Transient receptor potential channels are a family of cation channels involved in diverse cellular functions. Most of these channels are expressed in the nervous system and play a key role in sensory physiology. TRPM8 (transient receptor potential melastatine 8), a member of this family, is activated by cold, cooling substances such menthol and icilin and voltage. Although TRPM8 is a thermosensitive channel highly expressed in cold sensory neurons, the mechanisms underlying its temperature sensitivity are still poorly understood. Here we show that, in sensory neurons, TRPM8 channel is localized in cholesterolrich specialized membrane domains known as lipid rafts. We also show that, in heterologous expression systems, lipid raft segregation of TRPM8 is favored by glycosylation at the Asn 934 residue of the polypeptide. In electrophysiological and imaging experiments, using cold and menthol as agonists, we also demonstrate that lipid raft association modulates TRPM8 channel activity. We found that menthol-and cold-mediated responses of TRPM8 are potentiated when the lipid raft association of the channel is prevented. In addition, lipid raft disruption shifts the threshold for TRPM8 activation to a warmer temperature. In view of these data, we suggest a role for lipid rafts in the activity and temperature sensitivity of TRPM8. We propose a model wherein different lipid membrane environments affect the cold sensing properties of TRPM8, modulating the response of cold thermoreceptors.Ambient temperature detection is a critical biological process carried out by terminals of primary afferent sensory neurons of the dorsal root (DRG) 2 and trigeminal ganglia in the mammalian sensory system (1). Thermosensitive nerves express a subset of proteins of the transient receptor potential (TRP) ion channel family that are activated at different temperatures, making these cationic ion channels central elements in the temperature sensing machinery of peripheral nerve endings (2). Among thermoTRPs, TRPM8 (transient receptor potential melastatine 8) is characterized by its enhanced activity at low temperatures (threshold, ϳ25°C) and by application of cooling compounds such as menthol and icilin (3, 4). These properties, and its selective expression in a subset of small sensory neurons, make TRPM8 an excellent candidate for transducing cold temperatures at nerve endings, a view supported by behavioral findings in TRPM8 knock-out mice (5-7). In addition to mild cold temperature detection, several recent studies (8, 9) have attributed a role to TRPM8 in noxious cold transduction and nociception.Functional studies in chimeric channels suggest that the C terminus of TRPM8 contains structural elements important in temperature-dependent gating (10). However, the molecular mechanisms underlying temperature sensitivity of TRPM8 are still unknown. It has been hypothesized that TRP channels might sense temperature-mediated changes in lipid bilayer tension (11,12). Whereas at physiological temperatures most of the plasma membrane remains in a l...

Alterations in astrocyte function that may affect neuronal viability occur with brain aging. In this study, we evaluate the neuroprotective capacity of astrocytes in an experimental model of in vitro aging. Changes in oxidative stress, glutamate uptake and protein expression were evaluated in rat cortical astrocytes cultured for 10 and 90 days in vitro (DIV). Levels of glial fibrillary acidic protein and S100b increased at 90 days when cells were positive for the senescence b-galactosidase marker. In long-term astrocyte cultures, the generation of reactive oxygen species was enhanced and mitochondrial activity decreased. Simultaneously, there was an increase in proteins that stained positively for nitrotyrosine. The expression of Cu/Zn-superoxide dismutase (SOD-1) and haeme oxygenase-1 (HO-1) proteins and inducible nitric oxide synthase (iNOS) increased in aged astrocytes. Glutamate uptake in 90-DIV astrocytes was higher than in 10 DIV ones, and was more vulnerable to inhibition by H 2 O 2 exposure. Enhanced glutamate uptake was probably because of up-regulation of the glutamate/aspartate transporter protein. Aged astrocytes had a reduced ability to maintain neuronal survival. These findings indicate that astrocytes may partially loose their neuroprotective ability during aging. The results also suggest that aged astrocytes may contribute to exacerbating neuronal injury in age-related neurodegenerative processes.

Background: TRPM8 channel is N-glycosylated, a post-translational modification affecting trafficking and gating properties of other TRP channels. Results: Preventing N-glycosylation reduces responses of TRPM8 to agonists (cold and menthol) due to a change in its biophysical properties. Conclusion: N-Glycosylation is an important determinant of TRPM8 sensitivity to chemical and thermal stimuli. Significance: N-Glycosylation of TRPM8 could play a modulatory role in cold thermoreceptor activity.