A cation-pi interaction between extracellular TEA and an aromatic residue in potassium channels

Abstract

Open-channel blockers such as tetraethylammonium (TEA) have a long history as probes of the permeation pathway of ion channels. High affinity blockade by extracellular TEA requires the presence of an aromatic amino acid at a position that sits at the external entrance of the permeation pathway (residue 449 in the eukaryotic voltage-gated potassium channel Shaker). We investigated whether a cation-pi interaction between TEA and such an aromatic residue contributes to TEA block using the in vivo nonsense suppression method to incorporate a series of increasingly fluorinated Phe side chains at position 449. Fluorination, which is known to decrease the cation-pi binding ability of an aromatic ring, progressively increased the inhibitory constant K(i) for the TEA block of Shaker. A larger increase in K(i) was observed when the benzene ring of Phe449 was substituted by nonaromatic cyclohexane. These results support a strong cation-pi component to the TEA block. The data provide an empirical basis for choosing between Shaker models that are based on two classes of reported crystal structures for the bacterial channel KcsA, showing residue Tyr82 in orientations either compatible or incompatible with a cation-pi mechanism. We propose that the aromatic residue at this position in Shaker is favorably oriented for a cation-pi interaction with the permeation pathway. This choice is supported by high level ab initio calculations of the predicted effects of Phe modifications on TEA binding energy.

Figure 1.

Two orientations of Tyr82 residues of KcsA. Atomic coordinates (2BOC and 2ATK) were obtained from the Protein Data Bank. 2BOC (blue) is wild-type KcsA cocrystallized with TEAs (Lenaeus et al., 2005). 2ATK (orange) is one of two crystal forms of the E71A mutant (Cordero-Morales et al., 2006). (A) Side view of two opposing subunits of 2BOC with TEAs. (B) Top view of 2BOC aligned with 2ATK, showing the approximate en face orientation of Tyr82 in 2ATK.

Figure 2.

Functional expression of unnatural amino acids in Shaker at position 449. (A–E) Representative families of potassium currents elicited by test depolarizations for 10-mV increments between −60 and 50 mV from a holding potential of −80 mV. Leak and capacitance currents were subtracted online with a –P/8 protocol. The label beneath each panel indicates the introduced amino acid. In each case, the inset shows the 6-31G** electrostatic potential surface of benzene derivatives, with red and blue corresponding to −20 and 20 kcal/mol, respectively (Mecozzi et al., 1996a). The numbered positions of the fluorine atoms are shown with respect to the C_γ carbon of Phe. (F) Lack of potassium currents originating from cellular tRNA acylation when T449UAG mRNA was coinjected with an uncharged tRNA.

Figure 3.

Evidence for cation–π energetics in TEA block. (A and B) Reversible TEA inhibition for Phe and Cha currents at 50 mV. (C) TEA inhibition plots for Shaker channels containing the indicated residue at position 449. Curves are standard binding isotherms fitted to the data. Increased fluorination monotonically increases the dissociation constant K _i (Table I). Cha, which is devoid of aromatic character, renders the channel nearly insensitive to TEA.

Figure 4.

Voltage dependence of block is similar for all fluorinated derivatives. (A–D) Natural logarithm of the relative fraction of unblocked channels (F_un) versus blocked channels is plotted against membrane potential. [TEA] = 1, 1, 10, and 10 mM for Phe, 4F-Phe, 3,5F₂-Phe, and 3,4,5F₃-Phe, respectively. Error bars represent SEM.

Figure 5.

Thermodynamic and ab initio calculations support an en face model of TEA binding to Shaker. (A) Relative binding energy of TEA to Phe derivatives plotted against that calculated for Na⁺ binding to benzene derivatives. Data obtained experimentally for Shaker (squares) display a linear change (black line; slope = 0.19 ± 0.01, R ² = 0.88) in binding energy as π electrons are withdrawn by fluorine substitutions. Ab initio calculations for the binding energetics of a reduced system comprised of four aromatics and a single TEA molecule are shown on the same plot for comparison. (B) The reduced system (shown without hydrogen atoms) based on the coordinates of KcsA and TEAs (Lenaeus et al., 2005). These coordinates predict enhanced binding (diamonds in A) as π electrons are removed from the aromatic face, which is a trend inconsistent with experimental data. (C) Conversely, a 60° rotation qualitatively reproduces (triangles in A) the trend of TEA binding energetics obtained experimentally from Shaker.