Abstract
Recent molecular dynamic simulations and electrostatic calculations suggested that the external TEA binding site in K+ channels is outside the membrane electric field. However, it has been known for some time that external TEA block of Shaker K+ channels is voltage dependent. To reconcile these two results, we reexamined the voltage dependence of block of Shaker K+ channels by external TEA. We found that the voltage dependence of TEA block all but disappeared in solutions in which K+ ions were replaced by Rb+. These and other results with various concentrations of internal K+ and Rb+ ions suggest that the external TEA binding site is not within the membrane electric field and that the voltage dependence of TEA block in K+ solutions arises through a coupling with the movement of K+ ions through part of the membrane electric field. Our results suggest that external TEA block is coupled to two opposing voltage-dependent movements of K+ ions in the pore: (a) an inward shift of the average position of ions in the selectivity filter equivalent to a single ion moving ∼37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located ∼13% into the membrane electric field measured from the internal surface. The minimal voltage dependence of external TEA block in Rb+ solutions results from a minimal occupancy of the vestibule site by Rb+ ions and because the energy profile of the selectivity filter favors a more inward distribution of Rb+ occupancy.
Highlights
TEA ions have, for many years, been useful probes of the structure and function of Kϩ channels (Armstrong, 1966; Hille, 1967; Armstrong and Hille, 1972; Kirsch et al, 1991), perhaps because TEA is positively charged, like Kϩ ions, and about the same size as a hydrated Kϩ ion
The results of this study showed that block of the Shaker pore by external TEA was voltage dependent only in solutions with internal Kϩ ions
External TEA block was essentially insensitive to membrane voltage in Rbϩ solutions and insensitive to changes in internal Rbϩ concentration
Summary
TEA ions have, for many years, been useful probes of the structure and function of Kϩ channels (Armstrong, 1966; Hille, 1967; Armstrong and Hille, 1972; Kirsch et al, 1991), perhaps because TEA is positively charged, like Kϩ ions, and about the same size as a hydrated Kϩ ion. External TEA blocks many types of Kϩ channels but with Ͼ1,000-fold range of effective concentrations (Kavanaugh et al, 1991) Much of this difference can be attributed to the amino acid at a single position in the outer entrance to the pore (MacKinnon and Yellen, 1990; Gomez-Hernandez et al, 1997). Amino acids with an aromatic side chain at this location (residue 449 in Shaker Kϩ channels) confer high sensitivity to block by external TEA ions. The nature of this external TEA binding site has been the subject of several studies designed to probe the structure of the outer entrance to the pore.
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