Roles of Conserved Ectodomain Cysteines of the Rat P2X4 Purinoreceptor in Agonist Binding and Channel Gating

Mammalian P2X receptors contain 10 conserved cysteine residues in their ectodomains, which form five disulfide bonds (SS1-5). Here, we analyzed the relevance of these disulfide pairs in rat P2X4 receptor function by replacing one or both cysteines with alanine or threonine, expressing receptors in HEK293 cells and studying their responsiveness to ATP in the absence and presence of ivermectin, an allostenic modulator of these channels. Response to ATP was not altered when both cysteines forming the SS3 bond (C132-C159) were replaced with threonines. Replacement of SS1 (C116-C165), SS2 (C126-C149) and SS4 (C217-C227), but not SS5 (C261-C270), cysteine pairs with threonines resulted in decreased sensitivity to ATP and faster deactivation times. The maximum current amplitude was reduced in SS2, SS4 and SS5 double mutants and could be partially rescued by ivermectin in SS2 and SS5 double mutants. This response pattern was also observed in numerous single residue mutants, but receptor function was not affected when the 217 cysteine was replaced with threonine or arginine or when the 261 cysteine was replaced with alanine. These results suggest that the SS1, SS2 and SS4 bonds contribute substantially to the structure of the ligand binding pocket, while the SS5 bond located towards the transmembrane domain contributes to receptor gating

Correlation of RET somatic mutations with clinicopathological features in sporadic medullary thyroid carcinomas

Screening of REarranged during Transfection (RET) gene mutations has been carried out in different series of sporadic medullary thyroid carcinomas (MTC). RET-positive tumours seem to be associated to a worse clinical outcome. However, the correlation between the type of RET mutation and the patients' clinicopathological data has not been evaluated yet

Electrophysiological characterization of α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to 4BP-TQS.

<p>A) A bar chart illustrates responses (mean ± SEM) from α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (10 µM) concentration of the allosteric agonist 4BP-TQS at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 5–22 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. B) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. C) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p

Amplitude and desensitization of nAChR responses examined at different temperatures.

†, ††<p>Data for desensitization of all receptor and agonist combinations (with the exception of wild-type α7, activated by ACh) are expressed as the percentage of decay from the peak response in 5 seconds. Due to the rapid rate of desensitization for wild-type α7 activated by ACh, these values are expressed as the time required for the response to decay to 50% of the peak response.</p><p>Data are means ± SEM. Significant differences to responses recorded at RT are indicated (* = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001).</p

Electrophysiological characterization of nAChRs expressed in <i>Xenopus</i> oocytes examined at different temperatures.

<p>Representative traces are shown illustrating responses obtained at RT (black), 4°C (blue) and 37°C (red). Current traces obtained at each temperature have been normalized to the same peak response. In each case, the response showing the fastest rate of desensitization was observed at 37°C and the slowest rate of desensitization was observed at 4°C. Responses are from α7 nAChRs with 3 mM acetylcholine (A), α4β2 nAChRs with 1 mM acetylcholine in calcium-containing saline (B), α7^L247T nAChRs with 30 µM acetylcholine (C) and α7 nAChRs with 10 µM 4BP-TQS (D). Rates of receptor deactivation after removal of agonist were also influenced in a consistent manner by changes in temperature (faster at 37°C and slower at 4°C). Representative traces from α7^L247T nAChRs with 30 µM acetylcholine are illustrated (E) and are typical of results from all receptor/agonist combinations studied (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032073#pone-0032073-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032073#pone-0032073-t002" target="_blank">2</a> for details).</p

Electrophysiological characterization of α7^L247T nAChRs expressed in <i>Xenopus</i> oocytes in response to acetylcholine.

<p>Bar charts illustrate responses (mean ± SEM) from α7^L247T nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (30 µM) and <i>EC</i>₅₀ (0.4 µM) concentration of acetylcholine (A and B, respectively) at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 5–9 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. C) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. D) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p

Electrophysiological characterization of α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to acetylcholine.

<p>Bar charts illustrate responses (mean ± SEM) from α7 nAChRs expressed in <i>Xenopus</i> oocytes in response to a maximal (3 mM) and <i>EC</i>₅₀ (100 µM) concentration of acetylcholine (A and B, respectively) at room temperature (RT; 21°C), higher temperature (37°C) and lower temperature (4°C). Data are means of 7–11 responses, each from a different oocyte, in which responses obtained at either 4°C or 37°C are normalized to responses obtained from the same oocyte at RT. C) Representative traces illustrating responses obtained at RT (upper trace) and 4°C (lower trace) from a single oocyte. D) Representative traces illustrating responses obtained at RT (upper trace) and 37°C (lower trace) from a single oocyte.</p