Atomic mutagenesis in ion channels with engineered stoichiometry

Abstract

C-type inactivation of potassium channels fine-tunes the electrical signaling in excitable cells through an internal timing mechanism that is mediated by a hydrogen bond network in the channels' selectively filter. Previously, we used nonsense suppression to highlight the role of the conserved Trp434-Asp447 indole hydrogen bond in Shaker potassium channels with a non-hydrogen bonding homologue of tryptophan, Ind (Pless et al., 2013). Here, molecular dynamics simulations indicate that the Trp434Ind hydrogen bonding partner, Asp447, unexpectedly 'flips out' towards the extracellular environment, allowing water to penetrate the space behind the selectivity filter while simultaneously reducing the local negative electrostatic charge. Additionally, a protein engineering approach is presented whereby split intein sequences are flanked by endoplasmic reticulum retention/retrieval motifs (ERret) are incorporated into the N- or C- termini of Shaker monomers or within sodium channels two-domain fragments. This system enabled stoichiometric control of Shaker monomers and the encoding of multiple amino acids within a channel tetramer.DOI: http://dx.doi.org/10.7554/eLife.18976.001

Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs

“…Hydrogen bonding between D447 and W434 in the Shaker Kv channel (D375 and W362 in Kv chimera, respectively) has been shown to prevent C-type inactivation ( Pless et al, 2013 ). Mutation of these residues profoundly speeds C-type inactivation in the Shaker Kv channel, strengthening the bond by incorporating fluorinated Trp slows C-type inactivation and weakening the bond by incorporating 2-amino-3-indol-1-yl-propionic acid speeds inactivation ( Lueck et al, 2016 ; Perozo et al, 1993 ; Pless et al, 2013 ). Mutation of W366 in the Kv1.2 channel, the equivalent of W434 in Shaker, also speed C-type inactivation, albeit more modestly compared to Shaker, and both external K + and TEA slow C-type inactivation in Kv1.2 ( Cordero-Morales et al, 2011 ; Ishida, 2014 ), suggesting that the mechanisms of inactivation are similar between these Kv channels.…”

Pore-modulating toxins exploit inherent slow inactivation to block K+channels

“…Basins at the free energy landscape of unmodified KcsA, corresponding to the asymmetrically constricted pore conformation were detected (Li et al, 2017). Second, a chemical modification of K v 1.2:Trp434 was shown to enhance slow inactivation by increasing water traffic through the D-W gate (Lueck et al, 2016). A key event that re-occurred in all Cs1bound simulations was the collapse of the hydrophobic cuff barrier that enabled water exchange between the peripheral pore and the central vestibule of the channel and between pockets of neighboring subunits (Fig.…”

Pore-modulating toxins exploit inherent slow inactivation to block K + channels

“…Basins at the free energy landscape of unmodified KcsA, corresponding to the asymmetrically constricted pore con-formation, were detected (47). Second, a chemical modification of Kv1.2:W434 was shown to enhance slow inactivation by increasing water traffic through the D-W gate (48). A key event that reoccurred in all Cs1-bound simulations was the collapse of the hydrophobic cuff barrier that enabled water exchange between the peripheral pore and the central vestibule of the channel and between pockets of neighboring subunits (SI Appendix, Fig.…”