• 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.

• The Korean Journal of Physiology
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• v.28 no.1
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• pp.27-35
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• 1994

We investigated the alterations in basal tone of aortic strips by changing the Ca concentration, basal $^{45}Ca$ uptake and $^3H-nitrendipine$ binding of the single cells of aortic smooth muscles in the spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. While the basal tone of the aortic strips in WKY rats was not affected by alteration of Ca concentration, that in SHR was decreased by the removal of Ca from the bath solution and was recovered by the restoration of Ca to normal levels. This contraction increased in a Ca concentration-dependent manner and reached a maximum at 2 mM Ca. The basal tone of aorta in SHR was suppressed by verapamil $(10^{-6}M)$. The basal tone of aorta in SHR increased about 50% in the strips of endothelial rubbing, compared with that of intact endothelium. Basal $^{45}Ca$ uptake in the aortic single smooth muscle cells of SHR was greater than that of WKY (p<0.01), Specific bindings of $[^3H]nitrendipine$ in the aortic single smooth muscles of SHR and WKY were saturable. The dissociation constant $(K_d)\;was\;0.71{\pm}0.15\;and\;1.18{\pm}0.08nM$ SHR, respectively, and the difference in $K_d$ between two strains was statistically significant (p<0.03). The maximal binding capacity $(B_{max})\;was\;34.6{\pm}3.2\;and\;47.4{\pm}4.3\;fmol/10^6$ SHR respectively, and the difference of $(B_{max})$ between two strains was statistically significant (p<0.05). from the above results, it is suggested that the increase of Ca influx via potential-operated Ca channels and the increase of the number of dihydropyridine-sensitive Ca channels contribute to high basal tone of the aortic strips in SHR.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.43-43
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• 2003

Large-conductance $Ca^{2+}$-actived $K^{+}$ channels ($BK_{Ca}$ channels) play a key role in setting the pace of contractile activity in muscle and are involved in the regulation of neurotransmitter release in neuron. $BK_{Ca}$ channels are activated by depolarizing membrane potential and the elevated level of intracellular calcium. Using yeast-two hybrid assay, we have identified a novel protein interacting with the cytosolic carboxyl terminus of rSlo, the brain isoform of rat large-conductance $Ca^{2+}$-activated $K^{+}$ channel $\alpha$-subunit. The novel gene encodes 51 kDa protein and is named as SIRK(rSlo-interacting RGS-like protein). SIRK is expressed in various tissues and localized in the cytosolic and the membrane fraction. Biochemical and immunological studies indicated that SIRK physically interacted with the cytosolic region of rSlo. To investigate whether SIRK can modulate the activity of rSlo, GFP-fused SIRK and rSlo were transiently transfected into COS-7 cells and the effects of SIRK was studied using electrophysiological means. We concluded that the overexpression of SIRK alters the surface expression of rSlo channel with only a limited effect on the biophysical characteristics of the channel.the channel.

• 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.

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.34-34
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• 2002

Oxidative stress has been considered as a major cause of inducing cell damage, but it is recently recognized that mild oxidative stress or receptor-mediated production of ROS contributes to the regulation of various cellular functions. Several ion channels, such as L-type $Ca^{2＋}$ channels and $Ca^{2＋}$-activated $K^{＋}$ channels, have been shown to be regulated by oxidation of thiol group in their structure, and are suggested to be involved in ROS-sensitive cellular signaling.(omitted)

• The Korean Journal of Physiology
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• v.27 no.1
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• pp.107-114
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• 1993

Most of voltage operated $Ca^{2+}$ channels can be divided into three types (T-, N-, and L-type), according to the electrical and pharmacological properties. Their distribution is closely related to cell specific functions. Properties of the voltage activated $Ca^{2+}$ current in mouse eggs were examined to classify channel types and to deduce the function by using whole cell voltage clamp technique. $Ca^{2+}$ currents appeared below -40 mV and reached a maximum at -15 mV (half maximum was -31 mV), then decayed rapidly (inactivation time constant ${\tau}=28.2{\pm}9.59$ ms at -10 mV within 50 ms after the onset of step depolarization. Activation and inactivation of the $Ca^{2+}$ channel was steeply dependent on voltage, in a relatively low range of $-70\;mV{\sim}-10 mV,$ half maximum of activation was -31 mV and that of inactivation was -39 mV, respectively. This current was not decreased significantly by nifedipine, a specific dihydropyridine $Ca^{2+}$ channel blocker in the range of $1\;{\mu}M\;to\;100{\mu}M.$ The inhibitory effect of $Ni^{2+}\;on\;Ca^{2+}$ current was greater than that of $Cd^{2+}.$ The conductance of $Ba^{2+}$ through the channel was equal to or lower than that of $Ca^{2+}$ These results implied that $Ca^{2+}$ current activated at a lower voltage in the mouse egg is carried via a $Ca^{2+}$ channel with similar properties that of the T-type channel.

• The Korean Journal of Physiology and Pharmacology
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• v.8 no.4
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• pp.227-235
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• 2004

The aim of the present study was to examine the effect of leptin on CA release from the isolated perfused model of the rat adrenal gland, and to establish its mechanism of action. Leptin $(1{\sim}100\;ng/ml)$, when perfused into an adrenal vein of the rat adrenal gland for 60 min, enhanced a dose-dependently the secretory responses of CA evoked by ACh $(5.32{\times}10^{-3}\;M)$, DMPP $(10^{-4}\;M)$ and McN-A-343 $(10^{-4}\;M)$, although it alone has weak effect on CA secretion. However, it did not affect the CA secretion evoked by excess $K^+\;(5.6{\times}10^{-2}\;M)$. Leptin alone produced a weak secretory response of the CA. Moreover, leptin (10 ng/ml) in to an adrenal vein for 60 min also augmented the CA release evoked by BAY-K-8644, an activator of the dihydropyridine L-type $Ca^{2+}$ channels, and cyclopiazonic acid, an inhibitor of cytoplasmic $Ca^{2+}$ ATPase. However, in the presence of U0126 $(1\;{\mu}M)$, an inhibitor of mitogen-activated protein kinase (MAPK), leptin no longer enhanced the CA secretion evoked by ACh and DMPP. Furthermore, in the presence of anti-leptin (10 ng/ml), an antagonist of Ob receptor, leptin (10 ng/ml) also no longer potentiated the CA secretory responses evoked by DMPP and Bay-K-8644. Collectively, these experimental results suggest that leptin enhances the CA secretion from the rat adrenal medulla evoked by cholinergic stimulation (both nicotininc and muscarinic receptors), but does not that by membrane depolarization. It seems that this enhanced effect of leptin may be mediated by activation of U0126-sensitive MAPK through the leptin receptors, which is probably relevant to the activation of the dihydropyridine L-type $Ca^{2+}$ channels located on the rat adrenomedullary chromaffin cells.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.3
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• pp.277-286
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• 2020

Polycystic kidney disease 2-like-1 (PKD2L1), also known as polycystin-L or TRPP3, is a non-selective cation channel that regulates intracellular calcium concentration. Calmodulin (CaM) is a calcium binding protein, consisting of N-lobe and C-lobe with two calcium binding EF-hands in each lobe. In previous study, we confirmed that CaM is associated with desensitization of PKD2L1 and that CaM N-lobe and PKD2L1 EF-hand specifically are involved. However, the CaM-binding domain (CaMBD) and its inhibitory mechanism of PKD2L1 have not been identified. In order to identify CaM-binding anchor residue of PKD2L1, single mutants of putative CaMBD and EF-hand deletion mutants were generated. The current changes of the mutants were recorded with whole-cell patch clamp. The calmidazolium (CMZ), a calmodulin inhibitor, was used under different concentrations of intracellular. Among the mutants that showed similar or higher basal currents with that of the PKD2L1 wild type, L593A showed little change in current induced by CMZ. Co-expression of L593A with CaM attenuated the inhibitory effect of PKD2L1 by CaM. In the previous study it was inferred that CaM C-lobe inhibits channels by binding to PKD2L1 at 16 nM calcium concentration and CaM N-lobe at 100 nM. Based on the results at 16 nM calcium concentration condition, this study suggests that CaM C-lobe binds to Leu-593, which can be a CaM C-lobe anchor residue, to regulate channel activity. Taken together, our results provide a model for the regulation of PKD2L1 channel activity by CaM.

• The Korean Journal of Physiology
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• v.27 no.1
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• pp.93-105
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• 1993

The present study was performed to investigate the properties of ionic currents elicited by voltage pulses in the unfertilized eggs of mouse and hamster by using the whole cell voltage clamp techniques and to find out if there are any differences in properties between eggs of the two rodents. In addition, the modulatory effect of G proteins and protein kinase C (PKC) on the ionic channels were observed. The inward current in hamster eggs was shown to be due to $Ca^{2+}\;current\;(i_{ca})$). The current voltage relations of these currents in hamster egg were analogous to those in mouse eggs. The amplitude of $i_{ca}$ in the hamster egg was larger than that in the mouse egg ($-3.12{\pm}1.07\;nA\;vs.\;-1.71{\pm}0.71\;nA,\;mean{\pm}\;SD$). These results suggest that the $Ca^{2+}$ channels in both kinds of eggs have similar channel properties but their density, and/or conduct ance per unit area is higher in hamster eggs than in mouse eggs. Outward currents in eggs of both mouse and hamster were carried by $K^+$. In hamster eggs, they appeared to comprise at least two components; a transient outward component ($i_{to}$) and a steady state component ($i_{\infty}.$ The $i_{to}$ was found to be dependent on intracellular $Ca^{2+}$ concentration; whereas on the other hand $i_{\infty}\;was\;Ca^{2+}$-independent. $Ca^{2+}$ currents were increased in eggs treated with GTP (or $GTP{\gamma}S$) or fluoroaluminate ($AIF_4^-$). In the hamster egg these increments were antagonized by GDP (or $GDP{\beta}S$) application. In contrast to the enhancement of $i_{ca},\;i_k$ was reduced following GTP (or $GTP{\gamma}S$) perfusion in mouse eggs. The transient component ($i_{to}$) in hamster eggs was increased by adding GTP but decreased by phorbol ester, TPA or dioctanoyl glycerol (DOG). Simultaneous application of $GTP{\gamma}S$ and DOG suppressed $i_{to}$ more effectively than a single application or DOG or TPA. From the above results, we have shown that ionic currents elicited by voltage pulses existed in the unfertilized eggs of mouse and hamster. There are at least two types of currents, $i_{ca}\;and\;i_k$ in mouse eggs, while three types, $i_{ca},\;Ca^{2+}$-dependent $i_k$ and $Ca^{2+}$-independent $i_k$ exist in hamster eggs. ionic channels in these eggs may be regulated either directly by GTP and PKC or indirectly by the substances linked with GTP and PKC.

• Biomolecules & Therapeutics
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• v.20 no.6
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• pp.550-555
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• 2012

TRPA1 and TRPV1 are members of the TRP superfamily of structurally related, nonselective cation channels. TRPA1 and TRPV1 are often co-expressed in sensory neurons and play an important role in somatosense such as cold, pain, and irritants. The first leaves of Kalopanax pictus Nakai (Araliaceae) have long been used as a culinary ingredient in Korea because of their unique chemesthetic flavor. In this study, we observed the intracellular $Ca^{2+}$ response to cultured cells expressing human TRPA1 (hTRPA1) and human TRPV1 (hTRPV1) by $Ca^{2+}$ imaging analysis to investigate the ability of the first leaves of K. pictus to activate the hTRPA1 and hTRPV1. An 80% ethanol extract of K. pictus (KPEx) increased intracellular $Ca^{2+}$ influx in a response time- and concentration-dependent manner via either hTRPA1 or hTRPV1. KPEx-induced response to hTRPA1 was markedly attenuated by ruthenium red, a general blocker of TRP channels, and HC-030031, a specific antagonist of TRPA1. In addition, the intracellular $Ca^{2+}$ influx attained with KPEx to hTRPV1 was mostly blocked by ruthenium red, and capsazepine, a specific antagonist of TRPV1. These results indicate that KPEx selectively activates both hTRPA1 and hTRPV1, which may provide evidence that the first leaves of K. pictus primarily activate TRPA1 and TRPV1 to induce their unique chemesthetic sense.