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.

T-type Ca2+ channels play essential roles in numerous cellular processes. Recently, we reported that phorbol-12-myristate-13-acetate (PMA) potently enhanced the current amplitude of Ca v 3.2 T-type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Ca v 3.1, Ca v 3.2 and Ca v 3.3 channels, and have investigated the underlying mechanism. PMA augmented the current amplitudes of the three T-type channel isoforms, but the fold stimulations and time courses differed. The augmentation effects were not mimicked by 4α-PMA, an inactive stereoisomer of PMA, but were abolished by preincubation with protein kinase C (PKC) inhibitors, indicating that PMA augmented T-type channel currents via activation of oocyte PKC. The stimulation effect on Ca v 3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Ca v 3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Ca v 3.1 T-type channels was not significantly changed by activation of PKC. The PKC effect on Ca v 3.1 was localized to the cytoplasmic II-III loop using chimeric channels with individual cytoplasmic loops of Ca v 3.1 replaced by those of Ca v 2.1.

Ca2ϩ influx through T-type Ca 2ϩ channels is crucial for important physiological activities such as hormone secretion and neuronal excitability. However, it is not clear whether these channels are regulated by cAMP-dependent protein kinase A (PKA). In the present study, we examined whether PKA modulates Ca v 3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 M forskolin, an adenylyl cyclase stimulant, increased Ca v 3.2 channel activity by 40 Ϯ 4% over 30 min and negatively shifted the steady-state inactivation curve (V 50 ϭ Ϫ61.4 Ϯ 0.2 versus Ϫ65.5 Ϯ 0.1 mV). Forskolin did not affect other biophysical properties of Ca v 3.2 channels, including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 M 8-bromocAMP, a membrane-permeable cAMP analog. The augmentation of Ca v 3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 M N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), and reversed by subsequent application of 500 nM protein kinase A inhibitor peptide. The stimulation of Ca v 3.2 channel activity by PKA was mimicked by serotonin when 5HT 7 receptor was coexpressed with Ca v 3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Ca v 3.2 with those of the Na v 1.4 channel, which is insensitive to PKA, we localized a region required for the PKA-mediated augmentation to the II-III loop of the Ca v 3.2.

We recently reported that a histidine (H191) in the S3-S4 loop of domain I is critical for nickel inhibition of the Ca v 3.2 T-type Ca 2+ channel. As in Ca v 3.2, two histidine residues are commonly found in the IS3-IS4 loops of mammalian Ca v 2.3 Ca 2+ channels, which are also blocked by low micromolar concentrations of nickel. We show here by site-directed mutagenesis and electrophysiology that both residues contribute to the nickel sensitivity of Ca v 2.3, with H183 being more critical than H179. These findings strongly suggest that both H179 and H183 in the IS3-IS4 loop are essential structural determinants required for nickel sensitive inhibition of the Ca v 2.3.