Cellular permeation of large molecules mediated by TRPM8 channels

Abstract

While most membrane channels are only capable of passing small ions, certain non-selective cation channels have been recently shown to have the capacity to permeate large cations. The mechanisms underlying large molecule permeation are unclear, but this property has been exploited pharmacologically to target molecules, such nerve conduction blockers, to specific subsets of pain-sensing neurons (nociceptors) expressing the heat-gated transient receptor potential (TRP) channel TRPV1. However, it is not clear if the principal mediator of cold stimuli TRPM8 is capable of mediating the permeation large molecules across cell membranes, suggesting that TRPM8-positive nerves cannot be similarly targeted. Here we show that both heterologous cells and native sensory neurons expressing TRPM8 channels allow the permeation of the large fluorescent cation Po-Pro3. Po-Pro3 influx is blocked by TRPM8-specific antagonism and when channel activity is desensitized. The effects of the potent agonist WS-12 are TRPM8-specific and dye uptake mediated by TRPM8 channels is similar to that observed with TRPV1. Lastly, we find that as with TRPV1, activation of TRPM8 channels can be used as a means to target intracellular uptake of cell-impermeable sodium channel blockers. In a neuronal cell line expressing TRPM8 channels, voltage-gated sodium currents are blocked in the presence of the cell-impermeable, charged lidocaine derivative QX-314 and WS-12. These results show that the ability of somatosensory TRP channels to promote the permeation of large cations also includes TRPM8, thereby suggesting that novel approaches to alter cold pain can also be employed via conduction block in TRPM8-positive sensory neurons.

Selective cold pain inhibition by targeted block of TRPM8-expressing neurons with quaternary lidocaine derivative QX-314

“…Moreover, cold hypersensitivity associated with inflammatory and neuropathic injury is diminished in the absence of this channel 24 , 27 , 28 . Two reports suggested that TRPM8 does not permeate large cations in vitro and in vivo 29 , 30 , whereas we and others have recently shown uptake of large cations in both heterologous cells and sensory neurons expressing TRPM8 channels, as well as found that co-application of TRPM8 agonists with QX-314 blocked Na + currents in these cells 6 , 31 . Nonetheless, while these latter studies showed that TRPM8 channels permeate large cations, it was unclear if they can be employed for cell-specific targeting of molecules in vivo.…”

“…As we find that large cations permeate TRPM8 channels expressed in heterologous and native cells, and that QX-314 blocks sodium currents in TRPM8-expressing cells stimulated with TRPM8 agonists 31 , we asked if TRPM8 activation in the presence of QX-314 can alter cold sensitivity in vivo. First, to confirm that in vivo thermal responses can be blocked in our hands using TRP channel activation, we examined heat responses in adult wild-type mice after injection of QX-314 and the TRPV1 agonist capsaicin 8 .…”

“…Menthol is a low-potency TRPM8 agonist and been shown to act nonspecifically at high concentrations, such as those required to elicit a behavioral response 22 , 34 . However, the TRPM8 agonist WS-12 has a >100-fold higher affinity than menthol and shown to act specifically on TRPM8 in vivo 31 , 34 . We find that intraplantar injection of 20 µg WS-12 transiently increases cold sensitivity postinjection (Fig.…”

Pore-Forming Proteins: Fluorescent Dyes to Study the Channel Functionality and Biophysical Properties

“…Anionics such as Lucifer Yellow (457 Da, 2 À ) Fluo-4 (736.63 Da, 4 À ), Fura-2 (832 Da, 5 À ), and fluorescein (332.31 Da, 1 À ) are usually ion markers or intracellular structures [27,59,60]. A study by McCoy et al [42] on the permeabilization of large molecules mediated by the TRPM8 receptor, the cationic dye PO-PRO-3 (605.30 Da, 2 + ), was used to measure the dilation of the pore. However, it is not common to use fluorophores other than those mentioned above, having a great scenario to be explored in ionic conductance tests.…”

Targeting dorsal root ganglia and primary sensory neurons for the treatment of chronic pain

“…Recent progress has been made in selective targeting of different subsets DRG neurons [42-47]. Local anesthetics are mostly uncharged molecules that penetrate the membrane of all neurons, blocking not only sensory neurons but also motor and autonomic neurons, generating undesired effects such as blood pressure changes and motor deficits.…”