title: Multi-ion occupancy alters gating in high-conductance, Ca(2+)-activated K+ channels.
abstract: In this study, single-channel recordings of high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayer were used to analyze the effects of two ionic blockers, Ba2+ and Na+, on the channel's gating reactions. The gating equilibrium of the Ba(2+)-blocked channel was investigated through the kinetics of the discrete blockade induced by Ba2+ ions. Gating properties of Na(+)-blocked channels could be directly characterized due to the very high rates of Na+ blocking/unblocking reactions. While in the presence of K+ (5 mM) in the external solution Ba2+ is known to stabilize the open state of the blocked channel (Miller, C., R. Latorre, and I. Reisin. 1987. J. Gen. Physiol. 90:427-449), we show that the divalent blocker stabilizes the closed-blocked state if permeant ions are removed from the external solution (K+ less than 10 microM). Ionic substitutions in the outer solution induce changes in the gating equilibrium of the Ba(2+)-blocked channel that are tightly correlated to the inhibition of Ba2+ dissociation by external monovalent cations. In permeant ion-free external solutions, blockade of the channel by internal Na+ induces a shift (around 15 mV) in the open probability--voltage curve toward more depolarized potentials, indicating that Na+ induces a stabilization of the closed-blocked state, as does Ba2+ under the same conditions. A kinetic analysis of the Na(+)-blocked channel indicates that the closed-blocked state is favored mainly by a decrease in opening rate. Addition of 1 mM external K+ completely inhibits the shift in the activation curve without affecting the Na(+)-induced reduction in the apparent single-channel amplitude. The results suggest that in the absence of external permeant ions internal blockers regulate the permeant ion occupancy of a site near the outer end of the channel. Occupancy of this site appears to modulate gating primarily by speeding the rate of channel opening.
authors.affiliation: Laboratoire de Neurobiologie (URA 295 CNRS), Ecole Normale Supérieure, Paris, France.
authors.forename: J
authors.initials: J
authors.lastname: Neyton
authors.forename: M
authors.initials: M
authors.lastname: Pelleschi
mesh_terms: D000818:Animals; D001464:Barium / Q000494:pharmacology; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D004553:Electric Conductivity; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers; D009132:Muscles / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000494:pharmacology
chemical_list: D008051:Lipid Bilayers; D015221:Potassium Channels; D001464:Barium; D012964:Sodium; D011188:Potassium; D002118:Calcium