• Animal cells and systems
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• v.15 no.2
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• pp.131-138
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• 2011

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 $Ca_v3.1$ and $Ca_v3.2$ T-type $Ca^{2+}$ channels expressed in Xenopus oocytes. Application of the reagent inhibited $Ca_v3.1$ and $Ca_v3.2$ currents in a dose-dependent manner. The oxidizing reagent (1 mM) reduced the peak amplitude of $Ca_v3.1$ and $Ca_v3.2$ 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 $Ca_v3.1$, $Ca_v3.2$, and $Ca_v3.3$ channel currents. Notably, the reagent positively shifted both activation and steady-state inactivation curves of $Ca_v3.1$, while it did not those of $Ca_v3.2$. Utilizing chimeric channels from $Ca_v3.1$ and $Ca_v3.2$, we localized the domains III and IV of $Ca_v3.1$ 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.

• Journal of Microbiology and Biotechnology
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• v.18 no.2
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• pp.365-368
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• 2008

Calcium entry through $Ca_v3.2Ca^{2+}$ channels plays essential roles for various physiological events including thalamic oscillation, muscle contraction, hormone secretion, and sperm acrosomal reaction. In this study, we examined how protein tyrosine phosphatases or protein tyrosine kinases affect $Ca_v3.2Ca^{2+}$ channels reconstituted in Xenopus oocytes. We found that $Ca_v3.2$ channel activity was reduced by 25% in response to phenylarsine oxide (tyrosine phosphatase inhibitor), whereas it was augmented by 19% in response to Tyr A47 or herbimycin A (tyrosine kinase inhibitors). However, other biophysical properties of $Ca_v3.2$ currents were not significantly changed by the drugs. These results imply that $Ca_v3.2$ channel activity is capable of being increased by activation of tyrosine phosphatases, but is decreased by activation of tyrosine kinases.

• Animal cells and systems
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• v.12 no.3
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• pp.137-141
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• 2008

Molecular cloning revealed the three isoforms($Ca_v3.1,\;Ca_v3.2,\;and\;Ca_v3.3$) of the T-type calcium channel subfamily. Expression studies exhibited their distinctive electrophysiological and pharmacological properties, accounting for diverse properties of T-type calcium channel currents previously characterized from isolated cells. However, electrophysiological properties of ion channels have shown to be more diversified by their splice variants. We here searched splice variants of rat $Ca_v3.1$ T-type channel by reverse-transcription-polymerase chain reaction(RT-PCR) to further explore diversity of $Ca_v3.1$. Interestingly, analyses of cloned RT-PCR products displayed that there were at least four splicing variants of rat $Ca_v3.1$ in the loop connecting repeats III and IV. Southern blot analyses indicated that the predominantly detected variant in brain was $Ca_v3.1a$(492 bp), which were rarely detected in most of peripheral tissues. Other two variants($Ca_v3.1c$, 546 bp; $Ca_v3.1d$, 525 bp) were detected in most of the tissues examined. The smallest isoform($Ca_v3.1b$, 471 bp) was rarely detected all the tissues. Electrophysiological characterization of the splicing variants indicated that the splice variants differ in inactivation kinetics and the voltage dependence of activation and inactivation as well.

• Molecules and Cells
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• v.39 no.4
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• pp.322-329
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• 2016

Voltage-gated $Ca^{2+}$ ($Ca_V$) channels are dynamically modulated by Gprotein-coupled receptors (GPCR). The $M_1$ muscarinic receptor stimulation is known to enhance $Ca_V2.3$ channel gating through the activation of protein kinase C (PKC). Here, we found that $M_1$ receptors also inhibit $Ca_V2.3$ currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated $Ca_V2.3$ currents by ~two-fold. After the PMA-induced potentiation, stimulation of $M_1$ receptors decreased the $Ca_V2.3$ currents by $52{\pm}8%$. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate ($PI(4,5)P_2$) is responsible for the muscarinic suppression of $Ca_V2.3$ currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of $PI(4,5)P_2$ to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed $Ca_V2.3$ currents, by $53{\pm}3%$. Next, dephosphorylation of both PI(4)P and $PI(4,5)P_2$ to PI by PJ translocation further decreased the current by up to $66{\pm}3%$. The results suggest that $Ca_V2.3$ currents are modulated by the $M_1$ receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane $PI(4,5)P_2$. Our results also suggest that there is rapid turnover between PI(4)P and $PI(4,5)P_2$ in the plasma membrane.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.2
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• pp.199-205
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• 1999

Vibrio vulnificus cytolysin caused platelet cytolysis and increased intracellular calcium concentration $([Ca^{2+}]_i)$ of rat platelets in a concentration-dependent manner. In the presence of V. vulnificus cytolysin (3 HU/ml), lactate dehydrogenase (LDH) activity was increased from $1.3{\pm}0.4%$ of control to $64.3{\pm}3.4%$ in platelet suspension buffer. In $Ca^{2+}-free$ platelet suspension buffer, however, V. vulnificus cytolysin did not induce $[Ca^{2+}]_i$ increase and LDH release. Addition of EGTA (2 mM) to suspension buffer after the initial $Ca^{2+}$ influx reversed $[Ca^{2+}]_i$ to the control level. However, a $Ca^{2+}$ channel blocker verapamil $(20\;{\mu}M)$ or mefenamic acid $(20\;{\mu}M)$ did not inhibit V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. Divalent cations such as $Co^{2+},\;Cd^{2+}\;or\;Mn^{2+}$ (2 mM each) also did not alter V. vulnificus cytolysin-induced $[Ca^{2+}]_i$ increase and LDH release. V. vulnificus cytolysin (3 HU/ml)-induced calcium influx was completely blocked by lanthanum (2 mM). Lanthanum (2 mM) also completely blocked V. vulnificus cytolysin (3 HU/ml)-induced LDH release. Osmotic protectants such as, raffinose, sucrose or PEG600 (50 mM each) did not inhibit the lytic activity of V. vulnificus cytolysin. In conclusion, lanthanum sensitive $Ca^{2+}$ influx plays a significant role in Vibrio vulnificus cytolysin-induced platelet cytolysis and thrombocytopenia in V. vulnificus infection.

• The Korean Journal of Physiology and Pharmacology
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• v.14 no.1
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• pp.45-49
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• 2010

R-type $Ca_v2.3$ high voltage-activated $Ca^{2+}$ channels in peripheral sensory neurons contribute to pain transmission. Recently we have demonstrated that, among the six $Ca_v2.3$ isoforms ($Ca_v2.3a{\sim}Ca_v2.3e$), the $Ca_v2.3e$ isoform is primarily expressed in trigeminal ganglion (TG) nociceptive neurons. In the present study, we further investigated expression patterns of $Ca_v2.3$ isoforms in the dorsal root ganglion (DRG) neurons. As in TG neurons, whole tissue RT-PCR analyses revealed the presence of two isoforms, $Ca_v2.3a$ and $Ca_v2.3e$, in DRG neurons. Single-cell RT-PCR detected the expression of $Ca_v2.3e$ mRNA in 20% (n=14/70) of DRG neurons, relative to $Ca_v2.3a$ expression in 2.8% (n=2/70) of DRG neurons. $Ca_v2.3e$ mRNA was mainly detected in small-sized neurons (n=12/14), but in only a few medium-sized neurons (n=2/14) and not in large-sized neurons, indicating the prominence of $Ca_v2.3e$ in nociceptive DRG neurons. Moreover, $Ca_v2.3e$ was preferentially expressed in tyrosine-kinase A (trkA)-positive, isolectin B4 (IB4)-negative and transient receptor potential vanilloid 1 (TRPV1)-positive neurons. These results suggest that $Ca_v2.3e$ may be the main R-type $Ca^{2+}$ channel isoform in nociceptive DRG neurons and thereby a potential target for pain treatment, not only in the trigeminal system but also in the spinal system.

• BMB Reports
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• v.40 no.3
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• pp.302-314
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• 2007

N type calcium channels (CaV2.2) play a key role in the gating of transmitter release at presynaptic nerve terminals. These channels are generally regarded as parts of a multimolecular complex that can modulate their open probability and ensure their location near the vesicle docking and fusion sites. However, the proteins that comprise this component remain poorly characterized. We have carried out the first open screen of presynaptic CaV2.2 complex members by an antibody-mediated capture of the channel from purified rat brain synaptosome lysate followed by mass spectroscopy. 589 unique peptides resulted in a high confidence match of 104 total proteins and 40 synaptosome proteome proteins. This screen identified several known CaV2.2 interacting proteins including syntaxin 1, VAMP, protein phosphatase 2A, $G_{o\alpha}$, G$\beta$ and spectrin and also a number of novel proteins, including clathrin, adaptin, dynamin, dynein, NSF and actin. The unexpected proteins were classified within a number of functional classes that include exocytosis, endocytosis, cytoplasmic matrix, modulators, chaperones, and cell-signaling molecules and this list was contrasted to previous reports that catalogue the synaptosome proteome. The failure to detect any postsynaptic density proteins suggests that the channel itself does not exhibit stable trans-synaptic attachments. Our results suggest that the channel is anchored to a cytoplasmic matrix related to the previously described particle web.

• International Journal of Oral Biology
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• v.31 no.2
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• pp.45-51
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• 2006

R-type($Ca_v2.3$) calcium channel contributes to pain sensation in peripheral sensory neurons. Six isoforms of $Ca_v2.3$ that result from combinations of presence or deletion of three inserts(insert I and insert in the II-III loop, and insert III in N-terminal regions) have been demonstrated to be present in different mammalian tissues. However, the molecular basis of $Ca_v2.3$ in trigeminal ganglion(TG) neurons is not known. In the present study, we determined which isoforms of $Ca_v2.3$ are expressed in rat TG neurons using the RT-PCR analysis. Whole tissue RT-PCR analyses revealed that only two isoforms, $Ca_v2.3a$ and $Ca_v2.3e$, were present in TG neurons. From single-cell RT-PCR, we found that $Ca_v2.3e$ rather than $Ca_v2.3a$ was the major isoform expressed in TG neurons, and $Ca_v2.3e$ was preferentially detected in small-sized neurons that express nociceptive marker, transient receptor potential vanilloid 1(TRPV1). Our results suggest that $Ca_v2.3e$ in trigeminal neurons may be a potential target for the pain treatment.

• The Korean Journal of Physiology
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• v.29 no.1
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• pp.13-27
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• 1995

single $K^{+}$ channels of skeletal muscle from the rat and frog were into planar lipid bilayers and their properties were studied. Fusion was induced by an osmotic gradient. Of the four types of $K^{+}$ channels recorded, the two most frequently observed were a voltage and $Ca^{2+}-activated$ $K^{+}$ channel and a $K^{+}$ channel with a prominent conductance substate. The first $K^{+}$ channel was identified as the large $Ca^{2+}-activated$ $K^{+}$ (BK) channel because the open-state probability was increased with depolarization (e-fold change per $10.6{\pm}3.5$ mV, n=8) and internal $Ca^{2+}$ (half-activation at $16.7{\pm}3.8$ mV, n=8, pCa 4) and its conductance was large ($247{\pm}4.9$ pS, n=24 in 0.1 M KCI). Lifetime distributions of open- and closed-states could be fitted with single exponentials of several milliseconds. The mean open- and closed-lifetimes were linearly dependent on the intracellular $[Ca^{2+}]$ and $1/[Ca^{2+}]$, respectively. The second $K^{+}$ channel showed a conductance substate at $30{\sim}60%$ of the open state. Its current-voltage relation was linear in the range of $-80\;{\sim}\;+80\;mV$. The slope conductance of the substate and open-state were 40 and 144 pS in 0.2 M KCl, respectively. The channel was highly selective for $K^{+}$ over Cl. The open-state probability was weakly voltage-dependent (e-fold change per 35 mV. The lifetime distributions of open- and closed-states were fitted with two exponentials and the major gating occurred slowly at several hundred milliseconds. Based on the above results, we think the second type of $K^{+}$ channel is the sarcoplasmic reticulum $K^{+}$ (SRK) channel. In addition, both types of channel were also incorporated into the lipids extracted from the skeletal muscle. The channel properties recorded in the bilayers termed from synthetic and extracted lipids were qualitatively similar. Our data indicate that BK and SRK channels are rich in the skeletal muscle and their properties and regulation could be effectively studied in planar lipid bilayer.

• The Korean Journal of Physiology and Pharmacology
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• v.3 no.2
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• pp.119-125
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• 1999

Mammalian gastric smooth muscles generate spontaneous rhythmic contractions which are associated with slow oscillatory potentials (slow waves) and spike potentials. Spike potentials are blocked by organic $Ca^{2+}-antagonists,$ indicating that these result from the activation of L-type $Ca^{2+}-channel.$ However, the cellular mechanisms underlying the generation of slow wave remain unclear. Slow waves are insensitive to $Ca^{2+}-antagonists$ but are blocked by metabolic inhibitors or low temperature. Recently it has been suggested that Interstitial Cells of Cajal (ICC) serve as pacemaker cells and a slow wave reflects the coordinated behavior of both ICC and smooth muscle cells. Small segments of circular smooth muscle isolated from antrum of the guinea-pig stomach generated two types of electrical events; irregular small amplitude (1 to 7 mV) of transient depolarization and larger amplitude (20 to 30 mV) of slow depolarization (regenerative potential). Transient depolarization occurred irregularly and membrane depolarization increased their frequency. Regenerative potentials were generated rhythmically and appeared to result from summed transient depolarizations. Spike potentials, sensitive to nifedipine, were generated on the peaks of regenerative potentials. Depolarization of the membrane evoked regenerative potentials with long latencies (1 to 2 s). These potentials had long partial refractory periods (15 to 20 s). They were inhibited by low concentrations of caffeine, perhaps reflecting either depletion of $Ca^{2+}$ from SR or inhibition of InsP3 receptors, by buffering $Ca^{2+}$ to low levels with BAPTA or by depleting $Ca^{2+}$ from SR with CPA. They persisted in the presence of $Ca^{2+}-sensitive$ $Cl^--channel$ blockers, niflumic acid and DIDS or $Co^{2+},$ a non selective $Ca^{2+}-channel$ blocker. These results suggest that spontaneous activity of gastric smooth muscle results from $Ca^{2+}$ release from SR, followed by activation of $Ca^{2+}-dependent$ ion channels other than $Cl^-$ channels, with the release of $Ca^{2+}$ from SR being triggered by membrane depolarization.