External TEA block of shaker K+ channels is coupled to the movement of K+ ions within the selectivity filter

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 approximately 37% into the pore from the external surface; and (b) a movement of internal K+ ions into a vestibule binding site located approximately 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.

Figure 1.

External TEA block of Shaker K⁺ channels. (A) TEA block in K⁺ solutions (5 mM external K⁺//135 mM internal K⁺). (Top) Raw current records obtained with step changes in membrane potential from −70 mV to the indicated voltages. Lower record of each pair recorded in the presence of 20 mM TEA. Superimposed on each set of records is the current at the indicated voltage recorded from an uninjected oocyte illustrating the lack of any significant endogenous currents. The current records have not been leak corrected- the small current from the uninjected oocyte is likely through the ∼7 GΩ seal resistance. (Main) Fraction of channel current block by external TEA at −10 (□) and 70 mV (•), respectively. Lines are fits of Eq. 1 to the data with K _app values of 12.3 ± 0.81 and 25.2 ± 1.4 mM at −10 and 70 mV, respectively. (B) TEA block in Rb⁺ solutions (5 mM external Rb⁺//135 mM internal Rb⁺). (Top) Raw current records obtained with step changes in membrane potential from −70 mV to the indicated voltages. Lower record of each pair recorded in the presence of 20 mM TEA. (Main) Fraction of channel current block by external TEA at −10 (□) and 70 mV (•), respectively. Lines are fits of Eq. 1 to the data with K _app values of 10.9 ± 0.74 and 12.5 ± 1.2 mM at −10 and 70 mV, respectively.

Figure 2.

Voltage dependence of TEA block in K⁺ and Rb⁺ solutions. The apparent K_d for external TEA block, K _app, is shown as a function of membrane potential for solutions containing K⁺ on both sides of the channel (▪), solutions with external and internal Rb⁺ (○), external Rb⁺ and internal K⁺ (▴), and external K⁺ with internal Rb⁺ (▿). Lines are fits of Eq. 2 to the data with the indicated value of δ.

Figure 3.

Sensitivity of apparent affinity for external TEA block to internal K⁺ and Rb⁺. (A) Internal K⁺ dependence of K _app for TEA block at the indicated membrane potentials. Lines are fits of Eq. 4 to the data with parameters as described in text. Data at 0 mV (•) from Thompson and Begenisich (2001). (B) Internal Rb⁺ dependence of K _app for TEA block at the indicated membrane potentials. Lines are simulations from Eq. 4 with parameters as described in text.

SCHEME I

Figure 4.

Voltage dependence of the selectivity filter effective K⁺ equilibrium, K _eq, and the inner vestibule K⁺ site affinity, K _K. The K _eq (▪) and K _K (○) data were obtained as described in text. The lines are fits of Eq. 2 to the data. The electrical distance parameter, δ, for the selectivity filter equilibrium is 0.37, measured from the external side. The electrical distance for the vestibule site is 0.13 into the membrane electric field from the inner side, indicated by the minus sign. Other parameters from the fit are described in the text.

SCHEME II