An electrostatic interaction between TEA and an introduced pore aromatic drives spring-in-the-door inactivation in Shaker potassium channels

Abstract

Slow inactivation of Kv1 channels involves conformational changes near the selectivity filter. We examine such changes in Shaker channels lacking fast inactivation by considering the consequences of mutating two residues, T449 just external to the selectivity filter and V438 in the pore helix near the bottom of the selectivity filter. Single mutant T449F channels with the native V438 inactivate very slowly, and the canonical foot-in-the-door effect of extracellular tetraethylammonium (TEA) is not only absent, but the time course of slow inactivation is accelerated by TEA. The V438A mutation dramatically speeds inactivation in T449F channels, and TEA slows inactivation exactly as predicted by the foot-in-the-door model. We propose that TEA has this effect on V438A/T449F channels because the V438A mutation produces allosteric consequences within the selectivity filter and may reorient the aromatic ring at position 449. We investigated the possibility that the blocker promotes the collapse of the outer vestibule (spring-in-the-door) in single mutant T449F channels by an electrostatic attraction between a cationic TEA and the quadrupole moments of the four aromatic rings. To test this idea, we used in vivo nonsense suppression to serially fluorinate the introduced aromatic ring at the 449 position, a manipulation that withdraws electrons from the aromatic face with little effect on the shape, net charge, or hydrophobicity of the aromatic ring. Progressive fluorination causes monotonically enhanced rates of inactivation. In further agreement with our working hypothesis, increasing fluorination of the aromatic gradually transforms the TEA effect from spring-in-the-door to foot-in-the-door. We further substantiate our electrostatic hypothesis by quantum mechanical calculations.

Figure 1.

Extracellular TEA accelerates slow inactivation in T449F Shaker channels. (A) KcsA crystal structure with Y82 shown in space filling representation; PDB, 1K4C. (B) Outward potassium currents in response to a 50-s depolarizing pulse to +40 mV from a holding potential of −100 mV for control and in the presence of the indicated TEA concentration. Traces are normalized to their peak current value to facilitate comparison of the inactivation kinetics. As opposed to the traditional slowing behavior of TEA on inactivation, here the accelerating effect motivates the terminology “spring-in-the-door.” (C) The rate of inactivation is the inverse of the time constant for a single-exponential fit to the relaxation and is well fit by a three-state model (inset) in which inactivation is more rapid from the blocked state. Let k_OI be the inactivation rate constant from the open state (O) to the inactivated state (I), and k_BI the inactivation rate constant from the blocked state (B). The equilibrium probability of fast block in the absence of inactivation is P_block =(1 + K_i/[TEA])⁻¹. The observed inactivation rate is then defined as ρ = k_BIP_block + k_OI(1 − P_block). Values of k_OI and P_block were estimated separately, leaving only k_BI to be estimated from the data in C. We propose that electrostatic attraction contributes to TEA collapse in the single mutant due to the en face orientation of the aromatic rings.

Figure 2.

The V438A mutation enhances slow inactivation, lowers TEA affinity, and restores foot-in-the-door blockade in Shaker T449F channels. (A) Potassium currents from the double V438A/T449F mutant in response to 1-s depolarizations from −80 to +100 mV in 20-mV steps. The inset shows the structure of KcsA (PDB, 1ZW1) with the side chains shown for the aromatic Y82 and E71A (highlighted by arrow). The corresponding Shaker residues are 449 and 438, respectively. (B) Inhibition of the mutants by extracellular TEA. Smooth lines represent standard binding isotherms with inhibitory constants of 0.39 and 1.97 mM for T449F and V438A/T449F, respectively. (C) Normalized outward potassium currents from V438A/T449F channels in increasing TEA concentrations. The V438A mutation speeds slow inactivation from τ_inact = 9.9 ± 0.4 s to τ_inact = 0.18 ± .01 s at +40 mV for T449F (n = 14) and V438A/T449F (n = 5) channels, respectively, with the number of cells in parentheses. (D) The speeding can also be explained by a three-state model in which inactivation only occurs from the open state. The smooth line through the data points is a theory curve generated from known values. See equations in the legend of Fig. 1, with k_BI = 0.

Figure 3.

Serial fluorination of phenylalanine at position 449 reveals a role for a cation–π interaction in the TEA effects on slow inactivation. (A) Normalized representative currents in response to a 10-s depolarization to +40 mV from a holding potential of −100 mV for Shaker channels with the indicated side chain at the 449 position. (B) Time to 50% inactivation for the indicated channel types (number of cells in parentheses) shows a gradual and stepwise accelerating trend in the slow inactivation with each added fluorine. (C–E) Representative currents for the indicated phenylalanine derivative at the 449 position with and without TEA. Each channel type was temporally “normalized” where the control recording reached 50% inactivation at the end of the pulse. Scale bars represent 1 s for each channel type. TEA concentrations used (in mM): 10 (3.3), 56 (48), and 116 (175) for 4F₁-Phe, 3,5F₂-Phe, and 3,4,5F₃-Phe, respectively, with the measured K_i for the channel type in parentheses. (F) Fractional change in the effect of TEA on the amount of current at 50% inactivation for the indicated channel types (number of cells in parentheses). We suggest the inversion of behavior from spring-in-the-door to foot-in-the-door is due to the reduction of negative electrostatic potential on the face of the four aromatic residues due to serial fluorination.

Figure 4.

Model of pore collapse with TEA for the en face and edge-on conformations. (A, left) A bird's eye view of two proposed conformations of the aromatic rings of T449F from the edge-on crystal structure of KcsA with TEAs (Lenaeus et al., 2005) and the en face conformation (Cordero-Morales et al., 2006b; Ahern et al., 2006). (Right) A putative “collapse” of this region during slow inactivation, as each aromatic ring was moved 1.5 Å toward the central axis of the pore. (B) The plot shows the effect of fluorinating the aromatic rings on TEA's contribution to the energetics of collapse. The ab initio calculations were done at the MP2 level. Tri-fluorination had little effect (<0.3 kcal/mol) for the edge-on conformation. Consistent with our experimental data, however, increased fluorination causes a monotonically inhibitory effect of TEA on collapse for the en face conformation, where tri-fluorination increases ΔΔE by 2.5 kcal/mol.