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.
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 (
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.
