Figure 1.
The proximal C-terminal domain of P2X subunits contains a semi-conserved PIPn-binding motif.
A) Sequence alignment of rat P2X C-termini proximal to the TM2 domain showing the two polybasic clusters (shaded area, 1 and 2). The left column summarizes, for each subunit, the presence (+) or absence (−) of binding of the GST-fusion C-terminal domain to PIPn in PIP strip assays. The second column shows the presence (+) or absence (−) of modulation by PIPn in functional assays. Basic residues are shown in red and acidic residues in blue. B) Sequences showing residues that were reported (here or previously) to be involved in PIPn regulation. Basic residues in red, acidic residues in blue and an uncharged serine in green. C) Schematic representation of the topology of a P2X subunit showing binding of two positively charged amino acid clusters to membrane-bound PIPn.
Figure 2.
Requirement of two polybasic clusters in the PIPn-regulated P2X4 subtype.
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 PIPn 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 PIPn (n = 3–6). C) Representative ATP-activated P2X4 current traces obtained on P2X4-expressing Xenopus oocytes showing the slower activation and desensitization rates induced by the K362Q-K363Q mutation decreasing PIPn-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 (2nd/1st 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 PIPn 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.
Figure 3.
Requirement of two polybasic clusters for PIPn-binding in P2X1 and P2X7.
A) The GST construct containing the WT P2X1 C-terminal domain (L352-E378) (basic residues in red, acidic in blue, neutral mutations in grey) binds various PIPn on a phospholipid strip assay, whereas disrupting the positive charge of the first or second polybasic cluster with K359Q and K364Q mutations suppresses binding (n = 3). B) The absence of two polybasic clusters in the C-terminus of P2X7 prevents its binding to PIPn on a phospholipid strip assay (n = 3). Shown in grey boxes are various GST-fusion peptides generated.
Figure 4.
Mutations creating two polybasic clusters on the P2X5 C-terminus lead to PIPn binding and a PIPn-regulated current phenotype.
A) Sequence of the P2X5 C-terminus showing mutations adding positive charges to one (S365K-E366Y, SE→KY) or two clusters (S365K-E366Y-E374K, SEE→KYK) (basic residues in red, acidic in blue, neutral mutations in grey). B) Representative ATP-activated current traces recorded in Xenopus oocytes expressing P2X5 WT or SEE→KYK mutant in control and wortmannin conditions. C) The GST construct containing the WT P2X5 proximal C-terminal domain L361-V376 does not bind to PIPn. Adding positive residues to one amino acid cluster with the SE→KY mutation induces binding to several PIPn. Creating a second positive cluster with the SEE→KYK mutation increases PIPn binding (n = 3–6). D) Quantitative data showing the SE→KY and SEE→KYK gain-of-binding mutations lead to increased current amplitude in rP2X5-expressing Xenopus oocytes (left graph; rP2X5 WT: 0.21±0.04 µA, SE→KY: 1.22±0.11 µA, SEE→KYY: 3.19±0.55 µA, n = 13–25). The gain-of-binding mutants are sensitive to intracellular PIPn levels as wortmannin-induced PIPn depletion leads to a decrease in current amplitude (right graph, post/pre-treatment amplitude; WT: 107.5±21.8%, SE→KY: 50.3±10.7%, SEE→KYY: 40.9±8.4%, n = 4–17). E) Human P2X5 channel currents are significantly inhibited by PIPn depletion (vehicle = 4.90±0.79 µA; post-wortmannin = 1.44±0.24 µA, n = 7–10). F) Differences in current rundown, activation rate and desensitization rate between WT P2X5 and SEE→KYK mutant under control and PIPn-depletion conditions. The current rundown between successive applications measured with the WT P2X5 is prevented by gain-of-binding mutations, and is partially restored after a wortmannin treatment of the mutant (2nd/1st application: WT: 40.0±4.4%, mutant control: 113.9±8.0%, mutant wortmannin: 83.7±9.8%, n = 5–10). The mutant P2X5 channel shows a faster current activation compared to WT, and it is slowed by PIPn depletion (10–90% rise time: WT: 3.77±0.69 s, mutant control: 0.86±0.08 s, mutant wortmannin: 2.31±0.25 s, n = 7–8). The gain-of-binding P2X5 mutant current desensitizes faster than WT, and its desensitization rate is slowed by PIPn depletion (decay slope: WT: −0.001±0.006, mutant control: 0.31±0.09, mutant wortmannin: 0.07±0.02, n = 7–8). *: p<0.05; **: p<0.01; ***: p<0.001.
Figure 5.
P2X C-terminal peptides compete with P2X channels for binding to intracellular PIPn.
A) Representative traces of patch-clamp recordings showing that intracellular injection of the P2X4 C-terminal (-CT) peptide leads to a rundown of the P2X4 current in HEK293 cells by competing for intracellular PIPn. B) Intracellular injection of the P2X5-CT peptide does not affect the P2X4 current phenotype. C) Effect on P2X4 current amplitude of injection of peptides from the P2X4 WT, P2X4 K362Q-K363Q (2M), P2X5 WT or P2X5 S365K-E366Y-E374K (3M) C-terminus. D) Competition for PIPn binding from P2X4-CT or P2X5-3M-CT peptide injection leads to a slower desensitization of the P2X4 current. Values were normalized to the initial recording value obtained immediately after whole-cell configuration was obtained (n = 4–5; *, p<0.05; **, p<0.01; ***, p<0.001, each group compared to control).