Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high-conductance Ca2+ -activated K+ channel.

Abstract

In this study, high-conductance Ca2+-activated K+ channels from rat skeletal muscle were incorporated into planar phospholipid bilayers, and discrete blockade of single channels by Ba2+ was studied. With 150 mM K+ held constant in the internal solution, increasing external K+ over the range 100-1,000 mM raises the rate of Ba2+ dissociation. This "enhancement effect," which operates at K+ concentrations 3-4 orders of magnitude higher than those required for the "lockin" effect described previously, depends on applied voltage, saturates with K+ concentration, and is not observed with Na+. The voltage dependence of the Ba2+ off-rate varies with external K+ in a way suggesting that K+, entering the channel from the external side, forces Ba2+ dissociation to the internal solution. With K+ held fixed in the external solution, the Ba2+ off-rate decreases as internal K+ is raised over the range 0-50 mM. This "lock-in" effect is similar to that seen on the external side (Neyton and Miller, 1988), except that the internal lock-in site is of lower affinity and shows only a fivefold preference for K+ over Na+. All the results taken together argue strongly that this channel's conduction pathway contains four sites of very high affinity for K+, all of which may be simultaneously occupied under normal conducting conditions. According to this view, the mutual destabilization resulting from this high ionic occupancy leads to the unusually high conductance of this K+-specific channel.

Keywords

Affiliated Institutions

• Brandeis University US

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