• The Korean Journal of Physiology and Pharmacology
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• v.7 no.3
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• pp.131-142
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• 2003

We have studied mutant forms of Kir2.1 in which an aspartate residue (D172), important for gating by intracellular polyamines, is replaced by one of three basic residues (Arg, Lys or His). Such channels are highly selective for $K^+$, but show inward rectification that is a shallow function of voltage compared with that found in wild type. This inward rectification occurs with a reduced affinity for spermine and persists in the absence of polyamines. Though the unitary current-voltage relation shows some inward rectification, it is insufficient to account for that seen under whole cell recording. Channels open and shut under single channel recording, and changes of $P_{open}$ appear to generate inward rectification. In D172H, the reduction in affinity for spermine is greater when His is protonated at low $pH_i$. The effective valency for spermine is reduced from $3.09{\pm}0.07$ in wild type to $1.95{\pm}0.09$ in D172H at $pH_i$ 6.3. In the presence of dual mutants of Kir2.1, where E224 is also replaced, spermine affinity becomes undetectable. However, channels still show inward rectification and open and shut under hyper- and depolarisation, respectively. We suggest that Kir2.1 channel are able to undergo conformation changes; these changes may be important physiologically in generating inward rectification, the normal parameters of which are set by the binding of polyamines such as spermine.

• Proceedings of the Korean Biophysical Society Conference
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• 2002.06b
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• pp.19-19
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• 2002

The gating kinetics of an inward-rectifier K$\^$＋/ channel, ROMK2 (Kir1.lb), were described by a model having one open state and two closed states. The long closed state was abolished by EDTA, suggesting that it was due to block by divalent cations. These closures exhibit a biphasic voltage-dependence, implying that the divalent blockers can permeate the channel.(omitted)

• BMB Reports
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• v.28 no.5
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• pp.382-386
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• 1995

One of the reaction pathways in light-invoked signal transduction can be initiated through ion fluxes across the plasma membrane in higher plants. We isolated protoplasts from oat coleoptile and examined the effects of light on the membrane potential using a membrane potential-sensitive fluorescent probe (bisoxonol). Both red and far-red light initially induced a hyperpolarization in oat cells. Red light-induced hyperpolarization was effectively dissipated by 100 mM $K^+$, but the hyperpolarization induced by far-red light was not depolarized by any of the cations ($K^+$, $Ca^{2+}$, $Li^+$, $Na^+$) tested. The depolarization induced by red light and $K^+$ was inhibited by 200 mM TEA, which is a $K^+$ channel blocker. These results suggest that $K^+$ influx through the inward $K^+$ channel may be a depolarization path in the phytochrome-mediated signal transduction.

• The Korean Journal of Physiology and Pharmacology
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• v.10 no.1
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• pp.7-11
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• 2006

Interstitial cells of Cajal (ICCs) are pacemakers in gastrointestinal tracts, regulating rhythmicity by activating nonselective cation channels (NSCCs). In the present study, we investigated the general characteristics and pH-mediated regulation of pacemaker activity in cultured interstitial cells of Cajal. Under voltage clamp mode and at the holding potential of -60 mV, the I-V relationships and difference current showed that there was no reversal potential and voltage-independent inward current. Also, when the holding potentials were changed from +20 mV to -80 mV with intervals of 20 mV, there was little difference in inward current. In pacemaker activity, the resting membrane potential (RMP) was depolarized (In pH 5.5, $23{\pm}1.5$ mV depolarized) and the amplitude was decreased by a decrease of the extracellular pH. However, in case of increase of extracellular pH, the RMP was slightly hyperpolarized and the amplitude was decreased a little. The melastatin type transient receptor potential (TRPM) channel 7 has been suggested to be required for intestinal pacemaking activity. TRPM7 produced large outward currents and small inward currents by voltage ramps, ranging from +100 to -100 mV from a holding potential of -60 mV. The inward current of TRPM7 was dramatically increased by a decrease in the extracellular pH. At pH 4.0, the average inward current amplitude measured at -100 mV was increased by about 7 fold, compared with the current amplitude at pH 7.4. Changes in the outward current (measured at +100 mV) were much smaller than those of the inward current. These results indicate that the resting membrane potential of pacemaking activity might be depolarized by external acidic pH through TRPM7 that is required for intestinal pacemaking activity.

• Korean Journal of Veterinary Research
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• v.38 no.2
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• pp.280-289
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• 1998

Voltage-sensitive ion channels contribute to establishment of the cell excitablity and the generation of the cellular function. At hamster oocytes in the primitive stage during developing process, an inward current elicited by voltage pulses was found to be carried mainly by $Ca^{2+}$. Even at present, $Ca^{2+}$ channels serve as the most probable route to pass this inward current but there is no evidence of the presence of this channels in eggs. To date, both the characteristic properties and the physiological role in the early stage of development remain unclear. Here we examined the characteristic properties of the inward current and changes in this currents at unfertilized oocytes, fertilized zygotes and two-cell embryos using whole-cell voltage clamp technique. The inward current carried reportedly by $Ca^{2+}$ was remained following removing external $Ca^{2+}$ but completely abolished by further replacement of impermeants such as tetramethylammonium ion ($TMA^+$) or $choline^+$ instead of $[Na^+]_0$. Tetrodotoxin did not affect on this inward current remained at $[Ca^{2+}]_0$-free condition. Removal of $Na^+$ ion out of the experimental solution clearly decreased the current. After adding 2mM $Ca^{2+}$ to the $Na^+$-free media, the inward current was restored. Interestingly, this current carried by either $Ca^{2+}$ or $Na^+$ was decreased by the reduction of intracellular $Cl^-$ concentration, or by $Cl^-$ channel blockers such as niflumic acid, DIDS and SITS. When $Cl^-$ concentration was lowered without changes in other ionic components, this inward current was reduced. At fertilized oocytes and two-cell embryos, the inward current carried by $Ca^{2+}$ and $Na^+$ was severely reduced. Also $Cl^-$ component could not be observed. From these results, the inward current is composed of $Ca^{2+}$, $Na^+$ and $Cl^-$ component, suggesting that the channel carrying this inward current is not selective specifically to $Ca^{2+}$. During early stage of development, the voltage-sensitive ion current seems not to contribute essentially to the cell cleavage and differentiation. The loss of $Cl^-$ component after fertilization suggests that $Cl^-$ may play a role in maintaining the viability of unfertilized ova.

• The Korean Journal of Physiology and Pharmacology
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• v.1 no.6
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• pp.741-748
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• 1997

In the present study, we have investigated the effect of metabolic inhibition on the inward rectifier K current ($I_{K1}$). Using whole cell patch clamp technique we applied voltage ramp from +80 mV to -140 mV at a holding potential of -30 mV and recorded the whole cell current in single ventricular myocytes isolated from the rabbit heart. The current-voltage relationship showed N-shape (a large inward current and little outward current with a negative slope) which is a characteristic of $I_{K1}$. Application of 0.2 mM dinitrophenol (DNP, an uncoupler of oxidative phosphorylation as a tool for chemical hypoxia) to the bathing solution with the pipette solution containing 5 mM ATP, produced a gradual increase of outward current followed by a gradual decrease of inward current with little change in the reversal potential (-80 mV). The increase of outward current was reversed by glibenclamide ($10\;{\mu}M$), suggesting that it is caused by the activation of $K_{ATP}$. When DNP and glibenclamide were applied at the same time or glibenclamide was pretreated, DNP produced same degree of reduction in the magnitude of the inward current. These results show that metabolic inhibition induces not only the increase of $K_{ATP}$ channel but also the decrease of $I_{K1}$. Perfusing the cell with ATP-free pipette solution induced the changes very similar to those observed using DNP. Long exposure of DNP (30 min) or ATP-free pipette solution produced a marked decrease of both inward and outward current with a significant change in the reversal potential. Above results suggest that the decrease of $I_{K1}$ may contribute to the depolarisation of membrane potential during metabolic inhibition.

• Animal cells and systems
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• v.2 no.4
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• pp.471-476
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• 1998

The G protein-activated inwardly rectifying $K^＋$ channel (GIRK1) was coex-pressed in Xenopus oocytes along with the $5-HT_{1A}$ receptor, a 7-helix receptor known to be coupled to $K^＋$ channels in many neural tissues. Thus, the activation of the $5-HT_{1A}$ receptor by its agonist leads to the opening of GIRK1. The GIRK1 current was measured using the two electrode voltage clamp technique with bath application of 5-HT in the presence of various external potassium concentrations $[K^＋]_0$. GIRK1 showed a strong inward rectification since only hyperpolarizing voltages evoked inward currents. $K^{+}$ was the major ion carrier as evidenced by about 44㎷ voltage shift corresponding to a 10-fold external 〔$K^＋$〕 change. 5-HT induced a concentration-dependent inward $K^＋$ current ($EC_{50}{\equation omitted}10.7nM$) which was blocked by $Ba^{2＋}$. Pertussis toxin (PTX) pre-treatment reduced the $K^＋$ current by as much as about 70%, suggesting that PTX-sensitive G protein ($G_i or G_o$ type) are involved in the $5-HT_{1A}$ receptor-GIRK1 coupling in Xenopus oocytes.

• Proceedings of the Korean Society of Crop Science Conference
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• 2017.06a
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• pp.110-110
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• 2017

Stomata are the main gateways for water and air transport between leaves and the environment. Inward-rectifying potassium channels regulate photo-induced stomatal opening. Rice contains three inward rectifying shaker-like potassium channel proteins, OsKAT1, OsKAT2 and OsKAT3. Among these, only OsKAT2 is specifically expressed in guard cells. Here, we investigated the functions of OsKAT2 in stomatal regulation using three dominant negative mutant proteins, OsKAT2(T235R), OsKAT2(T285A) and OsKAT2(T285D), which are altered in amino acids in the channel pore and at a phosphorylation site. Yeast complementation and patch clamp assays showed that all three mutant proteins lost channel activity. However, among plants overexpressing these mutant proteins, only plants overexpressing OsKAT2(T235R) showed significantly less water loss than the control. Moreover, overexpression of this mutant protein led to delayed photo-induced stomatal opening and increased drought tolerance. Our results indicate that OsKAT2 is an inward-rectifying shaker-like potassium channel that mainly functions in stomatal opening. Interestingly, overexpression of OsKAT2(T235R) did not cause serious defects in growth or yield in rice, suggesting that OsKAT2 is a potential target for engineering plants with improved drought tolerance without yield penalty.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.4
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• pp.371-376
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• 2017

The caudal subnucleus of the spinal trigeminal nucleus (medullary dorsal horn; MDH) receives direct inputs from small diameter primary afferent fibers that predominantly transmit nociceptive information in the orofacial region. Recent studies indicate that reactive oxygen species (ROS) is involved in persistent pain, primarily through spinal mechanisms. In this study, we aimed to investigate the role of xanthine/xanthine oxidase (X/XO) system, a known generator of superoxide anion ($O_2{^-}$), on membrane excitability in the rat MDH neurons. For this, we used patch clamp recording and confocal imaging. An application of X/XO ($300{\mu}M/30mU$) induced membrane depolarization and inward currents. When slices were pretreated with ROS scavengers, such as phenyl N-tert-butylnitrone (PBN), superoxide dismutase (SOD), and catalase, X/XO-induced responses decreased. Fluorescence intensity in the DCF-DA and DHE-loaded MDH cells increased on the application of X/XO. An anion channel blocker, 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS), significantly decreased X/XO-induced depolarization. X/XO elicited an inward current associated with a linear current-voltage relationship that reversed near -40 mV. X/XO-induced depolarization reduced in the presence of $La^{3+}$, a nonselective cation channel (NSCC) blocker, and by lowering the external sodium concentration, indicating that membrane depolarization and inward current are induced by influx of $Na^+$ ions. In conclusion, X/XO-induced ROS modulate the membrane excitability of MDH neurons, which was related to the activation of NSCC.

• The Korean Journal of Physiology and Pharmacology
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• v.24 no.1
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• pp.101-110
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• 2020

Transient receptor potential canonical 4 (TRPC4) channel is a nonselective calcium-permeable cation channels. In intestinal smooth muscle cells, TRPC4 currents contribute more than 80% to muscarinic cationic current (mIcat). With its inward-rectifying current-voltage relationship and high calcium permeability, TRPC4 channels permit calcium influx once the channel is opened by muscarinic receptor stimulation. Polyamines are known to inhibit nonselective cation channels that mediate the generation of mIcat. Moreover, it is reported that TRPC4 channels are blocked by the intracellular spermine through electrostatic interaction with glutamate residues (E728, E729). Here, we investigated the correlation between the magnitude of channel inactivation by spermine and the magnitude of channel conductance. We also found additional spermine binding sites in TRPC4. We evaluated channel activity with electrophysiological recordings and revalidated structural significance based on Cryo-EM structure, which was resolved recently. We found that there is no correlation between magnitude of inhibitory action of spermine and magnitude of maximum current of the channel. In intracellular region, TRPC4 attracts spermine at channel periphery by reducing access resistance, and acidic residues contribute to blocking action of intracellular spermine; channel periphery, E649; cytosolic space, D629, D649, and E687.