Fischer H, Machen T E
Department of Molecular and Cell Biology, University of California at Berkeley 94720.
J Gen Physiol. 1994 Sep;104(3):541-66. doi: 10.1085/jgp.104.3.541.
The patch-clamp technique in conjunction with current noise analysis was employed to clarify the events underlying the regulation of the CFTR (cystic fibrosis transmembrane conductance regulator) during cAMP-dependent stimulation. 3T3 fibroblast cells expressing the CFTR were stimulated in cell-attached mode with forskolin. The number (N) of activated channels per patch ranged from 1 to approximately 100. In true single-channel recordings, CFTR's gating was best described by two open states (approximately 5 and approximately 100 ms) and three closed states (< or = 5, approximately 100, and approximately 1,000 ms). Current noise analysis resulted in spectra containing two distinct Lorentzian noise components with corner frequencies of 1.3 Hz and approximately 50 Hz, respectively. Single-channel time constants were dependent on voltage. The fastest closed state increased its contribution from 48% at +100 mV to 87% at -100 mV, and the medium open state reduced its length to one half, resulting in gating dominated by fast events. Similarly, the fast Lorentzian increased its amplitude, and its corner frequency increased from 44 Hz at +100 mV to 91 Hz at -100 mV, while the slow Lorentzian was voltage independent. In multi-channel recordings N.Po (i.e., N times open probability) increased significantly, on average by 52% between -90 and +90 mV. Stimulation with forskolin increased Po of CFTR to approximately 0.5, which resulted from a decrease of the longest closed state while the faster open and closed states were unaffected. Neither corner frequency was affected during stimulation. Recordings from multichannel patches revealed in addition, unique, very long channel openings (high Po mode, average 13 s). Channels exhibiting high Po (i.e., Po approximately 1.0) or low Po (i.e., Po approximately 0.5) gating modes were both present in multichannel recordings, and CFTRs switched modes during stimulation. In addition, the switch to the high Po mode appeared to be a cooperative event for channel pairs. High forskolin concentration (i.e., 10 microM) favored transition into the high Po mode, suggesting a cellularly mediated regulation of model switching due to a fundamental change in configuration of the CFTR. Thus, during stimulation the CFTR increased its activity through two distinct effects: the reduction of the long closed state and modal switching to the high Po mode.
采用膜片钳技术结合电流噪声分析，以阐明环磷酸腺苷（cAMP）依赖性刺激过程中CFTR（囊性纤维化跨膜传导调节因子）调控的潜在机制。用福斯高林以细胞贴附模式刺激表达CFTR的3T3成纤维细胞。每个膜片上激活通道的数量（N）范围为1至约100个。在真正的单通道记录中，CFTR的门控最好用两种开放状态（约5毫秒和约100毫秒）和三种关闭状态（≤5毫秒、约100毫秒和约1000毫秒）来描述。电流噪声分析得到的频谱包含两个不同的洛伦兹噪声成分，其拐角频率分别为1.3赫兹和约50赫兹。单通道时间常数取决于电压。最快的关闭状态在+100毫伏时的贡献从48%增加到-100毫伏时的87%，中等开放状态的持续时间缩短至一半，导致门控由快速事件主导。同样，快速洛伦兹成分的幅度增加，其拐角频率从+100毫伏时的44赫兹增加到-100毫伏时的91赫兹，而慢速洛伦兹成分与电压无关。在多通道记录中，N·Po（即N乘以开放概率）显著增加，在-90毫伏至+90毫伏之间平均增加52%。用福斯高林刺激使CFTR的Po增加到约0.5，这是由于最长关闭状态的减少，而较快的开放和关闭状态未受影响。刺激过程中两个拐角频率均未受影响。此外，多通道膜片的记录还显示出独特的、非常长的通道开放（高Po模式，平均13秒）。在多通道记录中同时存在表现出高Po（即Po约为1.0）或低Po（即Po约为0.5）门控模式的通道，并且CFTR在刺激过程中会切换模式。此外，向高Po模式的切换似乎是通道对的协同事件。高浓度福斯高林（即10微摩尔）有利于转变为高Po模式，这表明由于CFTR构象的根本变化，存在细胞介导的模式切换调节。因此，在刺激过程中，CFTR通过两种不同的效应增加其活性：长关闭状态的减少和向高Po模式的模式切换。
