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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.

In skeletal muscle, extracellular ATP arises locally from released synaptic vesicles and broadly from active muscle fibers. We examined the direct effect of ATP on developed, innervated mammalian skeletal muscle (ex vivo rat levator auris longus) using measurements of individual muscle fibers with two intracellular microelectrodes. Near the neuromuscular junction (NMJ), 20μM ATP prolonged the decay of miniature endplate potentials (mEPP tau) by 31 ± 7.5% and extended the membrane potential responses induced by step current pulses (ΔEm(Ipulse)) by 59 ± 3.0%. These responses correlated with an increased input resistance (Rin) of 31 ± 2.0%. Analogous increases in non-synaptic regions reveal that ATP acts throughout the muscle fiber. In contrast, 50μM adenosine, a well-characterized metabolite of ATP, induced no apparent increase in mEPP tau, ΔEm(Ipulse) or Rin. Applying established pharmacology showed that the ATP receptor mediating these effects is likely the G-protein-coupled P2Y1, since 20μM ATPγS and ADPβS (slowly hydrolysable analogs of ATP and ADP) and 10μM 2-methylthioadenosine-5′-O-diphosphate (2MeSADP) mirrored the effect of ATP. Furthermore, 20μM MRS2179 blocked activation by 20μM ADPβS. The significant effect of ATP on Rin was presumably achieved by closing chloride channels, which maintain the largest conductance of resting muscle. This was confirmed with the chloride channel blocker anthracene-9-carboxylic acid (200-500μM), which mimicked ATP and prevented additional increases by 20μM ATPγS. This ATP response appears unique to mammals, as 50μM ATP induced no analogous increase in the archetypical frog NMJ. Our work outlines a novel mechanism by which physiological levels of ATP regulate synaptic transmission and dramatically alter the resting membrane properties of mammalian skeletal muscle. This has potential implications for the physiology of muscle excitability and fatigue, and the pathophysiology of Thomsen and Becker myotonias.

Preconditioning is a powerful form of cardioprotection whereby a brief ischemic episode, or a brief exposure to drugs such as volatile anesthetics, can protect the myocardium from a subsequent prolonged ischemia. It triggers an intracellular signaling cascade that leads to the delay in the opening of the mitochondrial permeability transition pore (mPTP). Depolarization of the inner membrane of mitochondria (IMM) can delay mPTP opening. Ion channels have been identified on the IMM that may play key roles in this depolarization. Yet their molecular identities and detailed electrophysiological characterizations have been elusive. In the present study, we recorded ion channel activities on the IMM isolated from guinea pig hearts. Mitoplasts (mitochondria sans the outer membrane) were formed by incubating mitochondria in a hypotonic buffer. The inside-out configuration of the patch clamp technique was utilized. We have identified a channel with a primary conductance of 109 ± 5 pS (n=9) in equimolar 150 mM KCl. The channel exhibited voltage-dependent behavior, with activity being more prominent at positive membrane potentials. When the 150 mM KCl bath solution that corresponded to the mitochondrial matrix side was replaced with 150 mM K-glutamate, channel activity was abolished. When TEA-Cl substituted for KCl, channel activity was not significantly affected. These results suggested an anion channel permeable to chloride. This was confirmed by DIDS (100 μM), a chloride channel blocker, which abolished channel activity. However, bongkrekic acid (100 nM), a specific inhibitor of the mitochondrial adenine nucleotide translocase, failed to inhibit channel activity. In addition, the presence of 2 mM Mg2+ in the buffer solution, a concentration that blocks IMAC, the inner membrane anion channel, did not prevent channel opening. Experiments are currently underway to further characterize and identify this anion channel on the IMM.