The transient receptor potential channel vanilloid type 1 (TRPV1) is activated by a variety of endogenous and exogenous stimuli and is involved in nociception and body temperature regulation. Although the structure of TRPV1 has been experimentally determined in both the closed and open states, very little is known about its activation mechanism. In particular, the conformational changes that occur in the pore domain and result in ionic conduction have not yet been identified. Here we suggest a hypothetical molecular mechanism for TRPV1 activation, which involves rotation of a conserved asparagine in S6 from a position facing the S4–S5 linker toward the pore. This rotation is associated with hydration of the pore and dehydration of the four peripheral cavities located between each S6 and S4–S5 linker. In light of our hypothesis, we perform bioinformatics analyses of TRP and other evolutionary related ion channels, evaluate newly available structures, and reexamine previously reported water accessibility and mutagenesis experiments. These analyses provide several independent lines of evidence to support our hypothesis. Finally, we show that our proposed molecular mechanism is compatible with the prevailing theory that the selectivity filter acts as a secondary gate in TRPV1.

Phosphoinositides are emerging as general regulators of the functionally diverse transient receptor potential (TRP) ion channel family. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) has been reported to positively regulate many TRP channels, but in several cases phosphoinositide regulation is controversial. TRP melastatin 3 (TRPM3) is a heat-activated ion channel that is also stimulated by chemical agonists, such as pregnenolone sulfate. Here, we used a wide array of approaches to determine the effects of phosphoinositides on TRPM3. We found that channel activity in excised inside-out patches decreased over time (rundown), an attribute of PI(4,5)P 2 -dependent ion channels. Channel activity could be restored by application of either synthetic dioctanoyl (diC 8 ) or natural arachidonyl stearyl (AASt) PI(4,5)P 2 . The PI(4,5)P 2 precursor phosphatidylinositol 4-phosphate (PI(4)P) was less effective at restoring channel activity. TRPM3 currents were also restored by MgATP, an effect which was inhibited by two different phosphatidylinositol 4-kinase inhibitors, or by pretreatment with a phosphatidylinositol-specific phospholipase C (PI-PLC) enzyme, indicating that MgATP acted by generating phosphoinositides. In intact cells, reduction of PI(4,5)P 2 levels by chemically inducible phosphoinositide phosphatases or a voltage-sensitive 5′-phosphatase inhibited channel activity. Activation of PLC via muscarinic receptors also inhibited TRPM3 channel activity. Overall, our data indicate that TRPM3 is a phosphoinositide-dependent ion channel and that decreasing PI(4,5)P 2 abundance limits its activity. As all other members of the TRPM family have also been shown to require PI(4,5)P 2 for activity, our data establish PI(4,5)P 2 as a general positive cofactor of this ion channel subfamily.