• Korean Journal of Veterinary Research
• /
• v.32 no.2
• /
• pp.157-164
• /
• 1992

Changes in the both inward current and conductance of membrane by the fertilization were observed using the one microelectrode voltage clamp(or switch clamp) technique. Unfertilized eggs and both 1- and 2-cell stage eggs after fertilization were donated from the superovulated mouse (ICR, more than 6 weeks old) treated with PMSG(pregnant mare serum gonadotropin, Sigma) and HCG(human chorionic gonadotropin, Sigma) and naturally mated ones, respectively in this experiment. Membrane potential was held at -90mV and the voltage step was applied from -80mV to 50mV with interval of 10mV or 20mV for 300ms. since both of amplitudes and time courses in the membrane currents were various according to the states of cells and clamping condition, results were presented by their $averages{\pm}SEM$(standard mean error)and ratios or percentages. Inward currents began to appear in response to the step depolarization from -60mV and reached its maximum at -50mV. However, since the potential was not clamped evenly during the voltage step, current-voltage(I-V) relationship might be positively shifted 10 or 20mV. From the steady-state currents plotted in the I-V curve, outward rectification was markedly observed. Peak inward currents$(i_{in})$ at -50mV were $-0.62{\pm}0.23nA$(n=4),$-0.52{\pm}0.25nA$(n=5) and $-0.37{\pm}0.25nA$(n=6), in the 1-cell stage, 2-cell stage fertilized eggs and in the unfertilized eggs, respectively. Pure inward current (difference between steady-state and peak, $i_{in. pure}$) were $-1.01{\pm}0.23nA$, $-0.69{\pm}0.43nA$ and $-0.68{\pm}0.29nA$, respectively in the 1-cell stage fertilized eggs, unfertilized eggs and 2-cell stage fertilized eggs. These results suggested that the outward current in fertilized eggs of 2-cell stage was more increased than those in the unfertilized eggs. Pure inward currents in the all stages of eggs showed a similar fashion in the I-V relationship from -50mV to 50mV and reversal potential at 50mV. Time constant of inactivation$({\tau})$ in the inward current was decreased as the membrane potential was depolarized in the unfertilized and 2-cell stage eggs but in the 1-cell stage eggs t was not likely to be affected significantly. Slope conductances were 14.2nS, 8.9n5 and 7.7nS in the 1-cell, 2-cell stage fertilized eggs and the unfertilized eggs, respectively. Membranes between two cells within a zona pellucida seem to be electrical-connected in the 2-cell stage eggs from the observation made in the analysis for the electronic spread and decay to the current stimuli. Both of inward current and membrane conductance were increased after fertilization in the mouse eggs. Inward current seems to be carried by the same ion or through the same channels up to the 2-cell stage and ion that carried inward current was thought to play important function after fertilization in the mouse eggs.

• The Korean Journal of Physiology and Pharmacology
• /
• v.8 no.4
• /
• pp.219-225
• /
• 2004

The pelvic ganglia provide autonomic innervations to the various urogenital organs, such as the urinary bladder, prostate, and penis. It is well established that both sympathetic and parasympathetic synaptic transmissions in autonomic ganglia are mediated mainly by acetylcholine (ACh). Until now, however, the properties of ACh-induced currents and its receptors in pelvic ganglia have not clearly been elucidated. In the present study, biophysical characteristics and molecular nature of nicotinic acetylcholine receptors (nAChRs) were studied in sympathetic and parasympathetic major pelvic ganglion (MPG) neurons. MPG neurons isolated from male rat were enzymatically dissociated, and ionic currents were recorded by using the whole cell variant patch clamp technique. Total RNA from MPG neuron was prepared, and RT-PCR analysis was performed with specific primers for subunits of nAChRs. ACh dose-dependently elicited fast inward currents in both sympathetic and parasympathetic MPG neurons $(EC_{50};\;41.4\;{\mu}M\;and\;64.0\;{\mu}M,\;respectively)$. ACh-induced currents showed a strong inward rectification with a reversal potential near 0 mV in current-voltage relationship. Pharmacologically, mecamylamine as a selective antagonist for ${\alpha}3{\beta}4$ nAChR potently inhibited the ACh-induced currents in sympathetic and parasympathetic neurons $(IC_{50};\;0.53\;{\mu}M\;and\;0.22\;{\mu}M,\;respectively)$. Conversely, ${\alpha}-bungarotoxin$, ${\alpha}-methyllycaconitine$, and $dihydro-{\beta}-erythroidine$, which are known as potent and sensitive blockers for ${\alpha}7$ or ${\alpha}4{\beta}2$ nAChRs, below micromolar concentrations showed negligible effect. RT-PCR analysis revealed that ${\alpha}3$ and ${\beta}4$ subunits were predominantly expressed in MPG neurons. We suggest that MPG neurons have nAChRs containing ${\alpha}3$ and ${\beta}4$ subunits, and that their activation induces fast inward currents, possibly mediating the excitatory synaptic transmission in pelvic autonomic ganglia.

• The Korean Journal of Physiology and Pharmacology
• /
• v.3 no.1
• /
• pp.1-10
• /
• 1999

We sought to find out the mechanism of vascular relaxation by extracellular $K^+$ concentration $([K^+]_o)$ in the cerebral resistant arteriole from rabbit. Single cells were isolated from the cerebral resistant arteriole, and using voltage-clamp technique barium-sensitive $K^+$ currents were recorded, and their characteristics were observed. Afterwards, the changes in membrane potential and currents through the membrane caused by the change in $[K^+]_o$ was observed. In the smooth muscle cells of cerebral resistant arteriole, ion currents that are blocked by barium, 4-aminopyridine (4-AP), and tetraethylammonium (TEA) exist. Currents that were blocked by barium showed inward rectification. When the $[K^+]_o$ were 6, 20, 60, and 140 mM, the reversal potentials were $-82.7{\pm}1.0,\;-49.5{\pm}1.86,\;-26{\pm}1.14,\;-5.18{\pm}1.17$ mV, respectively, and these values were almost identical to the calculated $K^+$ equilibrium potential. The inhibition of barium-sensitive inward currents by barium depended on the membrane potential. At the membrane potentials of -140, -100, and -60 mV, $K_d$ values were 0.44, 1.19, and 4.82 ${\mu}M,$ respectively. When $[K^+]_o$ was elevatedfrom 6 mM to 15 mM, membrane potential hyperpolarized to -50 mV from -40 mV. Hyperpolarization by $K^+$ was inhibited by barium but not by ouabain. When the membrane potential was held at resting membrane potential and the $[K^+]_o$ was elevated from 6 mM to 15 mM, outward currents increased; when elevated to 25 mM, inward currents increased. Fixing the membrane potential at resting membrane potential and comparing the barium-sensitive outward currents at $[K^+]_o$ of 6 and 15 mM showed that the barium- sensitive outward current increased at 15 mM $K^+.$ From the above results the following were concluded. Barium-sensitive $K^+$?channel activity increased when $[K^+]_o$ is elevated and this leads to an increase in $K^+-outward$ current. Consequently, the membrane potential hyperpolarizes, leading to the relaxation of resistant arteries, and this is thought to contribute to an increase in the local blood flow of brain.

• The Korean Journal of Physiology and Pharmacology
• /
• v.3 no.1
• /
• pp.101-117
• /
• 1999

Amyotrophic lateral sclerosis (ALS) is a degenerative neuromuscular disease of unknown etiology in which the upper and lower motor neurons are progressively destroyed. Recent evidences support the role of autoimmune mechanisms in the pathogenesis of ALS. This study investigated the effects of sera from ALS patients on neuromuscular transmission in phrenic nerve-hemidiaphragm preparations and on calcium currents of single isolated dorsal root ganglion (DRG) cells in mice. Mice were injected with either control sera from healthy adults or ALS sera from 18 patients with ALS of sporadic form, for three days. Miniature end plate potential (MEPP) and nerve-evoked end plate potential (EPP) were measured using intracellular recording technique and the quantal content was determined. Single isolated DRG cells were voltage-clamped with the whole-cell configuration and membrane currents were recorded. Sera from 14 of 18 ALS patients caused a significant increase in MEPP frequency in normal Ringer's solution $(4.62{\pm}0.14\;Hz)$ compared with the control $(2.18{\pm}0.15\;Hz).$ In a high $Mg^{2+}/low\;Ca^{2+}$ solution, sera from 13 of 18 ALS patients caused a significant increase in MEPP frequency, from $2.18{\pm}0.31$ Hz to $6.09{\pm}0.38$ Hz. Sera from 11 of 18 patients produced a significant increase of nerve-evoked EPP amplitude, from $0.92{\pm}0.05$ mV to $1.30{\pm}0.04$ mV, while the other seven ALS sera did not alter EPP amplitude. In the ALS group, EPP quantal content was also elevated by the sera of 14 patients (from $1.49{\pm}0.07$ to $2.35{\pm}0.07).$ MEPP frequency and amplitude in wobbler mouse were $4.03{\pm}0.53$ Hz and $1.37{\pm}0.18$ mV, respectively, which were significantly higher than those of wobbler controls (wobblers without the symptoms of wobbler). Sera from ALS patients significantly reduced HVA calcium currents of DRG cells to 42.7% at -10 mV. Furthermore, the inactivation curve shifted to more negative potentials with its half-inactivation potential changed by 6.98 mV. There were, however, significant changes neither in the reversal potential of $I_{Ca}$ nor in the I-V curve. From these results it was concluded that: 1) The serum factors of sporadic ALS patients increase neuromuscular transmission and can alter motor nerve terminal presynaptic function. This suggests that ALS serum factors may play an important role in the early stage of ALS, and 2) Calcium currents in DRG cells were reduced and rapidly inactivated by ALS sera, suggesting that in these cells, ALS serum factors may exert interaction with the calcium channel.

• Journal of Embryo Transfer
• /
• v.19 no.2
• /
• pp.101-112
• /
• 2004

Chloride($Cl^-$) channels play critical roles in cell homeostasis and its specific functions such as volume regulation, differentiation, secretion, and membrane stabilization. The presence of these channels have been reported in all kinds of cells and even in frog oocytes. These essential role of $Cl^-$­ channels in cell homeostasis possibly play any role in egg homeostasis and in the early stage of development, however, there has been no report about the presence of $Cl^-$­ channel in the mammalian oocyte. This study was performed to elucidate the presence of $Cl^-$­ channels in hamster eggs. When allowing only $Cl^-$­ to pass through the channel of the egg membrane by using impermeant cation such as N-methyl-D-glucamine(NMDG), single channel currents were recorded. These channel currents showed typical long-lasted openings interrupted by rapid flickering. Mean open $time({\tau}o)$ was 43${\pm}$10.14 ms(n=9, at 50 mV). The open probability(Po) was decrease with depolarization. The current-voltage relation showed outward rectification. Outward slop conductance(32${\pm}$5.4 pS, n=22) was steeper than the inward slop conductance(10${\pm}$1.3 pS). Under the condition of symmetrical 140 mM NaCl, single channel currents were reversed at 0 mV(n=4). This reversal potential(Erev) was shifted from 0 mV at 140 mM concentration of internal NaCl(140 mM [Na+]i) to ­9.8${\pm}$0.5 mV(n=4) at 70 mM [Na+]i and 11.5${\pm}$1.9 mV at 280 mM [Na+]i(n=4) respectively, strongly suggesting that these are single $Cl^-$­ channel currents. To examine further whether this channel has pharmacological property of the $Cl^-$­ channel, specific Cl­ channel blockers, IAA-94(Indanyloxyacetic acid-94) and DIDS(4, 4'-diisothiocyan ostillben- 2-2'disulfonic acid) were applied. IAA-94 inhibited the channel current in a dose-dependent manner and revealed a rapid and flickering block. From these electrophysiological and pharmacological resluts, we found the novel $Cl^-$­ channel present in the hamster oocyte membrane. The first identification of $Cl^-$­ channel in the hamster oocyte may give a clue for the further study on the function of $Cl^-$­ channel in the fertilization and cell differentiation.

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

• Proceedings of the Korean Biophysical Society Conference
• /
• 2003.06a
• /
• pp.37-37
• /
• 2003

We have shown the $Ca^{2+}$-activated chloride current is present in cardiac myocyte in rabbit pulmonary vein (Kim et al., 2002). This current amplitude was increased as [N $a^{+}$]$_{i}$ was increased and we suggested this chloride current may be involve in the spontaneous action potential frequency change. Since this current is activated by the increase of intracellular $Ca^{2+}$, we would like to test what is the inducer of the increase of [C $a^{2+}$]$_{i}$ between a L-type $Ca^{2+}$-current or a reverse mode of N $a^{+}$-C $a^{2+}$ exchange current. White rabbit (1.5 kg) was used and anesthetized with Ketamin (100 mg/kg). Pulmonary vein (PV) was isolated and sleeve area between left atrium and PV was dissected. Using collagenase (Worthington 0.7 mg/cc), single cardiac myocytes were isolated. In the presence of 15 mM of N $a^{＋}$, three steps of voltage pulses were applied (holding potential : -40 ㎷, -80 ㎷ for 50 msec, 30 ㎷ for 5 msec, 10 ㎷ steps from -70 ㎷ to 60 ㎷). The inward and outward tail current was activated after brief 5 msec prepulse. The outward tail current was blocked by the removal of extracellular chloride substituted by glucuronic acid or by a chloride channel blocker, 5 mM 9-AC. But the inward tail current was still remained even though the amplitude was decreased. The reversal potentials were changed to the direction of the change of chloride equilibrium potential ( $E_{Cl}$ ) but the shift of equilibrium potential was not enough to match to the theoretical equilibrium potential shift. In the presence of L-type $Ca^{2+}$ channel blocker, nifedipine 1 uM, inward tail currents were greatly reduced but the outward current tail currents were still remained. In the presence of N $a^{+}$-C $a^{2+}$ exchange current blocker, 10 uM KB-R7943, the inward and outward tail currents were blocked almost completely. We tried to test the $Ca^{2+}$sensitivity of the chloride current with various [C $a^{2+}$]$_{i}$ in pipette solution from 100 nM to 1 uM but we failed to activate $Ca^{2＋}$-activated chloride currents even though the cell became contracted in the presence of 1 uM $Ca^{2+}$. From these results, we could conclude that the increase of [C $a^{2+}$]$_{i}$ to activate the outward $Ca^{2+}$-activated chloride current was mainly induced by the activation of the reverse mode of N $a^{+}$-C $a^{2+}$ exchanger, But for the increase of [C $a^{2+}$]$_{i}$ to activate the inward tail current, L-type $Ca^{2+}$ current may be the major provoking current. Since the cytosolic increase of [C $a^{2+}$]$_{i}$ through pipette solution have failed to activate $Ca^{2+}$-activated chloride current, this chloride current may have very low $Ca^{2+}$ sensitivity or a comparmental increase $Ca^{2+}$ such as in subsarcolemmal space may activate the chloride current. Since there are several reports and models that the increase of $Ca^{2+}$ in subsarcolemmal space would be over several to tens of uM, both possibility may be valid together.uM, both possibility may be valid together.