Activation of bovine tracheal chloride channels by amino group-specific reagents.
1. The predominant Cl- channel in bovine tracheal epithelial cells has a conductance of approximately 71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein-modifying reagents on channel function and found that amino groups are critically involved in gating. 2. Patch clamp studies showed that lysine-specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway. 3. Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time. 4. Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site-directed mutagenesis and pharmacological activation of Cl- channels in epithelial cells.
Duszyk
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Department of Physiology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. mduszyk@physio.med.ualberta.ca
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D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels / Q000187:drug effects / Q000502:physiology*; D004119:Dimethyl Adipimidate / Q000494:pharmacology*; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000187:drug effects / Q000502:physiology; D015640:Ion Channel Gating / Q000502:physiology; D007700:Kinetics; D008390:Markov Chains; D008565:Membrane Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D013050:Spectrometry, Fluorescence; D014132:Trachea / Q000502:physiology*
D018118:Chloride Channels; D008565:Membrane Proteins; D004119:Dimethyl Adipimidate