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.

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