TREK-1, TREK-2 and TRAAK are members of the two-pore domain K + (K 2P ) channel family and are activated by membrane stretch and free fatty acids. TREK-1 has been shown to be sensitive to temperature in expression systems. We studied the temperature-sensitivity of TREK-2 and TRAAK in COS-7 cells and in neuronal cells. In transfected COS-7 cells, TREK-2 and TRAAK whole-cell currents increased ∼20-fold as the bath temperature was raised from 24• C to 42• C. Similarly, in cell-attached patches of COS-7 cells, channel activity was very low, but increased progressively as the bath temperature was raised from 24• C to 42 • C. The thresholds for activation of TREK-2 and TRAAK were ∼25• C and ∼31 • C, respectively. Other K 2P channels such as TASK-3 and TRESK-2 were not significantly affected by an increase in temperature from 24• C to 37 • C. When the C-terminus of TREK-2 was replaced with that of TASK-3, its sensitivity to free fatty acids and protons was abolished, but the mutant could still be activated by heat. At 37• C, TREK-1, TREK-2 and TRAAK were sensitive to arachidonic acid, pH and membrane stretch in both cell-attached and inside-out patches. In cerebellar granule and dorsal root ganglion neurones, TREK-1, TREK-2 and TRAAK were generally inactive in the cell-attached state at 24• C, but became very active at 37• C. In cell-attached patches of ventricular myocytes, TREK-1 was also normally closed at 24• C, but was active at 37 • C. These results show that TREK-2 and TRAAK are also temperature-sensitive channels, are active at physiological body temperature, and therefore would contribute to the background K + conductance and regulate cell excitability in response to various physical and chemical stimuli.

Dorsal root ganglion (DRG) neurons express mRNAs for many two-pore domain K(+) (K(2P)) channels that behave as background K(+) channels. To identify functional background K(+) channels in DRG neurons, we examined the properties of single-channel openings from cell-attached and inside-out patches from the cell bodies of DRG neurons. We found seven types of K(+) channels, with single-channel conductance ranging from 14 to 120 pS in 150 mM KCl bath solution. Four of these K(+) channels showed biophysical and pharmacological properties similar to TRESK (14 pS), TREK-1 (112 pS), TREK-2 (50 pS), and TRAAK (73 pS), which are members of the K(2P) channel family. The molecular identity of the three other K(+) channels could not be determined, as they showed low channel activity and were observed infrequently. Of the four K(2P) channels, the TRESK-like (14 pS) K(+) channel was most active at 24 degrees C. At 37 degrees C, the 50-pS (TREK-2 like) channel was the most active and contributed the most (69%) to the resting K(+) current, followed by the TRESK-like 14-pS (16%), TREK-1-like 112-pS (12%), and TRAAK-like 73-pS (3%) channels. In DRG neurons, mRNAs of all four K(2P) channels, as well as those of TASK-1 and TASK-3, were expressed, as judged by RT-PCR analysis. Our results show that TREKs and TRESK together contribute >95% of the background K(+) conductance of DRG neurons at 37 degrees C. As TREKs and TRESK are targets of modulation by receptor agonists, they are likely to play an active role in the regulation of excitability in DRG neurons.

TRPA1 is an ion channel and has been proposed as a thermosensor across species. In invertebrate and ancestral vertebrates such as fly, mosquito, frog, lizard and snakes, TRPA1 serves as a heat receptor, a sensory input utilized for heat avoidance or infrared detection. However, in mammals, whether TRPA1 is a receptor for noxious cold is highly controversial, as channel activation by cold was observed by some groups but disputed by others. Here we attribute the discrepancy to species differences. We show that cold activates rat and mouse TRPA1 but not human or rhesus monkey TRPA1. At the molecular level, a single residue within the S5 transmembrane domain (G878 in rodent but V875 in primate) accounts for the observed difference in cold sensitivity. This residue difference also underlies the species-specific effects of menthol. Together, our findings identify the species-specific cold activation of TRPA1 and reveal a molecular determinant of cold-sensitive gating.

TASK-1 and TASK-3 are functional members of the tandem-pore K + (K 2P ) channel family, and mRNAs for both channels are expressed together in many brain regions. Although TASK-1 and TASK-3 subunits are able to form heteromers when their complementary RNAs are injected into oocytes, whether functional heteromers are present in the native tissue is not known. Using cultured cerebellar granule (CG) neurones that express mRNAs of both TASK-1 and TASK-3, we studied the presence of heteromers by comparing the sensitivities of cloned and native K + channels to extracellular pH (pH o ) and ruthenium red. The single-channel conductance of TASK-1, TASK-3 and a tandem construct (TASK-1/TASK-3) expressed in COS-7 cells were 14.2 ± 0.4, 37.8 ± 0.7 and 38.1 ± 0.7 pS (-60 mV), respectively. TASK-3 and TASK-1/TASK-3 (and TASK-3/TASK-1) displayed nearly identical single-channel kinetics. TASK-3 and TASK-1/ TASK-3 expressed in COS-7 cells were inhibited by 26 ± 4 and 36 ± 2 %, respectively, when pH o was changed from 8.3 to 7.3. In outside-out patches from CG neurones, the K + channel with single channel properties similar to those of TASK-3 was inhibited by 31 ± 7 % by the same reduction in pH o . TASK-3 and TASK-1/TASK-3 expressed in COS-7 cells were inhibited by 78 ± 7 and 3 ± 4 %, respectively, when 5 µM ruthenium red was applied to outside-out patches. In outside-out patches from CG neurones containing a 38 pS channel, two types of responses to ruthenium red were observed. Ruthenium red inhibited the channel activity by 77 ± 5 % in 42 % of patches (range: 72-82 %) and by 5 ± 4 % (range: 0-9 %) in 58 % of patches. When patches contained more than three 38 pS channels, the average response to ruthenium red was 47 ± 6 % inhibition (n = 5). These electrophysiological studies show that native 38 pS K + channels of the TASK family in cultured CG neurones consist of both homomeric TASK-3 and heteromeric TASK-1/TASK-3.