Bringing the Heat

From feeling the warmth of a fresh cup of tea to the furnace in your mouth following a particularly spicy dish, we are all familiar with the concept heat. What is less familiar are the physical and molecular mechanisms that enable our bodies to sense heat. The TrpV1 ion channels are directly regulated by heat (Caterina et al. 1997). As they warm, their pores open and allow various cations such as Ca2+, Na+, and K+ to pass. Members of the TrpV channel family, notably TrpV1, contribute to the feeling of hot by allowing an influx of cations into nociceptive (pain detecting) neurons. An increase of positive charge entering a neuron will depolarize it until the cell suddenly fires an action potential. This signal is propagated along your nervous system to relay the feeling of “hot” the brain. Chemicals such as capsaicin, that ingredient which makes chili peppers hot, can act as agonists to a member of this family of channels. This means capsaicin molecules force TRPV channels to open and make you feel heat even if your spicy salsa is served cold.

Another ion channel, TMEM16A, colocalizes with TrpV1 in pain-sensing dorsal root ganglion neurons. Like TrpV1, these channels are also proposed to be heat-activated (Cho et al. 2012). Unlike the cation-passing TrpV1 channel, a TMEM16A channel conducts Cl- currents. For these DRG neurons, the net result is the same whether a positive charge enters the cell through a TRPV channel or a negative charge leaves through a TMEM16A channel: the neuron is depolarized. Since TMEM16A and TrpV1 are found on the same neurons, when it fires the same signal, “I am warm!”, would be sent. Even so, the connection of TMEM16A to heat sensation remains incompletely characterized. For a 2015 manuscript, Farah Deba and Bret Bessac utilized chemical agonists and antagonists of ion channels to investigate how TMEM16A and TrpV1 may interact in living cells. For this publication, the researchers used a TMEM16A activator (E-Act) to open the Cl- channel, and Ano1-inhib to chemically inhibit the channel.

To study the how the concentration of Cl- within a mouse dorsal root ganglion neuron affects the ability of TMEM16A to help it fire, this research team used a clever trick. First, they inject a neuron with just enough current to depolarize a neuron without triggering the irreversible action potential. Think of this as akin to precariously perching a ball at the edge of a cliff. If there is a force that is further depolarizing the cell (like Cl- leaving or Na+ entering), then the neuron will suddenly fire and give a large easily measurable signal. The ball will fall quickly down to the ground. In the opposite case (like Cl- entering or Na+ leaving the cell), the neuron is being slightly repolarized towards its stable resting membrane potential. The metaphorical ball is nudged away from the cliff. At the end, whether the neuron fired or not, the cell is reset and brought back to the precarious state to test again every second to get many samples on a single cell.

So, what did they find? When the Cl- concentration was high in the cell, sometimes the neuron fired and sometimes it didn’t. But when they added E-Act to open TMEM16A channels, the neurons started firing every time. Cl- ions were leaving the cell through TMEM16A and gave just enough of a depolarizing nudge to make the neuron fire! As they lowered the Cl-concentration to more physiological levels, the neuron still fired pretty reliably when TMEM16A was opened. But when they made the Cl- concentration low, the opposite happened. There was too little Cl- to flood out and Cl- was actually entering the neurons, making them more negatively charged. This inhibited action potentials from being triggered when E-Act was applied. On the other hand, when TrpV1 channels were made to open with capsaicin, neurons almost always fired. It didn’t matter what the Cl- concentration was because TrpV1 does not let Cl- pass through, the influx of positive charges was enough to push the neuron over the edge. What was really interesting is that closing TMEM16A with Ano1-inhib made it harder for TrpV1 to cause neurons to fire only with higher intracellular Cl- concentrations. The researchers hypothesize that this could be due to TrpV1 letting Ca2+ into the cell. Some of the Ca2+ finds its way to TMEM16A channels and opens them. This would let Cl- ions leave and the combination of positive atoms moving in and negative atoms moving out enhances the depolarizing effect. This means TrpV1 and TMEM16A might be working together to make a neuron fire in response to heat!

Indeed, these results are even corroborated in vivo. When mice paws are injected with E-Act to make their TMEM16A channels open, they act as if their feet are hot. This effect is reduced when Ano1-inhib is applied alongside it to close some of the TMEM16A channels. Similarly, when capsaicin is injected alongside a TMEM16A inhibitor, the nocifensive (pain feeling) behaviors in mice decrease compared to capsaicin alone. Blocking TMEM16A reduced the ability of TrpV1 to cause pain.

Taken together the data presented by Farah Deba and Bret Bessac paint a picture of collaboration between TrpV1 and TMEM16A ion channels. Activation of TrpV1 may lead to the Ca2+-mediated activation of TMEM16A. With both channels open, the membrane of a neuron gains positive charge more rapidly and ends up more likely to fire. While TrpV1 has long been a target of analgesic pharmaceuticals, the discovery that TMEM16A may work with TrpV1 in heat sensation opens new possible routes for pain management. Future studies aimed at finding minimally toxic inhibitors of TMEM16A could provide a smorgasbord of novel analgesic drugs!

Post by Alex Francette and Anne Carlson