Requirement of two polybasic clusters in the PIP_n-regulated P2X4 subtype.

<p>A) Sequence of the P2X4 C-terminus showing lysine to glutamine mutations disrupting the positive charge of the first or second cluster (basic residues in red, acidic in blue, neutral mutations in grey). B) The GST construct containing the P2X4 C-terminal domain C360-V375 binds to several PIP_n including PIP₂ and PIP₃. Mutating the basic lysine residues K362 and K363, or K370 and K371 into neutral glutamine leads to a loss of binding to PIP_n (n = 3–6). C) Representative ATP-activated P2X4 current traces obtained on P2X4-expressing <i>Xenopus</i> oocytes showing the slower activation and desensitization rates induced by the K362Q-K363Q mutation decreasing PIP_n-binding affinity. D) Quantitative analysis of the functional changes induced by the K362Q-K363Q mutation on P2X4 current rundown (left), activation (middle) and desensitization (right). A larger rundown between agonist applications is observed with the mutant than with the WT (2^nd/1^st application: WT: 63.5±3.8%, mutant: 50.4±4.9%, n = 10–11). The mutant P2X4 channel shows a slower activation rate (10–90% rise time: WT: 0.67±0.03 s, mutant: 0.80±0.05 s, n = 70–80) and a slower desensitization rate (5-second decay %: WT: 55.6±3.1%, mutant: 30.1±2.6%, n = 55–61). E) Wortmannin-induced PIP_n depletion leads to a stronger inhibition of P2X4 current amplitude in the K362Q-K363Q mutant than in WT (post/pre-treatment: WT: 61.3±7.5%, mutant: 29.5±4.6%, n = 30–50). *: p<0.05; ***: p<0.001.