Structural insights into the competitive inhibition of the ATP-gated P2X receptor channel.

P2X receptors are non-selective cation channels gated by extracellular ATP, and the P2X7 receptor subtype plays a crucial role in the immune and nervous systems. Altered expression and dysfunctions of P2X7 receptors caused by genetic deletions, mutations, and polymorphic variations have been linked to various diseases, such as rheumatoid arthritis and hypertension. Despite the availability of crystal structures of P2X receptors, the mechanism of competitive antagonist action for P2X receptors remains controversial. Here, we determine the crystal structure of the chicken P2X7 receptor in complex with the competitive P2X antagonist, TNP-ATP. The structure reveals an expanded, incompletely activated conformation of the channel, and identified the unique recognition manner of TNP-ATP, which is distinct from that observed in the previously determined human P2X3 receptor structure. A structure-based computational analysis furnishes mechanistic insights into the TNP-ATP-dependent inhibition. Our work provides structural insights into the functional mechanism of the P2X competitive antagonist.P2X receptors are nonselective cation channels that are gated by extracellular ATP. Here the authors present the crystal structure of chicken P2X7 with its bound competitive antagonist TNP-ATP and give mechanistic insights into TNP-ATP dependent inhibition through further computational analysis and electrophysiology measurements.

Discovery of selective PDE4B inhibitors.

In this study the first PDE4B selective inhibitor is described. Optimization of lead 2-arylpyrimidine derivatives afforded a series of potent PDE4B inhibitors with >100-fold selectivity over the PDE4D isozyme. With a good pharmacokinetic profile, a selected compound exhibited potent anti-inflammatory effects in vivo and showed less emesis compared with Cilomilast.

Essential role of rho kinase in the Ca2+ sensitization of prostaglandin F(2alpha)-induced contraction of rabbit aortae.

Inhibition of dephosphorylation of the 20 kDa myosin light chain (MLC(20)) is an important mechanism for the Ca(2+)-induced sensitization of vascular smooth muscle contraction. We investigated whether this mechanism operates in prostaglandin F(2alpha) (PGF(2alpha))-induced contraction of rabbit aortic smooth muscle and, if so, whether protein kinase C (PKC) or rho-associated kinase (rho kinase) contribute to the inhibition of dephosphorylation. In normal medium, PGF(2alpha) (10 microM) increased the phosphorylation of MLC(20) and developed tension. The rho-kinase inhibitors fasudil and hydroxyfasudil inhibited these changes, despite having no effect on a phorbol-ester-induced MLC(20) phosphorylation. After treatment with verapamil or chelation of external Ca(2+) with EGTA, PGF(2alpha) increased the MLC(20) phosphorylation and tension without an increase in [Ca(2+)](i), all of which were sensitive to fasudil and hydroxyfasudil. ML-9, a MLC kinase inhibitor, quickly reversed the KCl-induced MLC(20) phosphorylation and contraction to the resting level. However, fractions of PGF(2alpha)-induced contraction and MLC(20) phosphorylation were resistant to ML-9 but were sensitive to fasudil. Ro31-8220 (10 microM), a PKC inhibitor, did not affect the phosphorylation of MLC(20) and the tension caused by PGF(2alpha), thus excluding the possibility of the involvement of PKC in the PGF(2alpha)-induced MLC(20) phosphorylation. PGF(2alpha) increased phosphorylation at Thr654 of the myosin binding subunit (MBS) of myosin phosphatase, which is a target of rho kinase, and fasudil decreased the phosphorylation. These data suggest that the PGF(2alpha)-induced contraction is accompanied by the inhibition of MLC(20) dephosphorylation through rho kinase-induced MBS phosphorylation, leading to Ca(2+) sensitization of contraction. An actin-associated mechanism may also be involved in the PGF(2alpha)-induced sensitization.

Synapsin I is phosphorylated at Ser603 by p21-activated kinases (PAKs) in vitro and in PC12 cells stimulated with bradykinin.

The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser(603) residue (site 3) is considered to be a pivotal site targeted by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser(603) residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser(603) without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser(603)-synapsin I (pS603-Syn I-Ab) and phospho-Thr(286/287)-CaMKII. The bradykinin-evoked phosphorylation of Ser(603) was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser(603) kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca(2+)/calmodulin-independent Ser(603) kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated (32)P into synapsin I in a Cdc42/GTPgammaS-dependent manner, and its phosphorylation site was confirmed as Ser(603) using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser(603) residue in the presence of Cdc42/GTPgammaS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat alphaPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser(603) even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative alphaPAK quenches the phosphorylation. These results raise the possibility that Ser(603) on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.