Mechanism for selectivity-inactivation coupling in KcsA potassium channels

Abstract

Structures of the prokaryotic K(+) channel, KcsA, highlight the role of the selectivity filter carbonyls from the GYG signature sequence in determining a highly selective pore, but channels displaying this sequence vary widely in their cation selectivity. Furthermore, variable selectivity can be found within the same channel during a process called C-type inactivation. We investigated the mechanism for changes in selectivity associated with inactivation in a model K(+) channel, KcsA. We found that E71A, a noninactivating KcsA mutant in which a hydrogen-bond behind the selectivity filter is disrupted, also displays decreased K(+) selectivity. In E71A channels, Na(+) permeates at higher rates as seen with and flux measurements and analysis of intracellular Na(+) block. Crystal structures of E71A reveal that the selectivity filter no longer assumes the "collapsed," presumed inactivated, conformation in low K(+), but a "flipped" conformation, that is also observed in high K(+), high Na(+), and even Na(+) only conditions. The data reveal the importance of the E71-D80 interaction in both favoring inactivation and maintaining high K(+) selectivity. We propose a molecular mechanism by which inactivation and K(+) selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.

Fig. 1.

The E71-D80 interaction is structurally analogous to the molecular bowstring in Kir channels and is disrupted in the E71A KcsA mutant. Images are taken from crystal structures of Kir2.2 (3JYC), KcsA (1K4C), and the flipped (yellow) and nonflipped (gray) structures of the E71A mutant of KcsA (1ZWI, 2ATK), showing the selectivity filter region from one subunit and highlighting the residues involved in a salt-bridge interaction in Kir2.2, and the hydrogen-bond interactions in KcsA. Black spheres represent potassium ions and the blue sphere represents water.

Fig. 2.

The E71A KcsA mutant is more permeable to Na⁺, Li⁺, Cs⁺, and . (A) Representative time courses of a liposomal flux assay of WT and E71A at pH 7 driven by intraliposomal K⁺ or Na⁺. Each time course is normalized to its own maximum level of uptake. (B) Same as A except assay performed at pH 4 for WT and pH 8 for E71A (n = 4, mean ± SEM). (C) Maximum uptake for WT and E71A at pH 7 driven by different intraliposomal cations and normalized to uptake driven by K⁺ (n = 5, mean ± SEM).

Fig. 3.

WT KcsA, but not E71A, becomes nonconductive in the absence of K⁺ and the presence of Na⁺. (A) Time courses of WT fluxes at pH 7, showing K⁺-driven uptake (black), K⁺-driven uptake (blue), and Na⁺-driven uptake (orange) (n = 3, mean ± SEM). (B) Same as A except for E71A (n = 3, mean ± SEM).

Fig. 4.

Na⁺ blocks E71A KcsA current in a voltage-dependent manner. (A) Open channel IV curves calculated from control (black circles, n = 8, mean ± SEM) and Na⁺-blocked (red triangles, n = 4, mean ± SEM) single-channel currents. The curve through the control points is a hyperbolic fit with no theoretical significance. The dotted blue curve is a fit with Eq. S1 with the fitted block parameters: K_B(0) = 573 mM and Z = 0.64 (compared with 500 mM and 0.55 for WT), and the smooth red line is a fit with the equation I(V) = I₀(V)(1 + B/((1 + K/Q_K)(K_B + Q_B)))^-1, where and Q_K and Q_B are Michaelis-type constants for K⁺ and Na⁺ (see ref. 28). Fitted parameters are as follows: Q_K is 6.1 exp(z₁FV/RT), where z₁ is 0.34, Q_B was set to 0.5 exp(-z₁FV/RT), and K_B is 283 exp(-z₂FV/RT), where z₂ is 0.9. (B) Plot of the ratio of Na⁺-blocked and control currents (I/I₀) for E71A KcsA (red) and WT (black) showing the much larger increase in Na⁺-modified E71A currents in the punch-through region.

Fig. 5.

Crystal structure of E71A KcsA features a noncollapsed selectivity filter. (A) Stick model of selectivity filter from E71A KcsA in the presence of 150 mM NaCl and the absence of K⁺ and accompanying electron density (2Fo-Fc) contoured at 1.0σ. Residues G77 and T75 are labeled. For comparison, (B) stick models of filters of the conductive (Left) and collapsed (Right) states of WT KcsA [1K4C and 1K4D (12)]. (C) Stick models of filters of the nonflipped (Left) and flipped (Right) structures of E71A KcsA [2ATK and 1ZWI (15)]. Bound ions are purple (Na⁺) or cyan (K⁺) spheres.