Unusually strong temperature dependence of p2×3 receptor traffic to the plasma membrane

Abstract

ATP-gated P2×3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2×3 receptors is strongly reduced at low tempera- tures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed tagged P2×3 receptors and studied the effects of temperature on perimem brane turnover of P2×3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2×3-DsRed receptors. By combiningTIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2×3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2×3-DsRed perimembrane turnover as measured by TIRF-FRAP had a time constant of ~2min. At 29 °C, receptor turnover was strongly acceler- ated (0.6min), yielding an extremely high temperature dependence coefficient Q 10 ~4.5. In comparison, AMPA receptor turnover measured with TIRF-FRAP was only moderately sensitive to temperature (Q 10 ~1.5).The traffic inhibitor Brefeldin A selectively decelerated P2×3-DsRed receptor turnover at 29 °C, but had no effect at 21 °C (Q 10 ~1.0).This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2×3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2×3-DsRed receptor turnover (Q 10 ~2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2×3 receptors to plasma membrane has an exceptionally high sensitivity to temperature.These findings suggest an important role of P2×3 receptor turnover in hypothermia-associated analgesia. © 2011 Pryazhnikov, Fayuk, Niittykoski, Giniatullin and Khiroug.