺ transients, it also contained 15 EGTA, 6 CaCl₂, and 0.1 rhod-2. For measurements with fluo-4, isolated muscle fibers were incubated for 30 min in the presence of Tyrode solution containing 10 μM fluo-4 AM. All solutions were adjusted to pH 7.20. The Ringer solution used for muscle force measurements contained (in mM) 140 NaCl, 6 KCl, 3 CaCl₂, 2 MgCl₂, and 10 HEPES, adjusted to pH 7.40.
Probenecid was prepared as a 0.3 M aliquoted stock solution in DMSO and used in the extracellular solution at 0.5, 1, or 2 mM. Carbenoxolone was prepared as a 10 mM stock solution in the extracellular solution and used at 0.1 mM. These concentrations were chosen on the basis of their effectiveness and wide use to block Panx1 channels throughout the literature (e.g., Dahl et al., 2013 (link)). When testing the effect of either probenecid or carbenoxolone using the preincubation protocol (Figs. 1 and 2), fibers were bathed in the drug-containing extracellular solution from the beginning of the intracellular dialysis with the rhod-2-containing solution (i.e., 30 min before taking measurements). The ¹⁰panx1 peptide and the scrambled control peptide (¹⁰panx1SCr) were tested under the same conditions at 200 µM while the P2Y2 antagonist AR-C 118925XX was tested at 10 µM. All chemicals and drugs were purchased from Sigma-Aldrich, except for tetrodotoxin (Alomone Labs), rhod-2 and fluo-4 (Thermo Fisher Scientific), and AR-C 118925XX (TOCRIS—Bio-Techne).
In vitro fluorescence measurements using droplets of a solution containing (in mM) 120 K-glutamate, 10 HEPES, 15 EGTA, 6 CaCl₂, and 0.1 rhod-2, with or without probenecid, showed that fluorescence intensity in the presence of 1 mM probenecid corresponded to 1.09 ± 0.12% (n = 6) the intensity in the absence of probenecid, excluding an interaction of the drug with the dye to explain the effect on resting fluorescence in muscle fibers.

Detection of fluorescence from the Ca²⁺-sensitive dye dialyzed into the fiber’s cytosol was achieved with a Zeiss LSM 800 microscope equipped with a 63× oil immersion objective (numerical aperture 1.4). Standard green and red configurations were used for detection of the fluorescence of fluo-4 and rhod-2, respectively. Voltage-activated fluorescence changes were imaged using the line-scan mode (x,t) of the system. They were expressed as F/F₀ with F₀ as the baseline fluorescence. In experiments designed to follow the effect of probenecid after acute application of the drug, the changes in resting fluorescence F₀ were normalized to F₀ in the control condition at the beginning of the experiment; this value is referred to as _intialF₀. In graphs presenting fluorescence transients, the y-scale bar corresponds to the indicated multiple of F₀. Quantification of the Ca²⁺ release flux (dCa_Tot/dt) underlying the rhod-2 Ca²⁺ transients was performed as previously described (Lefebvre et al., 2011 (link); Kutchukian et al., 2016 (link)). The resting Ca²⁺ concentration was assumed to be 100 nM in all conditions, except for results shown in Figs. 8 and 9, for which the change in resting Ca²⁺ concentration induced by probenecid was implemented in the Ca²⁺ release flux calculation. For this, the resting Ca²⁺ level in the presence of probenecid was calculated from the increase in resting fluorescence, assuming an initial resting [Ca²⁺] level of 0.1 µM, and F_max/F_min and K_D values for rhod-2 of 30 and 1.63 µM, respectively (Sanchez et al., 2021 (link)). The voltage-dependence of the peak Ca²⁺ release flux from each muscle fiber was fitted with a Boltzmann function: $\frac{dC{a}_{Tot}}{dt}=\frac{Max\frac{dC{a}_{Tot}}{dt}}{\left\{1+\exp \left[\left(V_{0.5}-V\right)/k\right]\right\}}\text{,}$ with Max dCa_Tot/dt the maximum Ca²⁺ release flux, V_0.5 the mid-activation voltage, and k the steepness factor.

Cell preparations were suspended in 1 ml loading buffer (DPBS supplemented with 1 mM CaCl₂, 1 mM MgSO₄, and 1% FBS) at a density of 1 × 10⁷ cells per ml and loaded with the calcium indicator dyes Fluo-4 (Thermo Fisher Scientific) and FuraRed (Thermo Fisher Scientific) at 37°C according to manufacturer’s directions. Cells were then washed and stained for flow cytometry at room temperature. Immediately before measurement, cells were incubated at 37°C for 5 min and then transferred to the flow cytometer for continuous monitoring. After obtaining a baseline measurement for 30 s, anti-CD3 antibodies (5 μg/ml) and IgG crosslinking antibodies (25 μg/ml) were added to the sample. After 180 s, ionomycin (1 μg/ml) was added to stimulate maximal cytosolic calcium influx.