Jung-Ha Lee

Reducing agents sensitize C-type nociceptors by relieving high-affinity zinc inhibition of T-type calcium channels

Recent studies have demonstrated an important role for T-type Ca2+ 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 Zn2+ ions off specific extracellular histidine residues on Cav3.2 T-channels, thus relieving tonic channel inhibition, enhancing Cav3.2 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 Zn2+, Zn2+-chelating amino acids, and Zn2+-chelating proteins as endogenous modulators of Cav3.2 and nociceptor excitability.

Molecular Mechanisms of Subtype-Specific Inhibition of Neuronal T-Type Calcium Channels by Ascorbate

T-type Ca2+ 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 Cav3.2 T-channels in peripheral and central neurons, as well as recombinant Cav3.2 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 Cav3.2 channels over a wide range of membrane potentials, and inhibits Cav3.2-dependent low-threshold-Ca2+ 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.

A molecular determinant of nickel inhibition in Cav3.2 T-type calcium channels.

Molecular cloning studies have revealed that heterogeneity of T-type Ca2+ currents in native tissues arises from the three isoforms of Cav3 channels: Cav3.1, Cav3.2, and Cav3.3. From pharmacological analysis of the recombinant T-type channels, low concentrations (<50 μm) of nickel were found to selectively block the Cav3.2 over the other isoforms. To date, however, the structural element(s) responsible for the nickel block on the Cav3.2 T-type Ca2+ channel remain unknown. Thus, we constructed chimeric channels between the nickel-sensitive Cav3.2 and the nickel-insensitive Cav3.1 to localize the region interacting with nickel. Systematic assaying of serial chimeras suggests that the region preceding domain I S4 of Cav3.2 contributes to nickel block. Point mutations of potential nickel-interacting sites revealed that H191Q in the S3–S4 loop of domain I significantly attenuated the nickel block of Cav3.2, mimicking the nickel-insensitive blocking potency of Cav3.1. These findings indicate that His-191 in the S3–S4 loop is a critical residue conferring nickel block to Cav3.2 and reveal a novel role for the S3–S4 loop to control ion permeation through T-type Ca2+ channels.

Activation of protein kinase C augments T-type Ca2+ channel activity without changing channel surface density.

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 Cav3.2 T‐type channels reconstituted in Xenopus oocytes. Here, we have compared PMA modulation of the activities of Cav3.1, Cav3.2 and Cav3.3 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 Cav3.1 channel activity by PKC was mimicked by endothelin when endothelin receptor type A was coexpressed with Cav3.1 in the Xenopus oocyte system. Pharmacological studies combined with fluorescence imaging revealed that the surface density of Cav3.1 T‐type channels was not significantly changed by activation of PKC. The PKC effect on Cav3.1 was localized to the cytoplasmic II–III loop using chimeric channels with individual cytoplasmic loops of Cav3.1 replaced by those of Cav2.1.

Molecular identification of Ca 2+ channels in human sperm

The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+)channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+)channels has been limited. Here we identified Ca(2+)channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+)channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed α1B, α1C, α1E, α1G, and α1H sequences. RT-PCR using specific primers repeatedly detected α1B, α1C, α1E, α1G, and α1H mRNAs, and additionally α1I mRNA. But α1A and α1D messages were not detected. Relative expression levels of the detected Ca(2+)channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: α1H> or =α1G> or =α1E> or =α1B>α1C>α1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+)channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.

Reversal of Neuropathic Pain in Diabetes by Targeting Glycosylation of Cav3.2 T-Type Calcium Channels

It has been established that CaV3.2 T-type voltage-gated calcium channels (T-channels) play a key role in the sensitized (hyperexcitable) state of nociceptive sensory neurons (nociceptors) in response to hyperglycemia associated with diabetes, which in turn can be a basis for painful symptoms of peripheral diabetic neuropathy (PDN). Unfortunately, current treatment for painful PDN has been limited by nonspecific systemic drugs with significant side effects or potential for abuse. We studied in vitro and in vivo mechanisms of plasticity of CaV3.2 T-channel in a leptin-deficient (ob/ob) mouse model of PDN. We demonstrate that posttranslational glycosylation of specific extracellular asparagine residues in CaV3.2 channels accelerates current kinetics, increases current density, and augments channel membrane expression. Importantly, deglycosylation treatment with neuraminidase inhibits native T-currents in nociceptors and in so doing completely and selectively reverses hyperalgesia in diabetic ob/ob mice without altering baseline pain responses in healthy mice. Our study describes a new mechanism for the regulation of CaV3.2 activity and suggests that modulating the glycosylation state of T-channels in nociceptors may provide a way to suppress peripheral sensitization. Understanding the details of this regulatory pathway could facilitate the development of novel specific therapies for the treatment of painful PDN.

Alternative splicing of the rat Cav3.3 T-type calcium channel gene produces variants with distinct functional properties1

Molecular diversity in T‐type Ca2+ channels is produced by expression of three genes, and alternative splicing of those genes. Prompted by differences noted between rat and human Cav3.3 sequences, we searched for splice variants. We cloned six variants, which are produced by splicing at exon 33 and exon 34. Expression of the variants differed between brain regions. The electrophysiological properties of the variants displayed similar voltage‐dependent gating, but differed in their kinetic properties. The functional impact of splicing was inter‐related, suggesting an interaction. We conclude that alternative splicing of the Cav3.3 gene produces channels with distinct properties.

Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A.

Ca2+ influx through T-type Ca2+ 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 Cav3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 μM forskolin, an adenylyl cyclase stimulant, increased Cav3.2 channel activity by 40 ± 4% over 30 min and negatively shifted the steady-state inactivation curve (V50 = -61.4 ± 0.2 versus -65.5 ± 0.1 mV). Forskolin did not affect other biophysical properties of Cav3.2 channels, including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 μM 8-bromo-cAMP, a membrane-permeable cAMP analog. The augmentation of Cav3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 μM N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), and reversed by subsequent application of 500 nM protein kinase A inhibitor peptide. The stimulation of Cav3.2 channel activity by PKA was mimicked by serotonin when 5HT7 receptor was coexpressed with Cav3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Cav3.2 with those of the Nav1.4 channel, which is insensitive to PKA, we localized a region required for the PKA-mediated augmentation to the II-III loop of the Cav3.2.

Modulation of Cav3.2 T-type Ca2+ channels by protein kinase C

Although T‐type Ca2+ channels have been implicated in numerous physiological functions, their regulations by protein kinases have been obscured by conflicting reports. We investigated the effects of protein kinase C (PKC) on Cav3.2 T‐type channels reconstituted in Xenopus oocytes. Phorbol‐12‐myristate‐13‐acetate (PMA) strongly enhanced the amplitude of Cav3.2 channel currents (∼3‐fold). The augmentation effects were not mimicked by 4α‐PMA, an inactive stereoisomer of PMA, and abolished by preincubation with PKC inhibitors. Our findings suggest that PMA upregulates Cav3.2 channel activity via activation of oocyte PKC.

Structural determinants of the high affinity extracellular zinc binding site on Cav3.2 T-type calcium channels

Cav3.2 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 Cav3.2 and modulation by redox agents. His191 is found on the extracellular face of the Cav3.2 channel on the IS3-S4 linker and is not conserved in other Cav3 channels. Mutation of the corresponding residue in Cav3.1 to histidine, Gln172, significantly enhances trace metal inhibition, but not to the level observed in wild-type Cav3.2, 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.