Jin-Yong Park

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.

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.

Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang.

Bacillus amyloliquefancies CH51 isolated from cheonggukjang, a traditional Korean fermented soy food, has strong fibrinolytic activity and produces several fibrinolytic enzymes. Among four different growth media, tryptic soy broth was the best in terms of supporting cell growth and fibrinolytic activity of this strain. A protein with fibrinolytic activity was partially purified from the culture supernatant by CMSephadex and Phenyl Sepharose column chromatographies. Tandem mass spectrometric analysis showed that this protein is a homolog of AprE from B. subtilis and it was accordingly named AprE51. The optimum pH and temperature for partially purified AprE51 activity were 6.0 and 45 degrees , respectively. A gene encoding AprE51, aprE51, was cloned from B. amyloliquefaciens CH51 genomic DNA. The aprE51 gene was overexpressed in heterologous B. subtilis strains deficient in fibrinolytic activity using an E.colo-Bacillus Shuttle vector, pHY300PLK.

A Post-Burst Afterdepolarization Is Mediated by Group I Metabotropic Glutamate Receptor-Dependent Upregulation of Cav2.3 R-Type Calcium Channels in CA1 Pyramidal Neurons

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.

Synergistic Actions of Metabotropic Acetylcholine and Glutamate Receptors on the Excitability of Hippocampal CA1 Pyramidal Neurons

A variety of neurotransmitters are responsible for regulating neural activity during different behavioral states. Unique responses to combinations of neurotransmitters provide a powerful mechanism by which neural networks could be differentially activated during a broad range of behaviors. Here, we show, using whole-cell recordings in rat hippocampal slices, that group I metabotropic glutamate receptors (mGluRs) and muscarinic acetylcholine receptors (mAChRs) synergistically increase the excitability of hippocampal CA1 pyramidal neurons by converting the post-burst afterhyperpolarization to an afterdepolarization via a rapidly reversible upregulation of Cav2.3 R-type calcium channels. Coactivation of mAChRs and mGluRs also induced a long-lasting enhancement of the responses mediated by each receptor type. These results suggest that cooperative signaling via mAChRs and group I mGluRs could provide a mechanism by which cognitive processes may be modulated by conjoint activation of two separate neurotransmitter systems.

Improvement of Fibrinolytic Activity of Bacillus subtilis 168 by Integration of a Fibrinolytic Gene into the Chromosome.

Fibrinolytic enzyme genes (aprE2, aprE176, and aprE179) were introduced into the Bacillus subtilis 168 chromosome without any antibiotic resistance gene. An integration vector, pDG1662, was used to deliver the genes into the amyE site of B. subtilis 168. Integrants, SJ3-5nc, SJ176nc, and SJ179nc, were obtained after two successive homologous recombinations. The integration of each fibrinolytic gene into the middle of the amyE site was confirmed by phenotypes (Amy(-), Spec(S)) and colony PCR results for these strains. The fibrinolytic activities of the integrants were higher than that of B. subtilis 168 by at least 3.2-fold when grown in LB broth. Cheonggukjang was prepared by inoculating each of B. subtilis 168, SJ3-5nc, SJ176nc, and SJ179nc, and the fibrinolytic activity of cheonggukjang was 4.6 ± 0.7, 10.8 ± 0.9, 7.0 ± 0.6, and 8.0 ± 0.2 (U/g of cheonggukjang), respectively at 72 h. These results showed that construction of B. subtilis strains with enhanced fibrinolytic activities is possible by integration of a strong fibrinolytic gene via a marker-free manner.

DTNB oxidation effects on T-type Ca2+ channel isoforms

Redox regulation is one of the ubiquitous mechanisms to modulate ion channels. We here investigated how 5,5′-dithio-bis (2-nitrobenzoic acid), a cysteine specific oxidizing reagent, modulates Cav3.1 and Cav3.2 T-type Ca2 + channels expressed in Xenopus oocytes. Application of the reagent inhibited Cav3.1 and Cav3.2 currents in a dose-dependent manner. The oxidizing reagent (1 mM) reduced the peak amplitude of Cav3.1 and Cav3.2 currents by ~50% over 2–3 minutes and the decreased currents were fully recovered upon washout of it. The reagent slowed the activation and inactivation kinetics of Cav3.1, Cav3.2, and Cav3.3 channel currents. Notably, the reagent positively shifted both activation and steady-state inactivation curves of Cav3.1, while it did not those of Cav3.2. Utilizing chimeric channels from Cav3.1 and Cav3.2, we localized the domains III and IV of Cav3.1 responsible for the positive shifts of channel activation and steady-state inactivation. These findings provide hints relevant to the electrophysiological and molecular mechanisms accounting for the oxidative regulation of T-type channels.