Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). I. Single-channel properties.
Ca(2+)-dependent Cl- channels in undifferentiated human colonic cells (HT-29). I. Single-channel properties.
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DOI：
10.1152/ajpcell.1993.264.4.c968
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发表时间：
1993-04
期刊：
影响因子：
--
通讯作者：
A. P. Morris;R. Frizzell
中科院分区：
文献类型：
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作者：
A. P. Morris;R. Frizzell
应用膜片钳技术结合细胞内Ca ~（2+）浓度（[Ca ~（2+）]i）照相成像技术，研究了人结肠腺癌细胞（HT-29）Ca ~（2+）依赖性Cl ~-电导的单通道基础。当将膜斑切离到含有高（1 μ M）Ca 2+浓度的溶液中时，Cl-通道被激活。通过幅度直方图分析测量的它们的单通道电导在-90 mV和+90 mV膜电位（MP）下平均为13 pS和16 pS。在多通道膜片钳中，Cl-电流表现出与Ca（2+）激活的全细胞电流相似的特性：在去极化MP时，外向整流和时间依赖性激活。通道活动消失后不久，补丁切除细胞。在细胞贴附的贴片中，静息[Ca 2 +]i值（96 +/- 18 nM）时Cl-通道开放不常见，但当[Ca 2 +]i被Ca 2+离子载体离子霉素（1 μ M）增加时，Cl-通道被激活，时间过程与[Ca 2 +]i升高一致。重复离子载体曝光产生等效上升[Ca 2 +]i，但相应的Cl-通道活性逐渐减少。Ca（2+）-介导的激动剂神经降压素（50 nM）引起在全身性细胞[Ca 2 +]i升高之前的瞬时Cl-通道激活。神经降压素的通道激活发生在吸管Ca ~（2+）的情况下，但通过用1，2-双（2-氨基苯氧基）乙烷-N，N，N ′，N ′-四乙酸预加载细胞而消除。因此，响应于Ca（2+）-介导的激动剂神经降压素，Cl-通道激活是由位于质膜附近的细胞内池的Ca 2+动员引起的。该15-pS Cl-通道的Ca ~（2+）依赖性、电压敏感性和动力学表明它是整个细胞Ca ~（2+）激活的Cl-电流的基础。
The patch-clamp technique was combined with camera-based intracellular Ca2+ concentration ([Ca2+]i) imaging to identify the single-channel basis of the Ca(2+)-dependent Cl- conductance in human colonic adenocarcinoma cells (HT-29). Cl- channels were activated when membrane patches were excised into solutions containing high (1 microM) Ca2+ concentrations. Their single-channel conductance, measured by amplitude histogram analysis, averaged 13 pS at -90 mV and 16 pS at +90 mV membrane potential (MP). In multiple channel patches, Cl- currents showed properties similar to Ca(2+)-activated whole cell currents: outward rectification and time-dependent activation at depolarizing MP. Channel activity disappeared shortly after patch excision from the cell. In cell-attached patches, Cl- channel opening was infrequent at resting [Ca2+]i values (96 +/- 18 nM), but when [Ca2+]i was increased by the Ca2+ ionophore ionomycin (1 microM), Cl- channels were activated with a time course that paralleled the [Ca2+]i rise. Repetitive ionophore exposure produced equivalent rises in [Ca2+]i, but the corresponding Cl- channel activity became progressively reduced. The Ca(2+)-mediated agonist neurotensin (50 nM) elicited a transient Cl- channel activation that preceded the generalized cellular [Ca2+]i rise. Channel activation with neurotensin occurred in the absence of pipette Ca2+ but was abolished by preloading cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus, in response to the Ca(2+)-mediated agonist neurotensin, Cl- channel activation results from Ca2+ mobilization from intracellular pools localized within the vicinity of the plasma membrane. The Ca2+ dependency, voltage sensitivity, and kinetics of this 15-pS Cl- channel indicate that it is the basis of the whole cell Ca(2+)-activated Cl- current.