Genomic organization of purinergic P2X receptors

"Purinergic P2X receptors are a family of ligand-gated cationic channels activated by extracellular ATP. P2X subunit protein sequences are highly conserved between vertebrate species. However, they can generate a great diversity of coding splicing variants to fulfill several roles in mammalian physiology. Despite intensive research in P2X expression in both central and peripheral nervous system, there is little information about their homology, genomic structure and other key features that can help to develop selective drugs or regulatory strategies of pharmacological value which are lacking today. In order to obtain clues on mammalian P2X diversity, we have performed a bioinformatics analysis of the coding regions and introns of the seven P2X subunits present in human, simian, dog, mouse, rat and zebrafish. Here we report the arrangements of exon and intron sequences, considering its number, size, phase and placement; proposing some ideas about the gain and loss of exons and retention of introns. Taken together, these evidences show traits that can be used to gain insight into the evolutionary history of vertebrate P2X receptors and better understand the diversity of subunits coding the purinergic signaling in mammals.

Heteromeric channels with different phenotypes are generated when coexpressing two P2X2 receptor isoforms

Tesis (Doctorado en Ciencias en Biología Molecular)"Para determinar si al coexpresar dos isoformas del receptor P2X2 en ovocitos de Xenopus laevis se forman canales heteroméricos con diferente estequiometria. Tomando ventaja de una mutante (P2X2-2bm) debido a que tiene una menor sensibilidad al ATP que el receptor P2X2-2b (wild type), y a su vez incrementa las diferencias entre esta y la variante P2X2-1a. Los canales P2X son triméricos con tres sitios de unión al agonista, dos posibles combinaciones pueden dar lugar a los canales heteroméricos: i) 2(P2X2-1a) +1(P2X2-2bm); o ii) 1(P2X2-1a) +2(P2X2- 2bm). Debido a que la apertura de los canales P2X se da por la unión de dos moléculas del ATP, se esperaría diferente sensibilidad al ATP entre estos heterómeros. En apoyo a esta hipótesis, la coexpresión de las dos isoformas de receptor P2X2 resultó en dos poblaciones de ovocitos con sensibilidad al ATP y coeficientes de Hill diferentes. Una población (P2X2-1a like), fue similar a los ovocitos en que solo se expresaron canales P2X2-1a, mientras la otra (P2X2-2bm like) fue similar a cuando solo se expresaron canales P2X2-2bm. Sin embargo, el análisis de sus cinéticas, descarta la expresión de canales homoméricos. Los datos también indican que, la heteromerización de los canales, provoca cambios en la cinética de desensibilización. En conclusión, cuando las isoformas de P2X2 son coexpresadas, los ovocitos expresan principalmente una de las dos estequiometrias de canales heteroméricos.""To investigate if channels with different stoichiometry are formed from two P2X2 receptor splice variants during their co-expression in Xenopus laevis oocytes. We used a mutant (P2X2-2bm) because it has a lower ATP sensitivity than the wild type receptor. P2X channels are trimeric proteins with three agonist binding sites; therefore, only two homomeric and two heteromeric stoichiometries are possible, the heteromeric channels might be formed by: i) 2(P2X2-1a) +1(P2X2-2bm); or ii) 1(P2X2-1a) +2(P2X2-2bm). Because P2X2 channels open when two binding sites are occupied, these stoichiometries are expected to have different sensitivities to ATP. In agreement with this, coexpressing both P2X2 isoforms, two oocyte populations were distinguished based on their channel sensitivities to ATP and Hill coefficients. For the first population (P2X2-1a like), such parameters were not different than those of homomeric P2X2-1a channels, and for the second population (P2X2-2bm like), these parameters were also not different than for homomeric P2X2- 2bm channels. Kinetics analysis indicates that heteromeric channel expression is occurring, and homomeric channel expression is not responsible for such differences. Our data indicate that oocytes expressed heteromeric channels with mainly one (of the two possible) stoichiometry when P2X2-1a and P2X2-2bm subunits are coexpressed, and the expression of homomeric channels is also not detectable.

Two P2X1 receptor transcripts able to form functional channels are present in most human monocytes

"To characterize the presence and general properties of P2X1 receptors in single human monocytes we used RT-PCR, flow cytometry, and the patch-clamp and the two-electrode voltage-clamp techniques. Most human monocytes expressed the canonical P2X1 (90%) and its splicing variant P2X1del (88%) mRNAs. P2X1 receptor immunoreactivity was also observed in 70% of these cells. Currents mediated by P2X1 (EC50=1.9±0.8 µm) and P2X1del (EC50 >1000 µm) channels, expressed in Xenopus leavis oocytes, have different ATP sensitivity and kinetics. Both currents mediated by P2X1 and P2X1del channels kept increasing during the continuous presence of high ATP concentrations. Currents mediated by the native P2X1 receptors in human monocytes showed an EC50=6.3±0.2 µm. Currents have kinetics that resemble those observed for P2X1 and P2X1del receptors in oocytes. Our study is the first to demonstrate the expression of P2X1 transcript and its splicing variant P2X1del in most human monocytes. We also, for the first time, described functional homomeric P2X1del channels and demonstrated that currents mediated by P2X1 or P2X1del receptors, during heterologous expression, increased in amplitude when activated with high ATP concentrations in a similar fashion to those channels that increase their conductance under similar conditions, such as P2X7, P2X2, and P2X4 channels.

Epithelial expression and function of trypsin-3 in irritable bowel syndrome.

ObjectivesProteases are key mediators of pain and altered enteric neuronal signalling, although the types and sources of these important intestinal mediators are unknown. We hypothesised that intestinal epithelium is a major source of trypsin-like activity in patients with IBS and this activity signals to primary afferent and enteric nerves and induces visceral hypersensitivity.DesignTrypsin-like activity was determined in tissues from patients with IBS and in supernatants of Caco-2 cells stimulated or not. These supernatants were also applied to cultures of primary afferents. mRNA isoforms of trypsin (PRSS1, 2 and 3) were detected by reverse transcription-PCR, and trypsin-3 protein expression was studied by western blot analysis and immunohistochemistry. Electrophysiological recordings and Ca2+ imaging in response to trypsin-3 were performed in mouse primary afferent and in human submucosal neurons, respectively. Visceromotor response to colorectal distension was recorded in mice administered intracolonically with trypsin-3.ResultsWe showed that stimulated intestinal epithelial cells released trypsin-like activity specifically from the basolateral side. This activity was able to activate sensory neurons. In colons of patients with IBS, increased trypsin-like activity was associated with the epithelium. We identified that trypsin-3 was the only form of trypsin upregulated in stimulated intestinal epithelial cells and in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons and mouse sensory neurons, and to induce visceral hypersensitivity in vivo, all by a protease-activated receptor-2-dependent mechanism.ConclusionsIn IBS, the intestinal epithelium produces and releases the active protease trypsin-3, which is able to signal to enteric neurons and to induce visceral hypersensitivity

Epithelial expression and function of trypsin-3 in irritable bowel syndrome

Objectives Proteases are key mediators of pain and altered enteric neuronal signalling, although the types and sources of these important intestinal mediators are unknown. We hypothesised that intestinal epithelium is a major source of trypsin-like activity in patients with IBS and this activity signals to primary afferent and enteric nerves and induces visceral hypersensitivity. Design Trypsin-like activity was determined in tissues from patients with IBS and in supernatants of Caco-2 cells stimulated or not. These supernatants were also applied to cultures of primary afferents. mRNA isoforms of trypsin (PRSS1, 2 and 3) were detected by reverse transcription-PCR, and trypsin-3 protein expression was studied by western blot analysis and immunohistochemistry. Electrophysiological recordings and Ca2+ imaging in response to trypsin-3 were performed in mouse primary afferent and in human submucosal neurons, respectively. Visceromotor response to colorectal distension was recorded in mice administered intracolonically with trypsin-3. Results We showed that stimulated intestinal epithelial cells released trypsin-like activity specifically from the basolateral side. This activity was able to activate sensory neurons. In colons of patients with IBS, increased trypsin-like activity was associated with the epithelium. We identified that trypsin-3 was the only form of trypsin upregulated in stimulated intestinal epithelial cells and in tissues from patients with IBS. Trypsin-3 was able to signal to human submucosal enteric neurons and mouse sensory neurons, and to induce visceral hypersensitivity in vivo, all by a protease-activated receptor-2-dependent mechanism. Conclusions In IBS, the intestinal epithelium produces and releases the active protease trypsin-3, which is able to signal to enteric neurons and to induce visceral hypersensitivity