Affinity and location of an internal K+ ion binding site in shaker K channels

Abstract

We have examined the interaction between TEA and K+ ions in the pore of Shaker potassium channels. We found that the ability of external TEA to antagonize block of Shaker channels by internal TEA depended on internal K+ ions. In contrast, this antagonism was independent of external K+ concentrations between 0.2 and 40 mM. The external TEA antagonism of internal TEA block increased linearly with the concentration of internal K+ ions. In addition, block by external TEA was significantly enhanced by increases in the internal K+ concentration. These results suggested that external TEA ions do not directly antagonize internal TEA, but rather promote increased occupancy of an internal K+ site by inhibiting the emptying of that site to the external side of the pore. We found this mechanism to be quantitatively consistent with the results and revealed an intrinsic affinity of the site for K+ ions near 65 mM located approximately 7% into the membrane electric field from the internal end of the pore. We also found that the voltage dependence of block by internal TEA was influenced by internal K+ ions. The TEA site (at 0 internal K+) appeared to sense approximately 5% of the field from the internal end of the pore (essentially colocalized with the internal K+ site). These results lead to a refined picture of the number and location of ion binding sites at the inner end of the pore in Shaker K channels.

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Figure 1

Only internal K⁺ ions allow external TEA to protect from internal TEA block. Currents recorded at 0 mV in the absence (Control) and in the presence of 1 mM internal TEA (smaller current in each panel, marked 1 mM TEA_i in the first panel) without (left column) and with (right column) 100 mM external TEA. External and internal concentrations of the indicated ions were 5 and 135 mM, respectively.

Figure 4

Internal K⁺ and block by external TEA. K^App (at 0 mV) for block by external TEA as a function of the concentration of internal K⁺ ions. The solid line is a fit of to the data with K_o and K _K values of 41 ± 3.9 and 64 ± 12 mM, respectively.

Figure 5

Voltage dependence of external TEA site and internal K⁺ site. Intrinsic affinity of external TEA site (K_o, ▪, left ordinate) and K⁺ site (K _K, ○, right ordinate) at the indicated membrane voltages. (solid line) Fit to the K_o data of the standard (Woodhull 1972) equation for block by an external monovalent cation: K_o = K_o(0)exp(δ_oV_mF/RT), where K_o(0) is the affinity at 0 mV, and δ_o is the fraction of the membrane voltage through which the external ion has to move to reach the site with values of 41 ± 0.11 mM and 0.29 ± 0.002 mM, respectively. (dashed line) Fit to the K _K data of the equivalent equation for block by an internal monovalent cation. (inset) Voltage dependence of the apparent affinity for external TEA block determined with 135 mM internal K⁺. Solid line is the fit of the Woodhull equation with a zero voltage affinity of 16 ± 0.8 mM and δ value of 0.24 ± 0.032.

Figure 6

Internal K⁺ ions affect the voltage sensitivity of internal TEA block. (A) Voltage dependence of the apparent affinity of block by internal TEA in solutions of 20 (▪) and 100 mM (○) internal K⁺. Values obtained from fits of to data consisting of at least three independent measurements from at least four different TEA concentrations. (lines) Fits to the Woodhull equation (see Fig. 5 legend) with δ as the fraction of the membrane voltage (measured from the inner surface) through which internal TEA moves to reach its blocking site. (B) Apparent electrical distance (δ) determined as in A at the indicated internal K⁺ concentrations.

Figure 7

Pictorial representation of ion binding sites in the pore of Shaker K channels. Shown is a representation of the KcsA crystal structure (Doyle et al. 1998) with two of the four chains removed for clarity. The KcsA structure was visualized by RasMol (see http://www.umass.edu/microbio/rasmol/index2.htm), exported to a drawing program, and (crudely) converted to the “bent S6” conformation of del Camino et al. 2000.

Figure 2

Effects of internal TEA in the presence and absence of external TEA with 20 mM intracellular K⁺ ions. Currents recorded at −30, −10, +10, and +30 mV (smallest to largest in each panel) in the absence (left column) and the presence (right column) of 1 mM internal TEA without (top row) and with (bottom row) 100 mM external TEA.

Figure 3

Actions of external TEA on block by internal TEA with different concentrations of intracellular K⁺. (A, top) Fraction of current blocked (at 0 mV) by the indicated internal TEA concentrations in a 20-mM internal K⁺ solution without (▪) and with (○) 100 mM external TEA. (A, bottom) Fraction of current blocked (at 0 mV) by the indicated internal TEA concentrations in a 135-mM internal K⁺ solution without (▪) and with (○) 100 mM external TEA. Includes data from Thompson and Begenisich 2000. (lines) Fits of to the data with the indicated K^App values: 0.43 ± 0.017 mM and 0.66 ± 0.031 mM in the absence and presence of 100 mM TEA_o with 20 mM internal K⁺ and 0.52 ± 0.022 mM (0 TEA_o) and 1.5 ± 0.12 mM (100 mM TEA_o) in 135 mM K⁺. (B) K^App (at 0 mV) for block by internal TEA (obtained as in A) as a function of the concentration of internal K⁺ ions without (▪) and with (○) 100 mM external TEA. The broken line represents the mean K^App value (0.45 mM) obtained in the absence of external TEA. The solid line is the fit of to the data with 100 mM external TEA with values for K_o and K _K of 0.49 ± 0.06 and 68 ± 14 mM, respectively.