Lipid Raft Segregation Modulates TRPM8 Channel Activity

Abstract

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...

N-Glycosylation of TRPM8 Ion Channels Modulates Temperature Sensitivity of Cold Thermoreceptor Neurons

“…Equal amounts of protein for each condition (15-30 g) were denatured at 95°C for 5 min, loaded onto a 7.5% SDS-polyacrylamide gel, and electrophoresed. Proteins were transferred to a nitrocellulose membrane, blocked with 10% skim milk in TBS, and incubated with antibodies against mouse TRPM8 (diluted 1:500) (14). Horseradish peroxidase (HRP)-coupled anti-rabbit secondary antibodies (Sigma) were used at a final concentration of 1:2000 for detection, and the signal was developed with an enhanced chemiluminescence kit (Amersham Biosciences) and recorded by using an image analyzer LAS-1000Plus (Fujifilm).…”

“…Studies in dorsal root ganglia neurons also confirmed that endogenous TRPM8 channels are highly glycosylated in vivo (14). To further study the N-glycosylation of TRPM8 in a native context, we cultured TG neurons that express TRPM8 endogenously and analyzed their immunoreactive profile to TRPM8.…”

“…Channel Activity-Previously, we reported that mature N-glycosylation of TRPM8 contains terminal sialic acid residues, which are negatively charged (14). This post-translational modification is located at a residue in the vicinity of the pore region.…”

“…To this end, we used tunicamycin, an antibiotic that blocks the addition of carbohydrate molecules to asparagine residues of potential glycoproteins (30), and brefeldin A, which inhibits transport of proteins from the ER to Golgi complex and leads to protein accumulation inside the ER (31). In a second strategy, we used single point mutation of the asparagine 934, the only residue in TRPM8 where the N-glycosylation takes place (12)(13)(14), to a glutamine residue. In both strategies, the tunicamycin treatment and the point mutation of N934Q resulted in the detection of a single band of ϳ128 kDa, corroborating that TRPM8 is N-glycosylated in both recombinant and native systems ( Fig.…”

“…Both studies favored impairment in the traffic of channels to the plasma membrane as causal to the reduced activity. Related with the role of N-glycosylation in traffic, we have recently described that glycosylation at the Asn-934 residue also facilitates segregation of TRPM8 into membrane lipid rafts (14).…”