A Chloride Channel Blocker Prevents Inorganic Phosphate Accumulation and Its Effects in the Sarcoplasmic Reticulum of Frog Permeabilized Skeletal Muscle Fibers

Abstract

In skeletal muscle intense activity is accompanied by an increase in intracellular inorganic phosphate (Pi) due to breakdown of ATP and phoshocreatine. It is widely accepted that Pi enters the sarcoplasmic reticulum (SR) where it precipitates as Ca2+ salt, thus contributing to the impairment of Ca2+ release in muscle fatigue (Fryer et al, J Physiol 1995; Launikonis et al., Biophys J 2005). We studied the effects of increasing Pi concentrations on elementary Ca2+ release events (sparks) in permeabilized fibers. Spark frequency F/F0 increased with [Pi] up to 7-10 mM/l and decreased monotonically reaching 0.1 at [Pi] = 55 mM. These changes were highly correlated with the level of [Ca2+]SR, monitored with Fluo5N or Mag-Fluo4. SR anion channels, probably chloride channels (Laver et al.,J Physiol2001), provide the principal entry path of Pi to the SR. The chloride channel blocker 9-anthracenecarboxylic acid (9AC, at 400 uM) increased spark frequency by about 15% and shifted the Pi effects to slightly higher concentrations. Because 9AC blocks the channel from the SR-luminal side another approach was taken to enhance its action. Fibers were incubated in the presence of 5 mM Mg2+ and 2 mM [9AC] for 30 minutes. There after 9AC was reduced to 400 uM and Mg2+ to 0.4 mM/l. After this protocol, Pi concentrations up to 55 mM/l had no effect on frequency or spark morphology. Correspondingly, [Ca2+]SR-monitoring signals were not affected. These results indicate that chloride channels constitute the main entrance of Pi to the SR, confirm that Pi impairs Ca2+ release by accumulating and precipitating with Ca2+ inside the SR and provide approaches to study these mechanisms quantitatively.Funded by CSIC and NIH.