Recent studies have demonstrated an important role for T-type Ca2ϩ channels (T-channels) in controlling the excitability of peripheral pain-sensing neurons (nociceptors). However, the molecular mechanisms underlying the functions of T-channels in nociceptors are poorly understood. Here, we demonstrate that reducing agents as well as endogenous metal chelators sensitize C-type dorsal root ganglion nociceptors by chelating Zn 2ϩ ions off specific extracellular histidine residues on Ca v 3.2 T-channels, thus relieving tonic channel inhibition, enhancing Ca v 3.2 currents, and lowering the threshold for nociceptor excitability in vitro and in vivo. Collectively, these findings describe a novel mechanism of nociceptor sensitization and firmly establish reducing agents, as well as Zn 2ϩ , Zn 2ϩ -chelating amino acids, and Zn 2ϩ -chelating proteins as endogenous modulators of Ca v 3.2 and nociceptor excitability.

T-type Ca2ϩ channels (T-channels) are involved in the control of neuronal excitability and their gating can be modulated by a variety of redox agents. Ascorbate is an endogenous redox agent that can function as both an anti-and pro-oxidant. Here, we show that ascorbate selectively inhibits native Ca v 3.2 T-channels in peripheral and central neurons, as well as recombinant Ca v 3.2 channels heterologously expressed in human embryonic kidney 293 cells, by initiating the metal-catalyzed oxidation of a specific, metal-binding histidine residue in domain 1 of the channel. Our biophysical experiments indicate that ascorbate reduces the availability of Ca v 3.2 channels over a wide range of membrane potentials, and inhibits Ca v 3.2-dependent low-threshold-Ca 2ϩ spikes as well as burst-firing in reticular thalamic neurons at physiologically relevant concentrations. This study represents the first mechanistic demonstration of ion channel modulation by ascorbate, and suggests that ascorbate may function as an endogenous modulator of neuronal excitability.

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Ca v 3.2 T-type channels contain a high affinity metal binding site for trace metals such as copper and zinc. This site is occupied at physiologically relevant concentrations of these metals, leading to decreased channel activity and pain transmission. A histidine at position 191 was recently identified as a critical determinant for both trace metal block of Ca v 3.2 and modulation by redox agents. His 191 is found on the extracellular face of the Ca v 3.2 channel on the IS3-S4 linker and is not conserved in other Ca v 3 channels. Mutation of the corresponding residue in Ca v 3.1 to histidine, Gln 172 , significantly enhances trace metal inhibition, but not to the level observed in wild-type Ca v 3.2, implying that other residues also contribute to the metal binding site. The goal of the present study is to identify these other residues using a series of chimeric channels. The key findings of the study are that the metal binding site is composed of a Asp-Gly-His motif in IS3-S4 and a second aspartate residue in IS2. These results suggest that metal binding stabilizes the closed conformation of the voltage-sensor paddle in repeat I, and thereby inhibits channel opening. These studies provide insight into the structure of T-type channels, and identify an extracellular motif that could be targeted for drug development.

The excitability of hippocampal pyramidal neurons is regulated by activation of metabotropic glutamate receptors, an effect that is mediated by modulation of R-type calcium channels.