BackgroundThe cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane.Methodology/Principal FindingsWe used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2–8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MβCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability.Conclusions/SignificanceThese results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons.

The spatial and temporal distribution of receptors constitutes an important mechanism for controlling the magnitude of cellular responses. Several members of the transient receptor potential (TRP) ion channel family can regulate their function by modulating their expression at the plasma membrane (PM) through rapid vesicular translocation and fusion. The mechanisms underlying this regulation are not completely understood, and the contribution of vesicular trafficking to physiological function is unknown. TRPM8 receptors are expressed in mammalian peripheral sensory neurons and are essential for the detection of cold temperatures. Previously, we showed that TRPM8-containing vesicles are segregated into three main pools, immobile at the PM, simple diffusive and corralled-hopping. Here, we show that channel expression at the PM is modulated by TRPM8 agonists in F11 and HEK293T cells. Our results support a model in which the activation of TRPM8 channels, located at the PM, induces a short-lived recruitment of a TRPM8-containing vesicular pool to the cell surface causing a transitory increase in the number of functional channels, affecting intrinsic properties of cold receptor responses. We further demonstrate the requirement of intact vesicular trafficking to support sustained cold responses in the skin of mice.

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laser heating to time-resolve the activation and deactivation of TRPM8. Our data reveal that temperature mainly drives the deactivation rate of the channel, which has Q10~10, while the activation rate has typical temperature dependence (Q10~2-3). That is, cooling mainly increases the open probability of the channel by slowing down channel closing. Thus the cold-sensitive channels work in an opposite way to the heat-sensitive channels in which temperature drives the opening rate while the closing rate is insensitive to temperature. But similar to heat activation of TRPV1, the temperature dependence of cold activation of TRPM8 is also strongly dependent on membrane potentials. At hyperpolarization (À60 mV) the deactivation involves an enthalpy change of~40 kcal/mol, while at depolarization (þ60 mV) the enthalpy is decreased to~20 kcal/mol. The reduction of enthalpy reaches~20 kcal/mol, which far exceeds the electrical energy due to charge movement (<2 kcal/mol for z~0.8), suggesting that the mechanisms of voltage-and temperature-sensing of the channel are coupled.

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