• Applied Biological Chemistry
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• v.47 no.4
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• pp.390-395
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• 2004

In order to characterize ion channels present in tomato roots, microsomes were incorporated into an artificial lipid bilayer arranged for electrophysiological analysis. Of the five different ion channels that could be found, a channel of 450 pS conductance was found most frequently. This channel displayed subconductance states of 450, 257 and 105 pS. All subconductance states showed linear current-voltage relationships. At positive holding potentials, high frequency of transient channel openings was observed; however, at negative potentials, the open times were long and open probability high. Po was 0.83 at -40 mV. When an additional 50 mM $K^+\;or\;Na^+$ was added to the cis side of bilayer, the reversal potentials shifted in the negative direction to near -10 mV. Thus, the 450 pS cation channel selects poorly between $K^+\;and\;Na^+$. In the presence of $100\;{\mu}M$ metal ions, the channel activity was severely inhibited by $La^{3+},\;Ba^{2+},\;and\;Zn^{2+}$, and Po was decreased to 0.2 or even less. However, $Al^{3+}\;and\;Cd^{2+}$ decreased the activity by only 20%. Interestingly, each metal ion showed different kinetics of channel inhibition. While $500\;{\mu}M\;La^{3+}$ inhibited the activities of all subconductance state, 1 mM $Zn^{2+}$ inhibited all except the 105 pS state. $Cd^{2+}$ changed the gating of the channel from a long-opening state to brief transient openings even at negative holding potentials. These data represent that the metal ions may have different binding sites on the channel protein and could be useful modulators and probes to investigate structural characteristics as well as the functional roles of the 450 pS channel on the root physiology.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.5
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• pp.343-348
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• 2012

Blocking or regulating $K^+$ channels is important for investigating neuronal functions in mammalian brains, because voltage-dependent $K^+$ channels (Kv channels) play roles to regulate membrane excitabilities for synaptic and somatic processings in neurons. Although a number of toxins and chemicals are useful to change gating properties of Kv channels, specific effects of each toxin on a particular Kv subunit have not been sufficiently demonstrated in neurons yet. In this study, we tested electro-physiologically if heteropodatoxin2 ($HpTX_2$), known as one of Kv4-specific toxins, might be effective on various $K^+$ outward currents in CA1 neurons of organotypic hippocampal slices of rats. Using a nucleated-patch technique and a pre-pulse protocol in voltage-clamp mode, total $K^+$ outward currents recorded in the soma of CA1 neurons were separated into two components, transient and sustained currents. The extracellular application of $HpTX_2$ weakly but significantly reduced transient currents. However, when $HpTX_2$ was added to internal solution, the significant reduction of amplitudes were observed in sustained currents but not in transient currents. This indicates the non-specificity of $HpTX_2$ effects on Kv4 family. Compared with the effect of cytosolic 4-AP to block transient currents, it is possible that cytosolic $HpTX_2$ is pharmacologically specific to sustained currents in CA1 neurons. These results suggest that distinctive actions of $HpTX_2$ inside and outside of neurons are very efficient to selectively reduce specific $K^+$ outward currents.

• BMB Reports
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• v.51 no.5
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• pp.236-241
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• 2018

Anoctamin 1 (ANO1) is an anion channel that is activated by changes in cytosolic $Ca^{2+}$ concentration and noxious heat. Although the critical roles of ANO1 have been elucidated in various cell types, the control of its gating mechanisms by $Ca^{2+}$ and heat remain more elusive. To investigate critical amino acid residues for modulation of $Ca^{2+}$ and heat sensing, we constructed a randomized mutant library for ANO1. Among 695 random mutants, reduced $Ca^{2+}$ sensitivity was observed in two mutants (mutant 84 and 87). Consequently, the E143A mutant showed reduced sensitivity to $Ca^{2+}$ but not to high temperatures, whereas the E705V mutant exhibited reduced sensitivity to both $Ca^{2+}$ and noxious heat. These results suggest that the glutamic acids (E) at 143 and 705 residues in ANO1 are critical for modulation of $Ca^{2+}$ and/or heat responses. Furthermore, these findings help to provide a better understanding of the $Ca^{2+}$-mediated activation and heat-sensing mechanism of ANO1.

• Molecules and Cells
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• v.27 no.5
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• pp.591-599
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• 2009

Nicotinic acetylcholine receptors (nAChRs) play important roles in nervous system functions and are involved in a variety of diseases. We previously demonstrated that ginsenosides, the active ingredients of Panax ginseng, inhibit subsets of nAChR channel currents, but not ${\alpha}7$, expressed in Xenopus laevis oocytes. Mutation of the highly conserved Leu247 to Thr247 in the transmembrane domain 2 (TM2) channel pore region of ${\alpha}7$ nAChR induces alterations in channel gating properties and converts ${\alpha}7$ nAChR antagonists into agonists. In the present study, we assessed how point mutations in the Leu247 residue leading to various amino acids affect 20(S)-ginsenoside $Rg_3$ ($Rg_3$) activity against the ${\alpha}7$ nAChR. Mutation of L247 to L247A, L247D, L247E, L247I, L247S, and L247T, but not L247K, rendered mutant receptors sensitive to $Rg_3$. We further characterized $Rg_3$ regulation of L247T receptors. We found that $Rg_3$ inhibition of mutant ${\alpha}7$ nAChR channel currents was reversible and concentration-dependent. $Rg_3$ inhibition was strongly voltage-dependent and noncompetitive manner. These results indicate that the interaction between $Rg_3$ and mutant receptors might differ from its interaction with the wild-type receptor. To identify differences in $Rg_3$ interactions between wild-type and L247T receptors, we utilized docked modeling. This modeling revealed that $Rg_3$ forms hydrogen bonds with amino acids, such as Ser240 of subunit I and Thr244 of subunit II and V at the channel pore, whereas $Rg_3$ localizes at the interface of the two wild-type receptor subunits. These results indicate that mutation of Leu247 to Thr247 induces conformational changes in the wild-type receptor and provides a binding pocket for $Rg_3$ at the channel pore.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.2
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• pp.259-265
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• 2017

Excessive influx and the subsequent rapid cytosolic elevation of $Ca^{2+}$ in neurons is the major cause to induce hyperexcitability and irreversible cell damage although it is an essential ion for cellular signalings. Therefore, most neurons exhibit several cellular mechanisms to homeostatically regulate cytosolic $Ca^{2+}$ level in normal as well as pathological conditions. Delayed rectifier $K^+$ channels ($I_{DR}$ channels) play a role to suppress membrane excitability by inducing $K^+$ outflow in various conditions, indicating their potential role in preventing pathogenic conditions and cell damage under $Ca^{2+}$-mediated excitotoxic conditions. In the present study, we electrophysiologically evaluated the response of $I_{DR}$ channels to hyperexcitable conditions induced by high $Ca^{2+}$ pretreatment (3.6 mM, for 24 hours) in cultured hippocampal neurons. In results, high $Ca^{2+}$-treatment significantly increased the amplitude of $I_{DR}$ without changes of gating kinetics. Nimodipine but not APV blocked $Ca^{2+}$-induced $I_{DR}$ enhancement, confirming that the change of $I_{DR}$ might be targeted by $Ca^{2+}$ influx through voltage-dependent $Ca^{2+}$ channels (VDCCs) rather than NMDA receptors (NMDARs). The VDCC-mediated $I_{DR}$ enhancement was not affected by either $Ca^{2+}$-induced $Ca^{2+}$ release (CICR) or small conductance $Ca^{2+}$-activated $K^+$ channels (SK channels). Furthermore, PP2 but not H89 completely abolished $I_{DR}$ enhancement under high $Ca^{2+}$ condition, indicating that the activation of Src family tyrosine kinases (SFKs) is required for $Ca^{2+}$-mediated $I_{DR}$ enhancement. Thus, SFKs may be sensitive to excessive $Ca^{2+}$ influx through VDCCs and enhance $I_{DR}$ to activate a neuroprotective mechanism against $Ca^{2+}$-mediated hyperexcitability in neurons.

• The Korean Journal of Physiology and Pharmacology
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• v.26 no.4
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• pp.277-285
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• 2022

To investigate the adverse effects of clozapine on cardiovascular ion channels, we examined the inhibitory effect of clozapine on voltage-dependent K⁺ (Kv) channels in rabbit coronary arterial smooth muscle cells. Clozapine-induced inhibition of Kv channels occurred in a concentration-dependent manner with an half-inhibitory concentration value of 7.84 ± 4.86 µM and a Hill coefficient of 0.47 ± 0.06. Clozapine did not shift the steady-state activation or inactivation curves, suggesting that it inhibited Kv channels regardless of gating properties. Application of train pulses (1 and 2 Hz) progressively augmented the clozapine-induced inhibition of Kv channels in the presence of the drug. Furthermore, the recovery time constant from inactivation was increased in the presence of clozapine, suggesting that clozapine-induced inhibition of Kv channels is use (state)-dependent. Pretreatment of a Kv1.5 subtype inhibitor decreased the Kv current amplitudes, but additional application of clozapine did not further inhibit the Kv current. Pretreatment with Kv2.1 or Kv7 subtype inhibitors partially blocked the inhibitory effect of clozapine. Based on these results, we conclude that clozapine inhibits arterial Kv channels in a concentration-and use (state)-dependent manner. Kv1.5 is the major subtype involved in clozapine-induced inhibition of Kv channels, and Kv2.1 and Kv7 subtypes are partially involved.

• Radiation Oncology Journal
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• v.28 no.4
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• pp.238-248
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• 2010

Purpose: To compare the dose distributions between three-dimensional (3D) and four-dimensional (4D) radiation treatment plans calculated by Ray-tracing or the Monte Carlo algorithm, and to highlight the difference of dose calculation between two algorithms for lung heterogeneity correction in lung cancers. Materials and Methods: Prospectively gated 4D CTs in seven patients were obtained with a Brilliance CT64-Channel scanner along with a respiratory bellows gating device. After 4D treatment planning with the Ray Tracing algorithm in Multiplan 3.5.1, a CyberKnife stereotactic radiotherapy planning system, 3D Ray Tracing, 3D and 4D Monte Carlo dose calculations were performed under the same beam conditions (same number, directions, monitor units of beams). The 3D plan was performed in a primary CT image setting corresponding to middle phase expiration (50%). Relative dose coverage, D95 of gross tumor volume and planning target volume, maximum doses of tumor, and the spinal cord were compared for each plan, taking into consideration the tumor location. Results: According to the Monte Carlo calculations, mean tumor volume coverage of the 4D plans was 4.4% higher than the 3D plans when tumors were located in the lower lobes of the lung, but were 4.6% lower when tumors were located in the upper lobes of the lung. Similarly, the D95 of 4D plans was 4.8% higher than 3D plans when tumors were located in the lower lobes of lung, but was 1.7% lower when tumors were located in the upper lobes of lung. This tendency was also observed at the maximum dose of the spinal cord. Lastly, a 30% reduction in the PTV volume coverage was observed for the Monte Carlo calculation compared with the Ray-tracing calculation. Conclusion: 3D and 4D robotic radiotherapy treatment plans for lung cancers were compared according to a dosimetric viewpoint for a tumor and the spinal cord. The difference of tumor dose distributions between 3D and 4D treatment plans was only significant when large tumor movement and deformation was suspected. Therefore, 4D treatment planning is only necessary for large tumor motion and deformation. However, a Monte Carlo calculation is always necessary, independent of tumor motion in the lung.