The ion channel TRPM8 is a direct target of the immunosuppressant rapamycin in primary sensory neurons.

BACKGROUND AND PURPOSE: The mechanistic target of rapamycin (mTOR) signalling pathway is a key regulator of cell growth and metabolism. Its deregulation is implicated in several diseases. The macrolide rapamycin, a specific inhibitor of mTOR, has immunosuppressive, anti-inflammatory and antiproliferative properties. Recently, we identified tacrolimus, another macrolide immunosuppressant, as a novel activator of TRPM8 ion channels, involved in cold temperature sensing, thermoregulation, tearing and cold pain. We hypothesized that rapamycin may also have agonist activity on TRPM8 channels. EXPERIMENTAL APPROACH: Using calcium imaging and electrophysiology in transfected HEK293 cells and wildtype or Trpm8 KO mouse DRG neurons, we characterized rapamycin's effects on TRPM8 channels. We also examined the effects of rapamycin on tearing in mice. KEY RESULTS: Micromolar concentrations of rapamycin activated rat and mouse TRPM8 channels directly and potentiated cold-evoked responses, effects also observed in human TRPM8 channels. In cultured mouse DRG neurons, rapamycin increased intracellular calcium levels almost exclusively in cold-sensitive neurons. Responses were markedly decreased in Trpm8 KO mice or by TRPM8 channel antagonists. Cutaneous cold thermoreceptor endings were also activated by rapamycin. Topical application of rapamycin to the eye surface evokes tearing in mice by a TRPM8-dependent mechanism. CONCLUSION AND IMPLICATIONS: These results identify TRPM8 cationic channels in sensory neurons as novel molecular targets of the immunosuppressant rapamycin. These findings may help explain some of its therapeutic effects after topical application to the skin and the eye surface. Moreover, rapamycin could be used as an experimental tool in the clinic to explore cold thermoreceptors.

The Immunosuppressant Macrolide Tacrolimus Activates Cold-Sensing TRPM8 Channels.

TRPM8 is a polymodal, nonselective cation channel activated by cold temperature and cooling agents that plays a critical role in the detection of environmental cold. We found that TRPM8 is a pharmacological target of tacrolimus (FK506), a macrolide immunosuppressant with several clinical uses, including the treatment of organ rejection following transplants, treatment of atopic dermatitis, and dry eye disease. Tacrolimus is an inhibitor of the phosphatase calcineurin, an action shared with cyclosporine. Tacrolimus activates TRPM8 channels in different species, including humans, and sensitizes their response to cold temperature by inducing a leftward shift in the voltage-dependent activation curve. The effects of tacrolimus on purified TRPM8 in lipid bilayers demonstrates conclusively that it has a direct gating effect. Moreover, the lack of effect of cyclosporine rules out the canonical signaling pathway involving the phosphatase calcineurin. Menthol (TRPM8-Y745H)- and icilin (TRPM8-N799A)-insensitive mutants were also activated by tacrolimus, suggesting a different binding site. In cultured mouse DRG neurons, tacrolimus evokes an increase in intracellular calcium almost exclusively in cold-sensitive neurons, and these responses were drastically blunted in Trpm8 KO mice or after the application of TRPM8 antagonists. Cutaneous and corneal cold thermoreceptor endings are also activated by tacrolimus, and tacrolimus solutions trigger blinking and cold-evoked behaviors. Together, our results identify TRPM8 channels in sensory neurons as molecular targets of the immunosuppressant tacrolimus. The actions of tacrolimus on TRPM8 resemble those of menthol but likely involve interactions with other channel residues.SIGNIFICANCE STATEMENT TRPM8 is a polymodal TRP channel involved in cold temperature sensing, thermoregulation, and cold pain. TRPM8 is also involved in the pathophysiology of dry eye disease, and TRPM8 activation has antiallodynic and antipruritic effects, making it a prime therapeutic target in several cutaneous and neural diseases. We report the direct agonist effect of tacrolimus, a potent natural immunosuppressant with multiple clinical applications, on TRPM8 activity. This interaction represents a novel neuroimmune interface. The identification of a clinically approved drug with agonist activity on TRPM8 channels could be used experimentally to probe the function of TRPM8 in humans. Our findings may explain some of the sensory and anti-inflammatory effects described for this drug in the skin and the eye surface.

Calcium-induced outgrowth of astrocytic peripheral processes requires actin binding by Profilin-1.

Peripheral astrocytic processes (PAPs) are highly motile structures that are strategically positioned in close proximity to synapses. Long-lasting PAP retraction in hypothalamus is known to alter synaptic transmission. The PAP motility is likely to be actin-based because they are known to contain actin-related proteins such as Ezrin. However, the link between dynamic activity-dependent changes in astrocytic morphology and the synaptic function has not been established experimentally, presumably due to lack of appropriate tools. To selectively suppress activity-dependent morphological plasticity of astrocytes, we developed a bicistronic construct that allows simultaneous tracing and manipulating the morphology of PAPs. The construct is designed for co-expression of (i) the mutant actin binding protein Profilin-1 (abdProf-1) with a single amino acid substitution (H119E) that prevents its binding to actin monomers with (ii) the membrane-targeted morphological tracer LckGFP. Cultured cortical astrocytes transfected with this construct showed abdProf-1 overexpression at a 5-fold level compared to the endogenous Profilin-1. The cells also expressed LckGFP at a level sufficient for precise morphological tracing. We found that photolysis of caged Ca²âº induced a pronounced outgrowth of PAPs, which was suppressed by abdProf-1 overexpression in terms of PAP number, growth rate and maximal length. In contrast, the morphological complexity of astrocytes, basal motility of their PAPs and major cytoskeletal structures were not affected by abdProf-1 overexpression. In summary, we identified the actin binding by Profilin-1 as a pivotal mechanism in activity-dependent morphological plasticity of PAPs in cultured astrocytes.