Korbmacher C, Segal A S, Fejes-Tóth G, Giebisch G, Boulpaep E L
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
J Gen Physiol. 1993 Oct;102(4):761-93. doi: 10.1085/jgp.102.4.761.
M-1 cells, derived from a microdissected cortical collecting duct of a transgenic mouse, grown to confluence on a permeable support, develop a lumen-negative amiloride-sensitive transepithelial potential, reabsorb sodium, and secrete potassium. Electron micrographs show morphological features typical of principal cells in vivo. Using the patch clamp technique distinct differences are detected in whole-cell membrane current and voltage (Vm) between single M-1 cells 24 h after seeding vs cells grown to confluence. (a) Under control conditions (pipette: KCl-Ringer; bath: NaCl-Ringer) Vm averages -42.7 +/- 3.4 mV in single cells vs -16.8 +/- 4.1 mV in confluent cells. Whole-cell conductance (Gcell) in confluent cells is 2.6 times higher than in single cells. Cell capacitance values are not significantly different in single vs confluent M-1 cells, arguing against electrical coupling of confluent M-1 cells. (b) In confluent cells, 10(-4)-10(-5) M amiloride hyperpolarizes Vm to -39.7 +/- 3.0 mV and the amiloride-sensitive fractional conductance of 0.31 shows a sodium to potassium selectivity ratio of approximately 15. In contrast, single cells express no significant amiloride-sensitive conductance. (c) In single M-1 cells, Gcell is dominated by an inwardly rectifying K-conductance, as exposure to high bath K causes a large depolarization and doubling of Gcell. The barium-sensitive fraction of Gcell in symmetrical KCl-Ringer is 0.49 and voltage dependent. (d) In contrast, neither high K nor barium in the apical bath affect confluent M-1 cells, showing that confluent cells lack a significant apical K conductance. (e) Application of 500 microM glibenclamide reduces whole-cell currents in both single and confluent M-1 cells with a glibenclamide-sensitive fractional conductance of 0.71 and 0.83 in single and confluent cells, respectively. Glibenclamide inhibition occurs slower in confluent M-1 cells than in single cells, suggesting a basolateral action of this lipophilic drug on ATP-sensitive basolateral K channels in M-1 cells. (f) A component of the whole-cell conductance in M-1 cells appears as a deactivating outward current during large depolarizing voltage pulses and is abolished by extracellular chloride removal. The deactivating chloride current averages 103.6 +/- 16.1 pA/cell, comprises 24% of the outward current, and decays with a time constant of 179 +/- 13 ms. The outward to inward conductance ratio obtained from deactivating currents and tail currents is 2.4, indicating an outwardly rectifying chloride conductance.
M-1细胞源自转基因小鼠经显微切割的皮质集合管，在可渗透支持物上生长至汇合状态，可产生管腔负性的阿米洛利敏感跨上皮电位，重吸收钠并分泌钾。电子显微镜照片显示出体内主细胞典型的形态特征。使用膜片钳技术检测到，接种24小时后的单个M-1细胞与生长至汇合状态的细胞相比，全细胞膜电流和电压（Vm）存在明显差异。（a）在对照条件下（移液管：KCl-林格液；浴槽：NaCl-林格液），单个细胞的Vm平均为-42.7±3.4 mV，而汇合细胞的Vm为-16.8±4.1 mV。汇合细胞的全细胞电导（Gcell）比单个细胞高2.6倍。单个M-1细胞与汇合M-1细胞的细胞电容值无显著差异，这排除了汇合M-1细胞存在电偶联的可能性。（b）在汇合细胞中，10⁻⁴ - 10⁻⁵ M的阿米洛利使Vm超极化至-39.7±3.0 mV，阿米洛利敏感的分数电导为0.31，表明钠钾选择性比约为15。相比之下，单个细胞未表现出明显的阿米洛利敏感电导。（c）在单个M-1细胞中，Gcell主要由内向整流钾电导主导，因为暴露于高浴槽钾会导致大幅度去极化并使Gcell加倍。在对称KCl-林格液中，Gcell对钡敏感的部分为0.49，且与电压有关。（d）相反，顶端浴槽中的高钾或钡均不影响汇合的M-1细胞，表明汇合细胞缺乏明显的顶端钾电导。（e）应用500 μM格列本脲可降低单个和汇合M-1细胞的全细胞电流，单个细胞和汇合细胞中格列本脲敏感的分数电导分别为0.71和0.83。格列本脲对汇合M-1细胞的抑制作用比对单个细胞的抑制作用出现得慢，这表明这种亲脂性药物对M-1细胞中ATP敏感的基底外侧钾通道具有基底外侧作用。（f）M-1细胞全细胞电导的一个成分在大幅度去极化电压脉冲期间表现为失活外向电流，且在去除细胞外氯化物后消失。失活氯化物电流平均为103.6±l6.1 pA/细胞，占外向电流的24%，并以179±l3 ms的时间常数衰减。从失活电流和尾电流获得的外向与内向电导比为2.4，表明存在外向整流氯化物电导。
