Fukushima Y, Hagiwara S
J Physiol. 1985 Jan;358:255-84. doi: 10.1113/jphysiol.1985.sp015550.
Membrane currents through the Ca2+ channel were studied in a hybridoma cell line (MAb-7B) constructed by fusion of S194 myeloma cells and splenic B lymphocytes from the mouse. The whole-cell variation of the patch-electrode voltage-clamp technique was used. When [Ca2+]o = 2.5 mM, [Na+]o = 150 mM and [Na+]i = 155 mM, the current reversed from inward to outward at 20.9 +/- 2.4 mV (mean +/- S.D., n = 62). Both inward and outward currents showed voltage-dependent inactivation with the same membrane potential dependence of steady-state inactivation. The decay time constant of the current decreased from about 27 ms at -44 mV to a saturation value of 16 ms at about -20 mV, and remained at this value even when the current became outward. From the above results both the inward and outward currents were considered to flow through Ca2+ channels. The inward current showed no change when the external Na+ was replaced with Cs+ or tetraethylammonium and increased when [Ca2+]o was increased. Also, the reversal potential became more positive with increasing [Ca2+]o with a slope of 29 mV/decade change of [Ca2+]o. Effects of different divalent cations examined at 10 mM concentration showed the reversal potential to become more positive in the order of Mn2+, Sr2+ approximately equal to Ba2+ and Ca2+ whereas the relative maximum amplitudes of peak inward current were 1.0 for Ca2+, 1.24 for Sr2+, 0.99 for Ba2+ and 0.07 for Mn2+. When [Ca2+]o or [Mg2+]o was reduced by chelators, monovalent cations became capable of carrying inward current through the Ca2+ channel. These monovalent currents share common kinetic properties with the Ca2+ current, as judged from the steady-state inactivation and the decay time constant of the current. The monovalent cation current was blocked by divalent cations in a voltage-dependent manner. The half-blocking concentrations of Ca2+ and Mg2+ at -45 mV were 2.0 X 10(-6) M and 3.0 X 10(-5) M respectively. The same voltage-dependent binding mechanism can explain the outward current carried by monovalent cations at large positive potentials at normal Ca2+ concentrations. The suppression of the monovalent currents by Ca2+ and Mg2+ showed different voltage dependences. The suppression by Ca2+ increased and then decreased as the membrane potential was made negative, whereas the suppression by Mg2+ increased monotonically. This difference can be explained by considering the fact the Ca2+ is permeant and Mg2+ is impermeant through the Ca2+ channel.
在由S194骨髓瘤细胞与小鼠脾B淋巴细胞融合构建的杂交瘤细胞系(MAb - 7B)中研究了通过Ca2+通道的膜电流。采用膜片钳技术的全细胞记录模式。当[Ca2+]o = 2.5 mM,[Na+]o = 150 mM且[Na+]i = 155 mM时,电流在20.9±2.4 mV(平均值±标准差,n = 62)处由内向转为外向。内向电流和外向电流均表现出电压依赖性失活,且稳态失活具有相同的膜电位依赖性。电流的衰减时间常数从-44 mV时的约27 ms降至约-20 mV时的饱和值16 ms,即使电流变为外向时也保持该值。根据上述结果,内向电流和外向电流均被认为是通过Ca2+通道流动的。当外部Na+被Cs+或四乙铵取代时,内向电流无变化,而当[Ca2+]o增加时,内向电流增加。此外,随着[Ca2+]o增加,反转电位变得更正,[Ca2+]o每变化一个数量级,斜率为29 mV。在10 mM浓度下检测不同二价阳离子的作用,结果显示反转电位按Mn2+、Sr2+≈Ba2+和Ca2+的顺序变得更正,而内向电流峰值的相对最大幅度分别为:Ca2+为1.0,Sr2+为1.24,Ba2+为0.99,Mn2+为0.07。当用螯合剂降低[Ca2+]o或[Mg2+]o时,单价阳离子能够通过Ca2+通道携带内向电流。从电流的稳态失活和衰减时间常数判断,这些单价电流与Ca2+电流具有共同的动力学特性。单价阳离子电流被二价阳离子以电压依赖性方式阻断。在-45 mV时,Ca2+和Mg2+的半阻断浓度分别为2.0×10(-6) M和3.0×10(-5) M。相同的电压依赖性结合机制可以解释在正常Ca2+浓度下,大的正电位时单价阳离子携带的外向电流。Ca2+和Mg2+对单价电流的抑制表现出不同的电压依赖性。Ca2+的抑制作用随着膜电位变负先增加后降低,而Mg2+的抑制作用单调增加。考虑到Ca2+可透过Ca2+通道而Mg2+不可透过这一事实,可以解释这种差异。