Probenecid, a gout remedy, inhibits pannexin 1 channels

Abstract

Probenecid is a well-established drug for the treatment of gout and is thought to act on an organic anion transporter, thereby affecting uric acid excretion in the kidney by blocking urate reuptake. Probenecid also has been shown to affect ATP release, leading to the suggestion that ATP release involves an organic anion transporter. Other pharmacological evidence and the observation of dye uptake, however, suggest that the nonvesicular release of ATP is mediated by large membrane channels, with pannexin 1 being a prominent candidate. In the present study we show that probenecid inhibited currents mediated by pannexin 1 channels in the same concentration range as observed for inhibition of transport processes. Probenecid did not affect channels formed by connexins. Thus probenecid allows for discrimination between channels formed by connexins and pannexins.connexin; transport; erythrocyte; ATP release PROBENECID HAS BEEN USED for decades for the treatment of gout. The mechanism of action of the drug is inhibition of a renal tubular transporter, thereby facilitating the excretion of the disease causative uric acid by blocking reuptake (5, 26, 37). Probenecid-sensitive transporters are widespread and are even found in plants (30,31,44,52,56). The inhibition of the transporter by probenecid is also exploited clinically to increase the effective concentrations of antibiotics, chemotherapeutics, and other medications.The inhibitory effect of probenecid on organic anion transporters is well established, and the effect is thought to be so specific that the drug is often used as a diagnostic tool, i.e., its effect is typically interpreted as an involvement of an anion transporter in the tested parameter. Accordingly, block of cAMP or cGMP release from erythrocytes (18, 25), ATP release from glia cells (1, 17), and block of dye loss in various cell types (20,21,23) by probenecid have been presented as evidence for a role of transporters in these phenomena. However, alternative pathways for the transit of these molecules across the plasma membrane have to be considered. Besides the well-documented vesicular release of ATP, a parallel release through membrane channels must exist, because the release is attenuated by drugs that do not interfere with vesicular release but affect gap junction proteins and because ATP release in several cell types is associated with uptake of dye from the extracellular medium (13).Special attention has to be given to pannexin 1 as an ATP release channel because of the specific properties of pannexin 1 channels and because of the expression pattern of pannexin 1 (2, 16, 28, 32-34, 55, 59). Pannexin 1 channels are highly permeable to ATP and to dyes typically used for dye flux measurements through gap junction channels. These dyes are in the same size range as the Ca 2ϩ indicator dyes whose loss is attenuated by probenecid. Pannexin 1 channels also are mechanosensitive, consistent with a role in Ca 2ϩ wave initiation. Expression of pannexin 1 is found in cells exhibiting ATP release, including er...

Hemolysis in human erythrocytes by Clostridium perfringens epsilon toxin requires activation of P2 receptors

“…Pannexin1 is expressed in human RBCs and has recently been suggested to operate as the ATP release channel in erythrocytes [12,28,29]. To assess the effect of pannexin 1 on ATP release, carbenoxolone and mefloquine were used as antagonists for pannexin 1 [30,31]. The results showed that inhibition of pannexin 1 did not decrease the release of ATP (Figure 6(e,f)), indicating that ATP was not released via pannexin1.…”

Alanine substitution scanning of pannexin1 reveals amino acid residues mediating ATP sensitivity

“…On the other hand, ATP released into narrow extracellular spaces as in the nervous system would be subject to the feedback inhibition. Thus activation of Panx1 channels by ATP through purinergic receptors would not necessarily result in cell death, a phenomenon nevertheless observed upon massive and prolonged stimulation and blockable by the Panx1 channel inhibitor probenecid [10,16].…”

“…Also, the pharmacology of Panx1 channels matches that of ATP release. Almost all compounds that are reported to inhibit non-vesicular ATP release are effective on the Panx1 channel including connexin mimetic peptides and the inhibitor of the organic anion transporter probenecid [15,16]. Finally, the Panx1 channel is controlled by ATP via a negative feedback loop [17].…”

Permeation of Fluorophore-Conjugated Phalloidin into Live Hair Cells of the Inner Ear Is Modulated by P2Y Receptors

“…In a pharmacological test for the involvement of OATs or OATPs in the permeation of phalloidin in the utricle, we used probenecid, which inhibits many OATs and OATPs (Sugiyama et al 2001;Yasui-Furukori et al 2005;Jorgensen et al 2007;Silverman et al 2008). Consistent with a potential involvement for OATPs or OATs in the permeation of phalloidin, probenecid significantly inhibited phalloidin permeation and decreased fluorescence by 33.9 % (Student's t-test, t(16)=3.472, p=0.0003, Fig.…”

“…Besides OATs and OATPs, probenecid also inhibits pannexin channels (Silverman et al 2008;Sandilos and Bayliss 2012). Pannexins are transmembrane proteins closely related to the gap-junction forming connexins (Baranova et al 2004).…”