We reported that P2X4 is a prototypical PIPn-dependent P2X subtype, being tightly regulated via direct binding to PIP2 and PIP3 [8]. We therefore aimed to neutralize the PIPn-binding site by mutating key lysine residues. We found that neutralizing the charge of either of the two clusters, by mutating lysines 362 and 363 or lysines 370 and 371 into neutral glutamines, leads to a loss of PIPn binding in an in vitro binding assay where a GST-fusion protein coding for a 16-amino acid sequence (Figure 2A,B) is applied to various PIPn. The lysine-to-glutamine mutations performed on residues 362 and 363 also induced significant changes in the P2X4 channel activity. Expressed in the Xenopus oocyte expression system, the P2X4 mutant with lower PIPn-binding affinity displayed a stronger current rundown upon repeated ATP applications as well as slower activation and desensitization current phases (Figure 2C,D), all these effects mimicking those brought by pharmacological PIPn depletion [8]. The K362Q-K363Q mutant receptor was more strongly inhibited by wortmannin-induced PIPn depletion than the wild-type (WT) receptor (Figure 2E), due to its lower affinity to PIPn. The mutation targetting the second basic cluster (K370Q-K371Q) could not be tested functionally as P2X4 channels with mutations on residue 371 are non-functional due to the role of conserved lysine 371 in receptor trafficking [11].
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