Pain is the most common presenting physical symptom and a primary reason for seeking medical care, which chronically affects people\'s mental health and social life. CaV3.2 channel plays an essential role in the peripheral processing maintenance of pain states. This study was designed to identify novel drug candidates targeting the CaV3.2 channel. Whole-cell patch-clamp, cellular thermal shift assay, FlexStation, in vivo and in vitro CaV3.2 knock-down, site-directed mutagenesis, and double-mutant cycle analysis were employed to explore the pain-related receptors and ligand-receptor direct interaction. We found that toddaculin efficiently inhibits the CaV3.2 channel and significantly reduced the excitability of dorsal root ganglion neurons and pain behaviors. The Carbonyl group of coumarins directly interacts with the pore domain of CaV3.2 via van der Waals (VDW) force. Docking with binding pockets further led us to identify glycycoumarin, which exhibited more potent inhibition on the CaV3.2 channel and better analgesic activity than the parent compound. Toddaculin and its analog showed beneficial therapeutic effects in pain models. Toddaculin binding pocket on CaV3.2 might be a promising docking site for the design of drugs.

T-type calcium (Ca2+) channels play important physiological functions in excitable cells including cardiomyocyte. Phosphatidylinositol-4,5-bisphosphate (PIP2) has recently been reported to modulate various ion channels\' function. However the actions of PIP2 on the T-type Ca2+ channel remain unclear. To elucidate possible effects of PIP2 on the T-type Ca2+ channel, we applied patch clamp method to investigate recombinant CaV3.1- and CaV3.2-T-type Ca2+ channels expressed in mammalian cell lines with PIP2 in acute- and long-term potentiation. Short- and long-term potentiation of PIP2 shifted the activation and the steady-state inactivation curve toward the hyperpolarization direction of CaV3.1-ICa.T without affecting the maximum inward current density. Short- and long-term potentiation of PIP2 also shifted the activation curve toward the hyperpolarization direction of CaV3.2-ICa.T without affecting the maximum inward current density. Conversely, long-term but not short-term potentiation of PIP2 shifted the steady-state inactivation curve toward the hyperpolarization direction of CaV3.2-ICa.T. Long-term but not short-term potentiation of PIP2 blunted the voltage-dependency of current decay CaV3.1-ICa.T. PIP2 modulates CaV3.1- and CaV3.2-ICa.T not by their current density but by their channel gating properties possibly through its membrane-delimited actions.

T-type calcium channels are prominently expressed in primary nociceptive fibers and well characterized in pain processes. Although itch and pain share many similarities including primary sensory fibers, the function of T-type calcium channels on acute itch has not been explored. We investigated whether T-type calcium channels expressed within primary sensory fibers of mouse skin, especially Cav3.2 subtype, involve in chloroquine-, endothelin-1- and histamine-evoked acute itch using pharmacological, neuronal imaging and behavioral analyses. We found that pre-locally blocking three subtypes of T-type calcium channels in the peripheral afferents of skins, yielded an inhibition in acute itch or pain behaviors, while selectively blocking the Cav3.2 channel in the skin peripheral afferents only inhibited acute pain but not acute itch. These results suggest that T-type Cav3.1 or Cav3.3, but not Cav3.2 channel, have an important role in acute itch processing, and their distinctive roles in modulating acute itch are worthy of further investigation.