Probenecid affects muscle Ca²⁺ homeostasis and contraction independently from pannexin channel block

Tight control of skeletal muscle contractile activation is secured by the excitation–contraction (EC) coupling protein complex, a molecular machinery allowing the plasma membrane voltage to control the activity of the ryanodine receptor Ca²⁺ release channel in the sarcoplasmic reticulum (SR) membrane. This machinery has been shown to be intimately linked to the plasma membrane protein pannexin-1 (Panx1). We investigated whether the prescription drug probenecid, a widely used Panx1 blocker, affects Ca²⁺ signaling, EC coupling, and muscle force. The effect of probenecid was tested on membrane current, resting Ca²⁺, and SR Ca²⁺ release in isolated mouse muscle fibers, using a combination of whole-cell voltage-clamp and Ca²⁺ imaging, and on electrically triggered contraction of isolated muscles. Probenecid (1 mM) induces SR Ca²⁺ leak at rest and reduces peak voltage-activated SR Ca²⁺ release and contractile force by 40%. Carbenoxolone, another Panx1 blocker, also reduces Ca²⁺ release, but neither a Panx1 channel inhibitory peptide nor a purinergic antagonist affected Ca²⁺ release, suggesting that probenecid and carbenoxolone do not act through inhibition of Panx1-mediated ATP release and consequently altered purinergic signaling. Probenecid may act by altering Panx1 interaction with the EC coupling machinery, yet the implication of another molecular target cannot be excluded. Since probenecid has been used both in the clinic and as a masking agent for doping in sports, these results should encourage evaluation of possible effects on muscle function in treated individuals. In addition, they also raise the question of whether probenecid-induced altered Ca²⁺ homeostasis may be shared by other tissues.