Epithelial cells of toad (Bufo bufo) skin were isolated by treatments of the epidermis with collagenase and trypsin. Cl- channels in the basolateral membrane from soma or neck of mitochondria-rich cells were studied in cell-attached and excised inside-out configurations. Of a total of 87 sealed patches only 28 (32%) were electrically active, and in these we identified four different types of Cl- channels. The two major populations constituted Ohmic Cl- channels with limiting conductance (γ125/125) of 10 pS and 30 pS, respectively. A much rarer 150 pS Ohmic Cl- channel was also characterized. From i/V relationships of individual channels the following Goldman-Hodgkin-Katz permeabilities were calculated, 2.2 (±0.1) × 10-14, 5.7 (±0.7) × 10-14, and 32 (±2) × 10-14 cm3/sec, for the 10, 30 and 150 pS Cl- channels, respectively. The 30 pS channel was activated by hyperpolarization. The gating kinetics of the 150 pS channel was complex with burstlike closures within openings of long duration. The fourth type of Cl- channel was studied in patches generating `noisy currents' with no discrete single-channel events, but with vanishing fluctuations at pipette potentials near E Cl. Noise analysis revealed a power spectrum with cutoff frequencies of 1.2 and 13 Hz, indicating that resolution of kinetic steps was limited by small channel currents rather than fast channel gating. From the background noise level we estimated the channel conductance to be less than 1.7 pS. Despite the fact that the majority of patches did not contain electrically active Cl- channels, patches being active, generally, contained more than a single active channel. Thus, for the above three types of resolvable channels, the mean number of active channels per patch amounted to 2.1, 1.4, and 2.0, respectively. This observation, like the finding of few patches with several unresolvable channels, indicates that electrically active Cl- channels are organized in clusters.
The Journal of Membrane Biology – Springer Journals
Published: Jun 1, 1997
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