• The Korean Journal of Physiology and Pharmacology
• /
• v.7 no.1
• /
• pp.25-28
• /
• 2003

Ion channel inhibitors are widely used for pharmacological discrimination between the different channel types as well as for determination of their functional role. In the present study, we tested the hypothesis that 4-aminopyridine (4-AP) could affect the large conductance $Ca^{2+}$-activated $K^+$ channel ($BK_{Ca}$) currents using perforated-patch or cell-attached configuration of patch-clamp technique in the rabbit pulmonary arterial smooth muscle. Application of 4-AP reversibly inhibited the spontaneous transient outward currents (STOCs). The reversal potential and the sensitivity to charybdotoxin indicated that the STOCs were due to the activation of $BK_{Ca}$. The $BK_{Ca}$ currents were recorded in single channel resolution under the cell-attached mode of patch-clamp technique for minimal perturbation of intracellular environment. Application of 4-AP also inhibited the single $BK_{Ca}$ currents reversibly and dose-dependently. The membrane potential of rabbit pulmonary arterial smooth muscle cells showed spontaneous transient hyperpolarizations (STHPs), presumably due to the STOC activities, which was also inhibited by 4-AP. These results suggest that 4-AP can inhibit $BK_{Ca}$ currentsin the intact rabbit vascular smooth muscle. The use of 4-AP as a selective voltage-dependent $K^+$ (KV) channel blocker in vascular smooth muscle, therefore, must be reevaluated.

• The Korean Journal of Physiology
• /
• v.25 no.1
• /
• pp.17-26
• /
• 1991

Single smooth muscle cells of the rabbit pulmonary artery were isolated by treatment with collagenase and elastase. Using the patch clamp technique, potassium channel activity was recorded from the inside-out membrane patch. The channel had a sin히e channel conductance of about 360 pS in symmetrical concentration of K on both sides of the patch, 150 mM, and had a linear current-voltage relationship. During the application of 10 mM tetraethylammonium (TEA) to the intracellular membrane surface, the amplitude of single channel current was reduced and very rapid flickering appeared. The open probability $(P_0)$ of this channel was increased by increasing positivity of the potential across the patch membrane, with e-fold increase by 20 mV depolarization, and by increasing the internal $Ca^{2+}$ concentration. These findings are consistent with those of large conductance Ca-activated K channels reported in other tissues. But the shortening of the mean open time by increasing $[Ca^{2+}]_i$, was an unexpected result and one additional closed state which might be arisen from a block of the open channel by Ca binding was suggested. The $P_0-membrane$ potential relationship was modulated by internal pH. Decreasing pH reduced $P_0$. Increasing pH not only increased $P_0$ but also weakened the voltage dependency of the channel opening. The modulation of Ca-activated K channel by pH was thought to be related to the mechanism of regulation of vascular tone by the pH change.

• The Korean Journal of Physiology and Pharmacology
• /
• v.20 no.1
• /
• pp.119-127
• /
• 2016

Dihydropyridine (DHP) calcium channel blockers (CCBs) have been widely used to treat of several cardiovascular diseases. An excessive shortening of action potential duration (APD) due to the reduction of $Ca^{2+}$ channel current ($I_{Ca}$) might increase the risk of arrhythmia. In this study we investigated the electrophysiological effects of nicardipine (NIC), isradipine (ISR), and amlodipine (AML) on the cardiac APD in rabbit Purkinje fibers, voltage-gated $K^+$ channel currents ($I_{Kr}$, $I_{Ks}$) and voltage-gated $Na^+$ channel current ($I_{Na}$). The concentration-dependent inhibition of $Ca^{2+}$ channel currents ($I_{Ca}$) was examined in rat cardiomyocytes; these CCBs have similar potency on $I_{Ca}$ channel blocking with $IC_{50}$ (the half-maximum inhibiting concentration) values of 0.142, 0.229, and 0.227 nM on NIC, ISR, and AML, respectively. However, ISR shortened both $APD_{50}$ and $APD_{90}$ already at $1{\mu}M$ whereas NIC and AML shortened $APD_{50}$ but not $APD_{90}$ up to $30{\mu}M$. According to ion channel studies, NIC and AML concentration-dependently inhibited $I_{Kr}$ and $I_{Ks}$ while ISR had only partial inhibitory effects (<50% at $30{\mu}M$). Inhibition of $I_{Na}$ was similarly observed in the three CCBs. Since the $I_{Kr}$ and $I_{Ks}$ mainly contribute to cardiac repolarization, their inhibition by NIC and AML could compensate for the AP shortening effects due to the block of $I_{Ca}$.

• BMB Reports
• /
• v.28 no.6
• /
• pp.499-503
• /
• 1995

In our previous studies (Chae et al., 1990; Chae et a1., 1993), we found that a phytochrome signal was clearly connected with the change in cytosolic free $Ca^{2+}$ concentration ($[Ca^{2+}]_i$) in oat cells. It was determined that the $[Ca^{2+}]_i$ change occured both by mobilization out of the intracellular $Ca^{2+}$ store and by influx from the medium. The specific aim of this work is to elucidate the processes connecting $Ca^{2+}$ mobilization and influx. The cells treated with thapsigargin (increasing $[Ca^{2+}]_i$ by inhibition of the $Ca^{2+}$-ATPase in the calcium pool) in the presence of external $Ca^{2+}$ showed the same increasing pattern (sustained increasing shape) of $[Ca^{2+}]_i$ as that measured in animal cells. Red light irradiation after thapsigargin treatment did not increase $[Ca^{2+}]_i$ These results suggest that thapsigargin also acts specifically in the processes of mobilization and influx of $Ca^{2+}$ in oat cells. When the cells were treated with TEA ($K^+$ channel blocker), changes in $[Ca^{2+}]_i$ were drastically reduced in comparison with that measured in the absence of TEA. The results suggest that the change in $[Ca^{2+}]_i$ due to red light irradiation is somehow related with $K^+$ channel opening to change membrane potential. The membrane potential change due to $K^+$ influx might be the critical factor in opening a voltage-dependent calcium channel for $Ca^{2+}$ influx.

• The Korean Journal of Pharmacology
• /
• v.30 no.1
• /
• pp.125-136
• /
• 1994

To characterize the SR Ca-release channel protein complex of crustacean, $^{45}Ca-release,\;[^3H]ryanodine$ binding, and immunoblot studies were carried out in the crayfish and/or lobster skeletal sarcoplasmic reticulum. Bmax and affinity of crayfish SR to ryanodine were lower than those of lobster SR. AMP (5mM) increased $[^3H]ryanodine$ binding significantly in both vesicles (P<0.05). $Mg^{2+}$(5mM) or tetracaine(1mM) inhibited $[^3H]ryanodine$ binding significantly in both vesicles (P<0.001), but ruthenium red $(10\;{\mu}M)$ inhibited it moderately. In SDS polyacrylamide gel electrophoretic analysis of crayfish SR vesicles, there was a high molecular weight band that showed similar mobility with Ca-release channel protein of lobster skeletal SR, but more rapid mobility (HMWBr) than that of rabbit skeletal SR (HMWBS). Immunoblot analysis showed that polyclonal Ab to lobster skeletal SR Ca-release channel protein was react with HMWBr of crayfish skeletal SR, but not with that of HMWBs of rabbit skeletal SR. ^{45}Ca-release from crayfish skeletal SR vesicles was increased by the increase of extravesicular calcium from $1{\mu}M$ to 1mM. This Ca-release phenomenon was similar, but more sensitive in the low concentration of $Ca^{2+}$, compared to that from lobster SR vesicles. AMP (5mM) or caffeine (10mM) did not affect to $^{45}Ca-release.\;^{45}Ca-release$ was inhibited slightly ($3{\sim}8%\;by\;Mg^{2+}$) (5mM) or tetracaine (1mM), and moderately (23%) by high concentration of ruthenium red $(300\;{\mu}M)$. From the above results, it is suggested that SR Ca-release channel protein of crustacean has different properties from that of the rabbit, and similar properties between crayfish and lobster in functional and immunological aspects, but Ca-release via crayfish channel may be more sensitive to calcium.

• Journal of Microbiology and Biotechnology
• /
• v.11 no.4
• /
• pp.614-618
• /
• 2001

Low-threshold T-type $Ca^{2+}$ channels are distinctive voltage-operated gates for external $Ca^{2+}$ entry around a resting membrane potential due to their low voltage activation. These phenomena have already been extensively studied due to their relevance in diverse physiological functions. Recently, three T-type $Ca^{2+}$ channel ${\alpha}$$_1$subunits were cloned and their biophysical properties were characterized after expression in mammalian expression systems. In this study, ${\alpha_IG} and {\alpha_IH}$ low-threshold $Ca^{2+}$ channels were expressed and characterized in Xenopus oocytes after adding 5' and 3'untranslated portions of a Xenopus ${\beta}$ globin to improve their expression levels. The added portions dramatically enhanced the expression levels of the ${\alpha_IG} and {\alpha_IH}$ T-type channels. When currents were recorded in 10 mM $Ba^{2+}$ as the charge carrier, the activation thresholds were about -60 mV, peak currents appeared at -20 mV, and the reversal potentials were between +40 and +45. The activation time constants were very similar to each other, while the inactivation time constants of the ${\alpha_IG}$ currents were smaller than those of ${\alpha_IH}$. Taken together, the electrophysiological properties of the ${\alpha_IG} and {\alpha_IH}$ channels expressed in Xenopus oocytes were similar to the previously reported characteristics of low-threshold $Ca^{2+}$ channel currents.

• Molecules and Cells
• /
• v.37 no.9
• /
• pp.656-663
• /
• 2014

Gintonin, a novel, ginseng-derived G protein-coupled lysophosphatidic acid (LPA) receptor ligand, elicits $[Ca^{2+}]_i$ transients in neuronal and non-neuronal cells via pertussis toxin-sensitive and pertussis toxin-insensitive G proteins. The slowly activating delayed rectifier $K^+$ ($I_{Ks}$) channel is a cardiac $K^+$ channel composed of KCNQ1 and KCNE1 subunits. The C terminus of the KCNQ1 channel protein has two calmodulin-binding sites that are involved in regulating $I_{Ks}$ channels. In this study, we investigated the molecular mechanisms of gintonin-mediated activation of human $I_{Ks}$ channel activity by expressing human $I_{Ks}$ channels in Xenopus oocytes. We found that gintonin enhances $I_{Ks}$ channel currents in concentration- and voltage-dependent manners. The $EC_{50}$ for the $I_{Ks}$ channel was $0.05{\pm}0.01{\mu}g/ml$. Gintonin-mediated activation 1 of the $I_{Ks}$ channels was blocked by an LPA1/3 receptor antagonist, an active phospholipase C inhibitor, an $IP_3$ receptor antagonist, and the calcium chelator BAPTA. Gintonin-mediated activation of both the $I_{Ks}$ channel was also blocked by the calmodulin (CaM) blocker calmidazolium. Mutations in the KCNQ1 $[Ca^{2+}]_i$/CaM-binding IQ motif sites (S373P, W392R, or R539W)blocked the action of gintonin on $I_{Ks}$ channel. However, gintonin had no effect on hERG $K^+$ channel activity. These results show that gintonin-mediated enhancement of $I_{Ks}$ channel currents is achieved through binding of the $[Ca^{2+}]_i$/CaM complex to the C terminus of KCNQ1 subunit.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.8
• /
• pp.1371-1379
• /
• 2015

The Ca_v1.2 Ca²⁺ channel is essential for cardiac and smooth muscle contractility and many physiological functions. We mutated single, double, and quadruple sites of the four potential Asn (N)-glycosylation sites in the rabbit Ca_v1.2 into Gln (Q) to explore the effects of Nglycosylation. When a single mutant (N124Q, N299Q, N1359Q, or N1410Q) or Ca_v1.2/WT was expressed in Xenopus oocytes, the biophysical properties of single mutants were not significantly different from Ca_v1.2/WT. In comparison, the double mutant N124,299Q showed a positive shift in voltage-dependent gating. Furthermore, the quadruple mutant (QM; N124,299,1359,1410Q) showed a positive shift in voltage-dependent gating as well as a reduction of current. We tagged EGFP to the QM, double mutants, and Ca_v1.2/WT to chase the mechanisms underlying the reduced currents of QM. The surface fluorescence intensity of QM was weaker than that of Ca_v1.2/WT, suggesting that the reduced current of QM arises from its lower surface expression than Ca_v1.2/WT. Tunicamycin treatment of oocytes expressing Ca_v1.2/WT mimicked the effects of the quadruple mutations. These findings suggest that Nglycosylation contributes to the surface expression and voltage-dependent gating of Ca_v1.2.

• Molecules and Cells
• /
• v.37 no.11
• /
• pp.804-811
• /
• 2014

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type $Ca^{2+}$ currents ($I_{Ca-N}$) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated $Ca^{2+}$ currents ($I_{Ca}$), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on $I_{Ca}$. This PAR-2-induced inhibition was almost completely prevented by ${\omega}$-CgTx, a potent N-type $Ca^{2+}$ channel blocker, suggesting the involvement of N-type $Ca^{2+}$ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited $I_{Ca-N}$ in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ${\omega}$-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type $Ca^{2+}$ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type $Ca^{2+}$ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals.

• The Korean Journal of Pharmacology
• /
• v.32 no.1
• /
• pp.39-49
• /
• 1996

In the present study, we observed change in intracellular $Ca^{2+}$$([Ca^{2+}]_i)$ as measured with the fluorescent $Ca^{2+}-indicator$ fura-2 in association with force development of the rat basilar arteries during activation by$K^+$ depolarizing solution and U46619, a thromboxane analogue, in the absence and the presence of calcitonin-gent related peptide (CGRP). CGRP (30 and 100 nM) caused a concentration-dependent inhibition of U46619-induced contraction with decrease in $[Ca^{2+}]_i$, whereas it did not exert any effect on the $K^+$ (90 mM)-induced contraction and increase in $[Ca^{2+}]_i$, Further, $[Ca^{2+}]_i-force$ relationships were determined by plotting the ratio of $F_{340}/F_{380}$ $([Ca^{2+}]_i)$ as a function of the force induced by U46619, and the results were compared with those obtained in the presence of CGRP. The curves obtained in the presence of CGRP (30 and 100 nM) were significantly moved to downward without right shift of the curves suggesting that CGRP inhibited the U46619-induced contraction only by mediation of reduction in $[Ca^{2+}]_i$ with out any change in the sensitivity of contractile apparatus to $Ca^{2+}$. The CGRP-induced attenuation of $[Ca^{2+}]_i$ and force development was significantly inhibited under pretreatment with CGRP $(8{\sim}37)$ fragment (100 nM), a CGRP1 receptor antagonist. Both the reduced contraction and reduction in $[Ca^{2+}]_i$ caused by CGRP were fully reversed by pretreatment with charybdotoxin (100 nM) and iberiotoxin (100 nM), large conductance $Ca^{2+}-activated$ $K^+$ channel blockers, but not by apamin (300 nM), a small conductance $Ca^{2+}-activated$ $K^+$ channel blocker, and glibenclamide ( 1 ${\mu}M$), an ATP-sensitive $K^+$ channel blocker. In conclusion, it is suggested that the CGRP1 receptor, upon activation by CGRP, are coupled to opening of $Ca^{2+}-activated$ $K^+$ channel and cause to decrease in $[Ca^{2+}]_i$, thereby leading to vasodilation of the rat basilar artery. However, it is not defined that the mechanism underlying vasodilation whether the $K^+$ channel blockers, charybdotoxin and iberiotoxin directly block the CGRP receptors and that CGRP-evoked relaxation is dependent on the cyclic AMP or $K^+$ channel opening or both actions.