Non-conventional Axonal Organelles Control TRPM8 Ion Channel Trafficking and Peripheral Cold Sensing.

TRPM8 is the main ion channel responsible for cold transduction in the somatosensory system. Nerve terminal availability of TRPM8 determines cold sensitivity, but how axonal secretory organelles control channel delivery remains poorly understood. Here we examine the distribution of TRPM8 and trafficking organelles in cold-sensitive peripheral axons and disrupt trafficking by targeting the ARF-GEF GBF1 pharmacologically or the small GTPase RAB6 by optogenetics. In axons of the sciatic nerve, inhibition of GBF1 interrupts TRPM8 trafficking and increases association with the trans-Golgi network, LAMP1, and Golgi satellites, which distribute profusely along the axonal shaft. Accordingly, both TRPM8-dependent ongoing activity and cold-evoked responses reversibly decline upon GBF1 inhibition in nerve endings of corneal cold thermoreceptors. Inhibition of RAB6, which also associates to Golgi satellites, decreases cold-induced responses in vivo. Our results support a non-conventional axonal trafficking mechanism controlling the availability of TRPM8 in axons and cold sensitivity in the peripheral nervous system.

Salsolinol and isosalsolinol: condensation products of acetaldehyde and dopamine. Separation of their enantiomers in the presence of a large excess of dopamine.

Dopamine (DA) condenses, at least in vitro, with acetaldehyde, the primary metabolite of ethanol, to form the regioisomers salsolinol (SAL) and isosalsolinol (isoSAL). An alternative in vivo route to SAL, requiring a decarboxylation step, has been suggested via condensation of DA with pyruvic acid. SAL has been proposed as a mediator of the rewarding effects of ethanol in the brain. We have now shown by HPLC, nuclear magnetic resonance (NMR) and mass spectrometry (MS) that the commercially available SAL contains about 10% of isoSAL, whose biological activity is unknown. If SAL is indeed the biologically active metabolite, rather than isoSAL, it is also unknown whether the rewarding molecule is (S)- or (R)-SAL. We have developed methodologies for the quantitative determination of DA, SAL and isoSAL using ion-pair reversed-phase HPLC, and for the separation of DA from (S)- and (R)-SAL and an isoSAL enantiomer on a ß-cyclodextrin-modified column, in both cases with electrochemical detection. A significant advance over earlier methods was achieved for the analysis of (S)- and (R)-SAL in the presence of a large excess of DA (100:1 DA-SAL ratio), as expected to occur in vivo, by suppressing the DA peak by selective derivatization with 2,3-naphthalenedicarboxaldehyde into a molecule that is electrochemically silent at the electrode potential used. The methodologies developed will allow the separation and determination of the pharmacological activity of these two products of condensation of acetaldehyde with DA. Further, the techniques for (S)- and (R)-SAL separation at a high DA:SA ratio will allow the existence of a putative (R)-SAL synthase to be determined and, if it exists, its role in alcoholism.