• The Korean Journal of Physiology and Pharmacology
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• v.23 no.5
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• pp.381-392
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• 2019

Sperm function and male fertility are closely related to pH dependent $K^+$ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

• Biomolecules & Therapeutics
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• v.31 no.2
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• pp.168-175
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• 2023

Tramadol is an opioid analog used to treat chronic and acute pain. Intradermal injections of tramadol at hundreds of millimoles have been shown to produce a local anesthetic effect. We used the whole-cell patch-clamp technique in this study to investigate whether tramadol blocks the sodium current in HEK293 cells, which stably express the pain threshold sodium channel Na_v1.7 or the cardiac sodium channel Na_v1.5. The half-maximal inhibitory concentration of tramadol was 0.73 mM for Na_v1.7 and 0.43 mM for Na_v1.5 at a holding potential of -100 mV. The blocking effects of tramadol were completely reversible. Tramadol shifted the steady-state inactivation curves of Na_v1.7 and Na_v1.5 toward hyperpolarization. Tramadol also slowed the recovery rate from the inactivation of Na_v1.7 and Na_v1.5 and induced stronger use-dependent inhibition. Because the mean plasma concentration of tramadol upon oral administration is lower than its mean blocking concentration of sodium channels in this study, it is unlikely that tramadol in plasma will have an analgesic effect by blocking Na_v1.7 or show cardiotoxicity by blocking Na_v1.5. However, tramadol could act as a local anesthetic when used at a concentration of several hundred millimoles by intradermal injection and as an antiarrhythmic when injected intravenously at a similar dose, as does lidocaine.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.79-89
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• 2001

Steroid hormones control the expression of many cellular regulators, and a role thor estrogen in mouse oocytes has been well documented. The preovulatory $E_2$increment is generally accepted as the endocrine process regulating induction of in vivo oocyte maturation To address whether the activity of the T-type $Ca^{2+}$ channel is altered by 17 beta-estradiol ( $E_2$), we examined the actions of $E_2$on the calcium channel of mouse oocytes and early embryos. Oocrtes were collected from the oviduct of mice treated with pregnant mare's serum gonadotropin (PMSG) and human choronic gonadotropin (hCG). Whole cell voltage clamp technique and confocal microscopy were used to examine that $E_2$increase intracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{i}$ ) via voltage dependent $Ca^{2+}$ channel (VDC) and estrogen receptor (FSR), and $E_2$concentration by the use of radioimmunoassay (RIA) were examined in mouse. The results obtained were as follows: The peak of $Ca^{2+}$ current induced by $E_2$increased 122% to 1.50$\pm$0.03 nA from 1.23$\pm$0.21 nA (n=15) in the presence of 5 mM extracellular $Ca^{2+}$ concentration ([C $a^{2+}$]$_{o}$ ). The increased $Ca^{2+}$ current was temporally associated with $Ca^{2+}$ transients. The intracellular $Ca^{2+}$ level increased 207%~30 s following the addition of 1${\mu}{\textrm}{m}$ $E_2$(relative fluorescence intensity: 836.4$\pm$131.2 for control, n=10, 1736.4$\pm$192.0 in the presence of $E_2$, n=10). $E_2$increased amplitude of $Ca^{2+}$ current and [C $a^{2+}$]$_{i}$ . $E_2$-induced $Ca^{2+}$ current and $E_2$concentration in blood were showed difference on the stage of embryo. These results suggest that $E_2$modulate $Ca^{2+}$ channel to increase $Ca^{2+}$ influx.$Ca^{2+}$ influx.

• The Korean Journal of Physiology and Pharmacology
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• v.28 no.4
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• pp.313-322
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• 2024

Mutations within the SCN5A gene, which encodes the α-subunit 5 (Na_V1.5) of the voltage-gated Na⁺ channel, have been linked to three distinct cardiac arrhythmia disorders: long QT syndrome type 3, Brugada syndrome (BrS), and cardiac conduction disorder. In this study, we have identified novel missense mutations (p.A385T/R504T) within SCN5A in a patient exhibiting overlap arrhythmia phenotypes. This study aims to elucidate the functional consequences of SCN5A mutants (p.A385T/R504T) to understand the clinical phenotypes. Whole-cell patch-clamp technique was used to analyze the Na_V1.5 current (I_Na) in HEK293 cells transfected with the wild-type and mutant SCN5A with or without SCN1B co-expression. The amplitude of I_Na was not altered in mutant SCN5A (p.A385T/R504T) alone. Furthermore, a rightward shift of the voltage-dependent inactivation and faster recovery from inactivation was observed, suggesting a gain-of-function state. Intriguingly, the co-expression of SCN1B with p.A385T/R504T revealed significant reduction of I_Na and slower recovery from inactivation, consistent with the loss-of-function in Na⁺ channels. The SCN1B dependent reduction of I_Na was also observed in a single mutation p.R504T, but p.A385T co-expressed with SCN1B showed no reduction. In contrast, the slower recovery from inactivation with SCN1B was observed in A385T while not in R504T. The expression of SCN1B is indispensable for the electrophysiological phenotype of BrS with the novel double mutations; p.A385T and p.R504T contributed to the slower recovery from inactivation and reduced current density of Na_V1.5, respectively.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.6
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• pp.437-442
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• 2009

A non-steroidal anti-inflammatory drug (NSAID) has many adverse effects including cardiovascular (CV) risk. Diclofenac among the nonselective NSAIDs has the highest CV risk such as congestive heart failure, which resulted commonly from the impaired cardiac pumping due to a disrupted excitationcontraction (E-C) coupling. We investigated the effects of diclofenac on the L-type calcium channels which are essential to the E-C coupling at the level of single ventricular myocytes isolated from neonatal rat heart, using the whole-cell voltage-clamp technique. Only diclofenac of three NSAIDs, including naproxen and ibuprofen, significantly reduced inward whole cell currents. At concentrations higher than $3\;{\mu}M$, diclofenac inhibited reversibly the $Na^+$ current and did irreversibly the L-type $Ca^{2+}$ channels-mediated inward current $(IC_{50}=12.89\pm0.43\;{\mu}M)$ in a dose-dependent manner. However, nifedipine, a well-known L-type channel blocker, effectively inhibited the L-type $Ca^{2+}$ currents but not the $Na^+$ current. Our finding may explain that diclofenac causes the CV risk by the inhibition of L-type $Ca^{2+}$ channel, leading to the impairment of E-C coupling in cardiac myocytes.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.3
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• pp.129-135
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• 2011

In this study we determined whether or not 5-hydroxytryptamine (5-HT) has an effect on the pacemaker activities of interstitial cells of Cajal (ICC) from the mouse small intestine. The actions of 5-HT on pacemaker activities were investigated using a whole-cell patch-clamp technique, intracellular $Ca^{2+}$ ($[Ca^{2+}]_i$) analysis, and RT-PCR in ICC. Exogenously-treated 5-HT showed tonic inward currents on pacemaker currents in ICC under the voltage-clamp mode in a dose-dependent manner. Based on RT-PCR results, we found the existence of 5-$HT_{2B,\;3,\;4,\;and\;7}$ receptors in ICC. However, SDZ 205557 (a 5-$HT_4$ receptor antagonist), SB 269970 (a 5-$HT_7$ receptor antagonist), 3-tropanylindole - 3 - carboxylate methiodide (3-TCM; a 5-$HT_3$ antagonist) blocked the 5-HT-induced action on pacemaker activity, but not SB 204741 (a 5-$HT_{2B}$ receptor antagonist). Based on $[Ca^{2+}]_i$ analysis, we found that 5-HT increased the intensity of $[Ca^{2+}]_i$. The treatment of PD 98059 or JNK II inhibitor blocked the 5-HT-induced action on pacemaker activity of ICC, but not SB 203580. In summary, these results suggest that 5-HT can modulate pacemaker activity through 5-$HT_{3,\;4,\;and\;7}$ receptors via $[Ca^{2+}]_i$ mobilization and regulation of mitogen-activated protein kinases.

• Progress in Medical Physics
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• v.15 no.4
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• pp.220-227
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• 2004

In N-type $Ca^{2＋}$ channels, the mechanism of inactivation - decline of inward current during a depolarizing voltage step- is still controversial between voltage-dependent inactivation and $Ca^{2＋}$ -dependent inactivation. In the previous paper we demonstrated that fast component of inactivation of N-type calcium channels does not involve classic $Ca^{2＋}$ -dependent mechanism and the slowly inactivating component could result from a $Ca^{2＋}$ -dependent process. However, there should be signal transduction pathway which enhances inactivation no matter what the inactivation mechanism is. We have investigated the effect of phosphorylation on calcium channels of rat sympathetic neurons. Intracellular dialysis with the phosphatase inhibitors okadaic acid markedly enhanced the inactivation. The rapidly inactivating component is N-type calcium current, which is blocked by $\omega$-conotoxin GVIA. Staurosporine, a nonselective protein kinase inhibitor, prevented the action of okadaic acid, suggesting that protein phosphorylation is involved. More specifically lavendustin C, inhibitor of CaM kinase II, prevented the action of okadaic acid, suggesting that calmodulin dependent pathway is involved in inactivation process. It is not certain to this point whether phosphorylation process is inactivation itself. Molecular biological research regarding binding site should be followed to address the question of how the divalent cation binding site is related to phoshorylation process.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.1
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• pp.111-119
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• 2020

In vascular smooth muscle, K⁺ channels, such as voltage-gated K⁺ channels (Kv), inward-rectifier K⁺ channels (Kir), and big-conductance Ca²⁺-activated K⁺ channels (BK_Ca), establish a hyperpolarized membrane potential and counterbalance the depolarizing vasoactive stimuli. Additionally, Kir mediates endothelium-dependent hyperpolarization and the active hyperemia response in various vessels, including the coronary artery. Pulmonary arterial hypertension (PAH) induces right ventricular hypertrophy (RVH), thereby elevating the risk of ischemia and right heart failure. Here, using the whole-cell patch-clamp technique, we compared Kv and Kir current densities (I_Kv and I_Kir) in the left (LCSMCs), right (RCSMCs), and septal branches of coronary smooth muscle cells (SCSMCs) from control and monocrotaline (MCT)-induced PAH rats exhibiting RVH. In control rats, (1) I_Kv was larger in RCSMCs than that in SCSMCs and LCSMCs, (2) I_Kv inactivation occurred at more negative voltages in SCSMCs than those in RCSMCs and LCSMCs, (3) I_Kir was smaller in SCSMCs than that in RCSMCs and LCSMCs, and (4) I_BKCa did not differ between branches. Moreover, in PAH rats, I_Kir and I_Kv decreased in SCSMCs, but not in RCSMCs or LCSMCs, and I_BKCa did not change in any of the branches. These results demonstrated that SCSMC-specific decreases in I_Kv and I_Kir occur in an MCT-induced PAH model, thereby offering insights into the potential pathophysiological implications of coronary blood flow regulation in right heart disease. Furthermore, the relatively smaller I_Kir in SCSMCs suggested a less effective vasodilatory response in the septal region to the moderate increase in extracellular K⁺ concentration under increased activity of the myocardium.

• The Korean Journal of Physiology and Pharmacology
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• v.9 no.2
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• pp.103-108
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• 2005

To study the direct effect of somatostatin (SS) on calcium channel current ($I_{Ba}$) in guinea-pig gastric myocytes, $I_{Ba}$ was recorded by using whole-cell patch clamp technique in single smooth muscle cells. Nicardipine ($1{\mu}M$), a L-type $Ca^{2+}$ channel blocker, inhibited $I_{Ba}$ by $98{\pm}1.9$% (n=5), however $I_{Ba}$ was decreased in a reversible manner by application of SS. The peak $I_{Ba}$ at 0 mV were decreased to $95{\pm}1.5$, $92{\pm}1.9$, $82{\pm}4.0$, $66{\pm}5.8$, $10{\pm}2.9$% at $10^{-10}$, $10^{-9}$, $10^{-8}$, $10^{-7}$, $10^{-5}$ M of SS, respectively (n=3∼6; $mean{\pm}SEM$). The steady-state activation and inactivation curves of $I_{Ba}$ as a function of membrane potentials were well fitted by a Boltzmann equation. Voltage of half-activation ($V_{0.5}$) was $-12{\pm}0.5$ mV in control and $-11{\pm}1.9$ mV in SS treated groups (respectively, n=5). The same values of half-inactivation were $-35{\pm}1.4$ mV and $-35{\pm}1.9$ mV (respectively, n=5). There was no significant difference in activation and inactivation kinetics of $I_{Ba}$ by SS. Inhibitory effect of SS on $I_{Ba}$ was significantly reduced by either dialysis of intracellular solution with $GDP_{\beta}S$, a non-hydrolysable G protein inhibitor, or pretreatment with pertussis toxin (PTX). SS also decreased contraction of guinea-pig gastric antral smooth muscle. In conclusion, SS decreases voltage-dependent L-type calcium channel current ($VDCC_L$) via PTXsensitive signaling pathways in guinea-pig antral circular myocytes.

• The Korean Journal of Physiology and Pharmacology
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• v.13 no.1
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• pp.61-70
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• 2009

We have examined the effects of certain mutations of the selectivity filter and of the membrane helix M2 on $Ba^{2+}$ blockage of the inward rectifier potassium channel, Kir 2.1. We expressed mutant and wild type murine Kir 2.1 in Chinese hamster ovary(CHO) cells and used the whole cell patch-clamp technique to record $K^+$ currents in the absence and presence of externally applied $Ba^{2+}$. Wild type Kir2.1 was blocked by externally applied $Ba^{2+}$ in a voltage and concentration dependent manner. Mutants of Y145 in the selectivity filter showed little change in the kinetics of $Ba^{2+}$ blockage. The estimated $K_d(0)$ was 108 ${\mu}M$ for Kir2.1 wild type, 124 ${\mu}M$ for a concatameric WT-Y145V dimer, 109 ${\mu}M$ for a WT-Y145L dimer, and 267 ${\mu}M$ for Y145F. Mutant channels T141A and S165L exhibit a reduced affinity together with a large reduction in the rate of blockage. In S165L, blockage proceeds with a double exponential time course, suggestive of more than one blocking site. The double mutation T141A/S165L dramatically reduced affinity for $Ba^{2+}$, also showing two components with very different time courses. Mutants D172K and D172R(lining the central, aqueous cavity of the channel) showed both a decreased affinity to $Ba^{2+}$ and a decrease in the on transition rate constant(${\kappa}_{on}$). These results imply that residues stabilising the cytoplasmic end of the selectivity filter(T141, S165) and in the central cavity(D172) are major determinants of high affinity $Ba^{2+}$ blockage in Kir 2.1.