Human P2X4 receptor belongs to the P2X family of ATP-gated non-selective cation channels. P2X4 is abundantly expressed in the apical membrane of cholangiocytes and may contribute to the local regulation of biliary secretion (1). Recently, we have demonstrated that bile acids stimulate several members of the epithelial sodium channel (ENaC)/degenerin (DEG) ion channel family including the acid-sensing ion channel 1 (ASIC1) (2-4). There are no significant amino acid sequence relationships between ASICs and P2X receptors. However, a comparison of their crystal structures revealed similarities of ASIC and P2X receptor ion channel domains (5). Therefore, we hypothesized that bile acids may also modulate P2X4. In the current study, we investigated whether unconjugated cholic (CA), deoxycholic (DCA) and chenodeoxycholic (CDCA) acid or their tauro-conjugated forms (t-CA, t-DCA, t-CDCA) may modulate human P2X4 heterologously expressed in Xenopus laevis oocytes. Whole-cell and single-channel currents were measured using the two-electrode voltage clamp and outside-out patch-clamp technique, respectively. Values are presented as mean ± S.E.M. Student’s t-test was used for statistical analysis. Application of DCA or t-DCA (250 μM) reduced ATP-activated P2X4-mediated currents by 65±3% (p<0.001, n=22) or 68±3% (p<0.001, n=22), respectively. In contrast, the inhibitory effect of t-CDCA was only 34±8% (p<0.05, n=20) and CDCA, CA or t-CA had no significant effect on P2X4-mediated currents (n=20). Inhibition of P2X4 by t-DCA was reversible. In the presence of t-DCA the affinity of P2X4 to ATP (EC50=2.4±1.3 μM) was similar to that under control conditions (EC50=4.8±1.2 μM). In outside-out patch-clamp recordings we observed that t-DCA significantly decreased the rate of P2X4 activation by ATP and increased the rate of subsequent channel inactivation. Moreover, the estimated channel open probability was significantly reduced in the presence of t-DCA. This can be attributed to a 3-fold increase of the apparent mean channel closed time from 13±4 ms (n=7) in the absence of t-DCA to 42±11 ms (n=6; p<0.01) in its presence. Finally, using site-directed mutagenesis we identified several amino acid residues involved in mediating the inhibitory effect of t-DCA on P2X4. We conclude that t-DCA may specifically interact with P2X4 and stabilize its closed state leading to the decrease of ATP-activated P2X4 currents.