CFTR DISPLAYS VOLTAGE-DEPENDENCE AND 2 GATING MODES DURING STIMULATION
CFTR DISPLAYS VOLTAGE-DEPENDENCE AND 2 GATING MODES DURING STIMULATION
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DOI：
10.1085/jgp.104.3.541
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发表时间：
1994-09-01
影响因子：
3.8
通讯作者：
MACHEN, TE
中科院分区：
文献类型：
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作者：
FISCHER, H;MACHEN, TE
膜片钳技术与电流噪声分析相结合，用于阐明 cAMP 依赖性刺激期间 CFTR（囊性纤维化跨膜电导调节器）调节的潜在事件。用毛喉素以细胞贴壁模式刺激表达 CFTR 的 3T3 成纤维细胞。每个补丁的激活通道数量 (N) 范围从 1 到大约 100。在真正的单通道录音中，CFTR 的门控最好通过两个开放状态（类似于 5 和类似于 100 毫秒）和三个关闭状态（小于或等于 5、类似于 100 和类似于 1,000 毫秒）来描述。当前噪声分析得出的频谱包含两个不同的洛伦兹噪声分量，其转角频率分别为 1.3 Hz 和类似于 50 Hz。单通道时间常数取决于电压。最快的闭合状态将其贡献从+100 mV 时的 48% 增加到-100 mV 时的 87%，而中等开放状态将其长度减少到一半，导致门控由快速事件主导。类似地，快速洛伦兹增加了其幅度，并且其转角频率从+100 mV 时的44 Hz 增加到-100 mV 时的91 Hz，而慢速洛伦兹则与电压无关。在多通道记录中，N.P-o（即 N 次开放概率）显着增加，在 -90 至 +90 mV 之间平均增加 52%。毛喉素刺激使 CFTR 的 P-o 增加到接近 0.5，这是由于最长闭合状态的减少而导致更快的打开和闭合状态不受影响。刺激期间两个角频率均不受影响。多通道音色的录音还揭示了独特的、非常长的通道开口（高 P-o 模式，平均 13 秒）。多通道记录中均存在表现出高 P-o（即 P-o 类似于 1.0）或低 P-o（即 P-o 类似于 0.5）门控模式的通道，并且 CFTR 在刺激期间切换模式。此外，切换到高 P-o 模式似乎是通道对的协作事件。高毛喉素浓度（即 10 μM）有利于向高 P-o 模式的转变，表明由于 CFTR 构型的根本变化而导致细胞介导的模型转换调节。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模式切换到高 P-o 模式。
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 similar to 100. In true single-channel recordings, CFTR's gating was best described by two open states (similar to 5 and similar to 100 ms) and three closed states (less than or equal to 5, similar to 100, and similar to 1,000 ms). Current noise analysis resulted in spectra containing two distinct Lorentzian noise components with corner frequencies of 1.3 Hz and similar to 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.P-o (i.e., N times open probability) increased significantly, on average by 52% between -90 and +90 mV. Stimulation with forskolin increased P-o of CFTR to similar to 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 P-o mode, average 13 s). Channels exhibiting high P-o (i.e., P-o similar to 1.0) or low P-o (i.e., P-o similar to 0.5) gating modes were both present in multichannel recordings, and CFTRs switched modes during stimulation. In addition, the switch to the high P-o mode appeared to be a cooperative event for channel pairs. High forskolin concentration (i.e., 10 mu M) favored transition into the high P-o 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 P-o mode.