Pon D C, Hill C E
Department of Physiology, Queen's University, Kingston, Ontario, Canada.
J Cell Physiol. 1997 Apr;171(1):87-94. doi: 10.1002/(SICI)1097-4652(199704)171:1<87::AID-JCP10>3.0.CO;2-L.
A large-conductance, Ca2+-activated K+ channel was identified and characterized in embryonic chick hepatocytes using the patch-electrode voltage-clamp technique. The channel conductance was 213 pS in excised patches bathed in symmetrical 145 mM KCI and 1 mM Ca2+. Current-voltage relationships were linear with high K+ on both sides of the membrane but showed constant field rectification as the K+ gradient was increased. The reversal potential shifted 58 mV per 10-fold change in the ratio of external to internal K+. Channel openings occurred at potentials higher than +50 mV in cell-attached patches. The open probability X voltage relationship shifted to more negative potentials in excised, inside-out patches exposed to a solution containing high Ca2+. The voltage sensitivity of the channel was not significantly affected by changes in internal Ca2+ concentration. Conversely, channel gating, reflected in the half-activation potential, shifted 118 mV per 10-fold change in internal Ca2+ at concentrations less than approximately 2 microM, although at higher Ca2+, this parameter was Ca2+ insensitive. Channel open probability in cell-attached patches increased significantly following exposure of the cells to either the Ca2+ ionophore A-23187 or L-alanine, a cell-volume modulator. Channel density increased with time spent in culture from no observations in 10-hr cells, through 13 and 80% of patches in 24-and 48-hr cultured cells, respectively. The implications of delayed functional expression for ion channel studies in acutely dissociated cells is discussed.
运用膜片钳电压钳技术,在胚胎期鸡肝细胞中鉴定并表征了一种大电导、Ca2+激活的K+通道。在对称的145 mM KCl和1 mM Ca2+溶液浸泡的膜片上,通道电导为213 pS。膜两侧均为高K+时,电流-电压关系呈线性,但随着K+梯度增加呈现恒定场整流。外部与内部K+比值每10倍变化,反转电位移动58 mV。在细胞贴附膜片中,通道开放发生在高于+50 mV的电位。在暴露于含高Ca2+溶液的切除的内向外膜片中,开放概率X电压关系向更负的电位移动。通道的电压敏感性不受内部Ca2+浓度变化的显著影响。相反,反映在半激活电位上的通道门控,在内部Ca2+浓度小于约2 microM时,每10倍变化移动118 mV,尽管在较高Ca2+浓度时,该参数对Ca2+不敏感。细胞贴附膜片中的通道开放概率在细胞暴露于Ca2+离子载体A-23187或细胞体积调节剂L-丙氨酸后显著增加。通道密度随着培养时间增加,10小时的细胞未观察到通道,24小时和48小时培养的细胞中分别有13%和80%的膜片观察到通道。本文讨论了急性解离细胞中离子通道研究中功能表达延迟的影响。