• The Korean Journal of Physiology and Pharmacology
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• v.17 no.3
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• pp.223-228
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• 2013

The calcium-activated $K^+$ ($BK_{Ca}$) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. $Ca^{2+}$ is the main regulator of $BK_{Ca}$ channel activation. The $BK_{Ca}$ channel contains two high affinity $Ca^{2+}$ binding sites, namely, regulators of $K^+$ conductance, RCK1 and the $Ca^{2+}$ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular $Ca^{2+}$ levels through diverse G proteins such as $G{\alpha}_{q/11}$, $G{\alpha}_i$, $G{\alpha}_{12/13}$, and $G{\alpha}s$ and the related signal transduction pathway. In the present study, we examined LPA effects on $BK_{Ca}$ channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated $BK_{Ca}$ channel activation was also attenuated by the PLC inhibitor U-73122, $IP_3$ inhibitor 2-APB, $Ca^{2+}$ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated $BK_{Ca}$ channel activation. The present study indicates that LPA-mediated activation of the $BK_{Ca}$ channel is achieved through the PLC, $IP_3$, $Ca^{2+}$, and PKC pathway and that LPA-mediated activation of the $BK_{Ca}$ channel could be one of the biological effects of LPA in the nervous and vascular systems.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.42-42
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• 2003

Large-conductance $Ca^{2+}$-activated potassium channels ($BK_{Ca}$ are a widely distributed and play key roles in various cell functions. In nerve cells, $BK_{Ca}$ channels shorten the duration of action potentials and block $Ca^{2+}$ entry thereby repolarizing excitable cells after excitation. $BK_{Ca}$ channel opening has been postulated to confer neuroprotection during stroke, and has attracted attention as a means for therapeutic intervention in asthma, hypertension, convulsions, and traumatic brain injury. Several natural and synthetic compounds including a steroid hormone, $\beta$-estradiol, have been identified as the activators of $BK_{Ca}$ channels. Based on the structural features of the previously reported activators of $BK_{Ca}$ channels, we designed several lead compounds, synthesized chemically, and tested their functional activity on cloned $BK_{Ca}$ channels. The $\alpha$ subunit of rat $BK_{Ca}$ channel was expressed alone or with different $\beta$ subunits in Xenopus oocytes and the effects of the compounds were tested electrophysiological means. One of the lead compounds affected the activity of the $\alpha$ subunit of $BK_{Ca}$ channel in a $\beta$ subunit-specific manner. While the activity of B $K_{ca}$ channel $\alpha$ subunit was Potentiated, the channel composed of $\alpha$ and $\beta$1 subunits were inhibited by this compound. We are currently investigating the mechanism of the $\beta$ subunit-dependent effects and planning to localize the receptor site of the lead compound.f the lead compound.

• The Korean Journal of Physiology and Pharmacology
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• v.7 no.1
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• pp.25-28
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• 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 and Pharmacology
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• v.9 no.2
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• pp.131-135
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• 2005

Potassium channels in human skin fibroblast have been studied as a possible site of Alzheimer disease pathogenesis. Fibroblasts in Alzheimer disease show alterations in signal transduction pathway such as changes in $Ca^{2+}$ homeostasis and/or $Ca^{2+}-activated$ kinases, phosphatidylinositol cascade, protein kinase C activity, cAMP levels and absence of specific $K^+$ channel. However, little is known so far about electrophysiological and pharmacological characteristics of large-conductance $Ca^{2+}$-activated $K^+$ ($BK_{Ca}$) channel in human fibroblast (CRL-1474). In the present study, we found Iberiotoxin- and TEA-sensitive outward rectifying oscillatory current with whole-cell recordings. Single channel analysis showed large conductance $K^{+}$ channels (106 pS of chord conductance at +40 mV in physiological $K^+$ gradient). The 106 pS channels were activated by membrane potential and $[Ca^{2+}]_i$, consistent with the known properties of $BK_{Ca}$ channels. $BK_{Ca}$ channels in CRL-1474 were positively regulated by adenylate cyclase activator ($10{\mu}M$ forskolin), 8-Br-cyclic AMP ($300{\mu}M$) or 8-Br-cyclic GMP ($300{\mu}M$). These results suggest that human skin fibroblasts (CR-1474) have typical $BK_{Ca}$ channel and this channel could be modulated by c-AMP and c-GMP. The electrophysiological characteristics of fibroblasts might be used as the diagnostic clues for Alzheimer disease.

• Proceedings of the Korean Biophysical Society Conference
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• 2003.06a
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• pp.44-44
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• 2003

$BK_{Ca}$ channels were suggested to contain one or more domains of the ‘regulator of K+ conductance’(RCK) in their cytosolic carboxyl termini (Jiang et al.2001). It was also shown that the RCK domain in mammalian $BK_{Ca}$ channels might sense the intracellular $Ca^{2+}$ with a low affinity (Xia et al. 2002). We aligned the amino acid sequence of the $\alpha$-subunit of rat $BK_{Ca}$ channels (rSlo) with known RCK domains and identified a second region exhibiting about 50％ homology. This putative domain, RCK2, contains the characteristic amino acids conserved in other RCK domains. We wondered whether this second domain is involved in the domain-domain interaction and the gating response to intracellular $Ca^{2+}$ for rSlo channel, as revealed in the structure of RCK domain of E. coli channel (Jiang et al.2001). In order to examine the possibility, site-directed mutations were introduced into the RCK2 domain of rSlo channel and the mutant channels were expressed in Xenopus oocytes for functional studies. One of such mutation, G772D, in the putative nucleotide-binding domain resulted in the enhanced $Ca^{2+}$ sensitivity and the channel gating of rSlo channel. These results suggest that this region of $BK_{Ca}$ channels is important for the channel gating and may form an independent domain in the cytosolic region of $BK_{Ca}$ channels. In order to obtain the mechanistic insights of these results, G772 residue was randomly mutagenized by site-directed mutagenesis and total 17 different mutant channels were constructed. We are currently investigating these mutant channels by electrophysiological techniques.ical techniques.

• The Korean Journal of Physiology and Pharmacology
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• v.11 no.5
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• pp.215-219
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• 2007

Small and large conductance $Ca^{2+}$-activated $K^+(SK_{Ca}\;and\;BK_{Ca})$ channels are implicated in the modulation of neuronal excitability. We investigated how changes in peripheral $K_{Ca}$ channel activity affect mechanical sensitivity as well as the afferent fiber type responsible for $K_{Ca}$ channel-induced mechanical sensitivity. Blockade of $SK_{Ca}$ and $BK_{Ca}$ channels induced a sustained decrease of mechanical threshold which was significantly attenuated by topical application of capsaicin onto afferent fiber and intraplantar injection of 1-ethyl-2-benzimidazolinone. NS1619 selectively attenuated the decrease of mechanical threshold induced by charybdotoxin, but not by apamin. Spontaneous flinching and paw thickness were not significantly different after $K_{Ca}$ channel blockade. These results suggest that mechanical sensitivity can be modulated by $K_{Ca}$ channels on capsaicin-sensitive afferent fibers.

• Journal of Ginseng Research
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• v.23 no.4
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• pp.230-234
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• 1999

Potassium channels play an important role in regulating vascular smooth muscle tone. Four types of $K^+$ channels areknown to be expressed in vascular smooth muscle cells, and maxi $Ca^{2+}-activated\;K^+$ channel $(BK_{Ca})$ is a dominant type of $K^+$ channels in these cells. Because total ginseng saponins and ginsenoside $Rg_3$ cause vasodilation with unclear mechanisms, we hypothesized that total ginseng saponins and ginsenoside $Rg_3$ induce vasodilation via activation of maxi $Ca^{2+}-activated\;K+$ channels. Whole-cell BKe. currents were voltage-dependent with half maximum activation at -14 mV, and the currents were sensitive to nanomolar ChTX and millimolar TEA. External application of total ginseng saponins increased the anlplitude of the whole-cell BKe. current in a concentration-dependent manner. Single-channel analysis indicates that total ginseng saponins caused the channel opening for a longer period of time. Ginsenoside $Rg_3$ increased the amplitude of whole-cell $K_{Ca}$ currents without affecting voltage dependence of the currents and increased single-channel open time. Hence, the results suggest that ginseng saponin-induced vasodilation may be due to activation of $K_{Ca}$.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.2
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• pp.95-99
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• 2006

Employing electrophysiological and cell proliferation assay techniques, we studied the effects of $Ca^{2+}$ -activated $K^+$ channel modulators on the proliferation of human dermal fibroblasts, which is important in wound healing. Macroscopic voltage-dependent outward $K^+$ currents were found at about -40 mV stepped from a holding potential of -70 mV. The amplitude of $K^+$ current was increased by NS1619, a specific large-conductance $Ca^{2+}$-activated $K^+$ (BK) channel activator, but decreased by iberiotoxin (IBTX), a specific BK channel inhibitor. To investigate the presence of an intermediate-conductance $Ca^{2+}$-activated $K^+$ (IK) channels, we pretreated the fibroblasts with low dose of TEA to block BK currents, and added 1-EBIO (an IK activator). 1-EBIO recovered the currents inhibited by TEA. When various $Ca^{2+}$-activated $K^+$ channel modulators were added into culture media for 1∼3 days, NS1619 or 1-EBIO inhibited the cell proliferation. On the other hand, IBTX, clotrimazole or apamin, a small conductance $Ca^{2+}$-activated $K^+$ channel (SK) inhibitor, increased it. These results suggest that BK, IK, and SK channels might be involved in the proliferation of human dermal fibroblasts, which is inversely related to the channel activation.

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.35-35
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• 2002

The large conductance $Ca^{2＋}$ -activated $K^{＋}$ channels ($BK_{Ca}$) in vascular smooth muscle have been considered to function as a negative feedback in pressure-induced vasoconstriction. In the present study, the function of cytoskeletons in the regulation of $BK_{Ca}$ and its stretch sensitivity was investigated. Using the inside-out patch clamp technique, we recorded single channel activities of $BK_{Ca}$ with 150 mM KCl in the bath solution (pCa=6.5).(omitted)itted)

• Proceedings of the PSK Conference
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• 2003.10b
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• pp.188.2-188.2
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• 2003

Large-conductance $Ca^{2+}$ activated potassium channels $(BK_{ca})$ are widely distributed and play key roles in various cell functions. In nerve cells, B $K_{ca}$ channels shorten the duration of action potentials and block $Ca^{2+}$ entry thereby repolarizing excitable cells after excitation. $(BK_{ca})$ channel opening has been postulated to confer neuroprotection during stroke and has attracted attention as a means for therapeutic intervention in asthma, hypertension, convulsion, and traumatic brain injury. (omitted)