Intracellular cross talk and physical interaction between two classes of neurotransmitter-gated channels

Fast chemical communications in the nervous system are mediated by several classes of receptor channels believed to be independent functionally and physically. We show here that concurrent activation of P2X 2 ATP-gated channels and 5-HT 3 serotonin-gated channels leads to functional interaction and nonadditive currents (47-73% of the predicted sum) in mammalian myenteric neurons as well as in Xenopus oocytes or transfected human embryonic kidney (HEK) 293 cell heterologous systems. We also show that these two cation channels coimmunoprecipitate constitutively and are associated in clusters. In heterologous systems, the inhibitory cross talk between P2X 2 and 5-HT 3 receptors is disrupted when the intracellular C-terminal domain of the P2X 2 receptor subunit is deleted and when minigenes coding for P2X 2 or 5-HT 3 A receptor subunit cytoplasmic domains are overexpressed. Injection of fusion proteins containing the C-terminal domain of P2X 2 receptors in myenteric neurons also disrupts the functional interaction between native P2X 2 and 5-HT 3 receptors. Therefore, activity-dependent intracellular coupling of distinct receptor channels underlies ionotropic cross talks that may significantly contribute to the regulation of neuronal excitability and synaptic plasticity

A dual polybasic motif determines phosphoinositide binding and regulation in the P2X channel family.

Phosphoinositides modulate the function of several ion channels, including most ATP-gated P2X receptor channels in neurons and glia, but little is known about the underlying molecular mechanism. We identified a phosphoinositide-binding motif formed of two clusters of positively charged amino acids located on the P2X cytosolic C-terminal domain, proximal to the second transmembrane domain. For all known P2X subtypes, the specific arrangement of basic residues in these semi-conserved clusters determines their sensitivity to membrane phospholipids. Neutralization of these positive charges disrupts the functional properties of the prototypical phosphoinositide-binding P2X4 subtype, mimicking wortmannin-induced phosphoinositide depletion, whereas adding basic residues at homologous positions to the natively insensitive P2X5 subtype establishes de novo phosphoinositide-mediated regulation. Moreover, biochemical evidence of in vitro P2X subunit-phospholipid interaction and functional intracellular phosphoinositide-binding assays demonstrate that the dual polybasic cluster is necessary and sufficient for regulation of P2X signaling by phospholipids

P2X C-terminal peptides compete with P2X channels for binding to intracellular PIP_n.

<p>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 PIP_n. 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 PIP_n 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).</p

Mutations creating two polybasic clusters on the P2X5 C-terminus lead to PIP_n binding and a PIP_n-regulated current phenotype.

<p>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 <i>Xenopus</i> 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 PIP_n. Adding positive residues to one amino acid cluster with the SE→KY mutation induces binding to several PIP_n. Creating a second positive cluster with the SEE→KYK mutation increases PIP_n 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 <i>Xenopus</i> 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 PIP_n levels as wortmannin-induced PIP_n 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 PIP_n 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 PIP_n-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 (2^nd/1^st 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 PIP_n 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 PIP_n 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.</p

The proximal C-terminal domain of P2X subunits contains a semi-conserved PIP_n-binding motif.

<p>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 PIP_n in PIP strip assays. The second column shows the presence (+) or absence (−) of modulation by PIP_n 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 PIP_n 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 PIP_n.</p

Requirement of two polybasic clusters for PIP_n-binding in P2X1 and P2X7.

<p>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 PIP_n 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 PIP_n on a phospholipid strip assay (n = 3). Shown in grey boxes are various GST-fusion peptides generated.</p

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.</p