Chike Cao

Gating of Transient Receptor Potential Melastatin 8 (TRPM8) Channels Activated by Cold and Chemical Agonists in Planar Lipid Bilayers

The transient receptor potential melastatin 8 (TRPM8) ion channel is a major sensor of environmental cold temperatures. It is activated by cold and chemical agonists, such as menthol and icilin. The activation of these channels both by cold and cooling agents requires the presence of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. The mechanism of TRPM8 activation by physical and chemical factors is unknown, and the involvement of cellular signaling pathways has been considered. Here we have characterized the gating mechanism of the rat TRPM8 reconstituted in planar lipid bilayers and its activation by different stimuli. In this system, the influence of cellular signaling pathways can be excluded. We found that TRPM8 activated by cold exhibits steep temperature dependence [temperature coefficient (Q 10) of ∼40], and the channel openings are accompanied by large changes in entropy and enthalpy, suggesting a substantial conformation change. TRPM8 channel behavior upon menthol and icilin activation was distinguishable, and the effect of icilin depended on the presence of calcium on the intracellular side of the protein. Here we also demonstrate that PI(4,5)P2 is the prime factor that impacts the gating of TRPM8 and that other phosphoinositides are less efficient in supporting channel activity. Menthol increases the potency of PI(4,5)P2 to activate the channels and increases binding of phosphoinositides to the full-length channel protein. Our data demonstrate conclusively that TRPM8 is gated by cold and its chemical agonists directly, and that dependence of its gating on PI(4,5)P2 is a result of direct specific interactions with the lipid.

Decrease in phosphatidylinositol 4,5-bisphosphate levels mediates desensitization of the cold sensor TRPM8 channels

Non‐technical summary  The transient receptor potential melastatin 8 (TRPM8) ion channel is a physiological sensor of environmental cold temperatures. This channel is also activated by menthol, which is responsible for the cooling sensation evoked by this compound. It is well known that we adapt to moderately cold temperatures, i.e. the same temperature feels less cold over time, and the cooling effects of menthol also wear off with time, presumably due to the calcium‐dependent desensitization of TRPM8 activity. The membrane phospholipid phosphatidylinositol 4,5‐bisphosphate (PIP2) is known to be required for TRPM8 activity. Our data support a model for desensitization, in which calcium influx through TRPM8 activates a phospholipase C enzyme, which breaks down PIP2, leading to decreased channel activity.

Intracellular ATP supports TRPV6 activity via lipid kinases and the generation of PtdIns(4,5)P2

Transient receptor potential vanilloid 6 (TRPV6) channels play an important role in Ca2+ absorption in the intestines. Both phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] and cytoplasmic ATP have been proposed to be important for maintaining TRPV6 activity. To evaluate whether PtdIns(4,5)P2 and ATP affect channel activity directly or indirectly, we have used a dual approach, examining channel activity in excised patches and planar lipid bilayers. In excised inside‐out patch‐clamp measurements, ATP reactivated the human TRPV6 channels after current rundown only in the presence of Mg2+. The effect of MgATP was inhibited by 3 structurally different compounds that inhibit type III phosphatidylinositol 4‐kinases (PI4Ks). PtdIns(4,5)P2 also activated TRPV6 in excised patches, while its precursor PtdIns(4)P had only minimal effect. These data demonstrate that MgATP provides substrate for lipid kinases, allowing the resynthesis of PtdIns(4,5)P2. To determine whether PtdIns(4,5)P2 is a direct activator of TRPV6, we purified and reconstituted the channel protein in planar lipid bilayers. The reconstituted channel showed high activity in the presence of PtdIns(4,5)P2, while PtdIns(4)P induced only minimal activity. Our data establish PtdIns(4,5)P2 as a direct activator of TRPV6 and demonstrate that intracellular ATP regulates the channel indirectly as a substrate for type III PI4Ks.—Zakharian, E., Cao, C., Rohacs, T. Intracellular ATP supports TRPV6 activity via lipid kinases and the generation of PtdIns(4,5)P2. FASEB J. 25, 3915–3928 (2011). www.fasebj.org

Interplay between calmodulin and phosphatidylinositol 4,5-bisphosphate in Ca2+-induced inactivation of Transient Receptor Potential Vanilloid 6 channels

Background: A large number of calmodulin-binding sites have been proposed in TRPV6. Results: We have identified the site that is responsible for inhibition of TRPV6 by calmodulin in excised inside-out patch clamp experiments. Conclusion: Calmodulin and PI(4,5)P2 antagonistically regulate TRPV6, but not through direct competition. Significance: This study provides mechanistic insight into Ca2+-induced inactivation of TRPV6. The epithelial Ca2+ channel transient receptor potential vanilloid 6 (TRPV6) undergoes Ca2+-induced inactivation that protects the cell from toxic Ca2+ overload and may also limit intestinal Ca2+ transport. To dissect the roles of individual signaling pathways in this phenomenon, we studied the effects of Ca2+, calmodulin (CaM), and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in excised inside-out patches. The activity of TRPV6 strictly depended on the presence of PI(4,5)P2, and Ca2+-CaM inhibited the channel at physiologically relevant concentrations. Ca2+ alone also inhibited TRPV6 at high concentrations (IC50 = ∼20 μm). A double mutation in the distal C-terminal CaM-binding site of TRPV6 (W695A/R699E) essentially eliminated inhibition by CaM in excised patches. In whole cell patch clamp experiments, this mutation reduced but did not eliminate Ca2+-induced inactivation. Providing excess PI(4,5)P2 reduced the inhibition by CaM in excised patches and in planar lipid bilayers, but PI(4,5)P2 did not inhibit binding of CaM to the C terminus of the channel. Overall, our data show a complex interplay between CaM and PI(4,5)P2 and show that Ca2+, CaM, and the depletion of PI(4,5)P2 all contribute to inactivation of TRPV6.