Design and characterization of a constitutively open KcsA

Abstract

The molecular nature of the structure responsible for proton sensitivity in KcsA has been identified as a charge cluster that surrounds the inner helical bundle gate. Here, we show that this proton sensor can be modified to engineer a constitutively open form of KcsA, amenable to functional, spectroscopic and structural analyses. By combining charge neutralizations for all acidic and basic residues in the cluster at positions 25, 117-122 and 124 (but not E118), a mutant KcsA is generated that displays constitutively open channel activity up to pH 9. The structure of this mutant revealed that full opening appears to be inhibited by lattice forces since the activation gate seems to be only on the early stages of opening.

Figure 1. Designing a constitutively open KcsA by electrostatic repulsion

A. Mutational strategy to generate a pH sensor with an individual ionizable side chain. Key charges in TM1 and TM2 were neutralized to glutamine (charge-less background) and this served as background to reintroduce the individual residues one-by one. Mutations represented by bold letters. B. Ribbon representation of the intracellular bundle region of the mutant H25Q-E118+, highlighting the cluster of charges responsible for pH-dependent activation. Neutralized residues are shown in green, residue E118 in red. This constitutively open mutant was created by combining systematically neutralization of residues H25 at the TM1 and R117, E120, R121, R122 and H124 at the TM2. C. Isopotential surface at the KcsA inner helical bundle calculated from the solution to the Poisson-Boltzmann equation [30]. Shown are slices through the center of the channel and across the pH sensor charge cluster (inset) for wt-KcsA at pH 7 (top) and the H25Q/E118+ mutant in 150 mM KCl. Calculated isopotential contours at 1kT/e (~25mV).

Figure 2. Macroscopic functional assays

A. LB 2003 E. coli strain complementation assay of KcsA mutants. KcsA mutants H25Q and H25Q-E118+ are capable of complement growth of the bacteria in a low potassium concentration media (1 mM KCl). KcsA-wt, H25R mutant and an empty expression vector fail to recovery the cells in the same growing conditions. B. ⁸⁶Rb uptake experiments of constitutively KcsA open mutants H25Q, E118+ and the combination H25Q-E118+. Activation pKa: KcsA-wt=5.89±0.2, H25Q=7.09±0.48, E118+6.78 and the combination H25Q-E118+=7.68±0.2

Figure 3. Single channel properties of H25Q/E118+

A. Single channel activity of H25Q-E118+ at different pH values. Overall activity in this multi-channel patch decreases slightly by changing from pH 8.0 to 4.0. Single channel currents were evoked at +100 mV in symmetric 200 mM KCl solution. B. All points histogram of the record in A. C. Estimated stationary NPo values for H25Q-E118+ at two pH values. Data shown as mean ± sd, for an n=4.

Figure 4. Conformation of the inner bundle gate

EPR spectra of the KcsA-G116C-SL (top) and H25Q-E118+-G116C-SL (bottom) depicting changes in dipolar spin-spin coupling at different pH values. The right panel shows the dependence of the relative extent of opening at the lower gate on pH for KcsA-G116C-SL (red) and H25Q-E118+-G116C-SL (black). For the open-mutant, the dipolar coupling is similar to that of KcsA at pH 3.0 for all pH values.

Figure 5. X-ray structure of H25Q/E118+

A. Structural alignment of wt-KcsA (1K4C=red ribbons) and KcsAH25Q-E118+ open mutant (white ribbons). Potassium ions (blue) and key residues (G99 in MthK and G104 in KcsA open-mutant) for TM2 hinge movement (gray) are represented as sphere. Black arrows indicate the direction of TM2 movement. B. Comparison of Cα-Cα RMSD between the closed structure (1K4C) and H25Q/E118+. In red are highlighted pivot points along the TM segment presumably important for the gating process. Major changes are seen before residue G30 and after residue G104 (open circles), while the core of the protein remain unchanged (filled circles). In the H25Q-E118+-KcsA mutant, the hinge point seems to be around G104 in contrast with the position G99 predicted based in MthK structure. C. Mapping of B-factors variation on top of wt-KcsA (1K4C) according a color code scale. High B-factor variances are clearly seen at the intracellular end of TM1 and TM2.