• 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.

• Molecules and Cells
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• v.22 no.3
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• pp.328-335
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• 2006

Imperatoxin A ($IpTx_a$), a 3.7 kDa peptide from the African scorpion Pandinus imperator, is an agonist of the skeletal muscle ryanodine receptor (RyR1). In order to study the structure of the toxin and its effect on RyR1, $IpTx_a$ cDNA was PCR-amplified using 3 pairs of primers, and the toxin was expressed in E. coli. The toxin was further purified by chromatography, and various point mutants in which basic amino acids were substituted by alanine were prepared by site-directed mutagenesis. Studies of single channel properties by the planar lipid bilayer method showed that the recombinant $IpTx_a$ was identical to the synthetic $IpTx_a$ with respect to high-performance liquid chromatography mobility, amino acid composition and specific effects on RyR1. Mutations of certain basic amino acids ($Lys^{19}$, $Arg^{23}$, and $Arg^{33}$) dramatically reduced the capacity of the peptide to activate RyRs. A subconductance state predominated when $Lys^8$ was substituted with alanine. These results suggest that some basic amino acid residues in $IpTx_a$ are important for activation of RyR1, and that $Lys^8$ plays an important role in regulating the gating mode of RyR1.

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