Morris A P, Frizzell R A
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005.
Am J Physiol. 1993 Apr;264(4 Pt 1):C968-76. doi: 10.1152/ajpcell.1993.264.4.C968.
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.
膜片钳技术与基于摄像的细胞内钙离子浓度（[Ca2+]i）成像相结合，以确定人结肠腺癌细胞（HT - 29）中钙依赖性氯电导的单通道基础。当膜片被切除并置于含有高浓度（1微摩尔）钙离子的溶液中时，氯通道被激活。通过幅度直方图分析测量，它们的单通道电导在 - 90 mV时平均为13 pS，在 + 90 mV膜电位（MP）时为16 pS。在多通道膜片中，氯电流表现出与钙激活的全细胞电流相似的特性：外向整流和在去极化MP时的时间依赖性激活。膜片从细胞上切除后不久，通道活性消失。在细胞贴附式膜片中，在静息[Ca2+]i值（96±18 nM）时氯通道开放很少见，但当[Ca2+]i通过钙离子载体离子霉素（1微摩尔）升高时，氯通道以与[Ca2+]i升高平行的时间进程被激活。重复暴露于离子霉素会使[Ca2+]i产生等效升高，但相应的氯通道活性会逐渐降低。钙介导的激动剂神经降压素（50 nM）引发了短暂的氯通道激活，该激活先于细胞内普遍的[Ca2+]i升高。神经降压素引起的通道激活在移液器中无钙离子时发生，但通过用1,2 - 双（2 - 氨基苯氧基）乙烷 - N,N,N',N' - 四乙酸预加载细胞可消除。因此，响应于钙介导的激动剂神经降压素，氯通道激活是由位于质膜附近的细胞内池释放钙离子导致的。这种15 - pS氯通道的钙依赖性、电压敏感性和动力学表明它是全细胞钙激活氯电流的基础。
