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.
Highlights
C-type inactivation, termed ‘slow-inactivation’ due to its delayed kinetics, in voltage-gated potassium channels describes a phenomenon by which ionic conductance through the channel pore is decreased in the presence of continued depolarizing stimulus (Choi et al, 1991; Hoshi et al, 1991)
A computational approach based on molecular dynamics (MD) simulations was employed to examine the local dynamics of the Ind sidechain and its relation to the intra-subunit hydrogen bond accepting side-chain Asp447
In the wild type (WT) a hydrogen bond is formed between Trp434 and Asp447 during a large fraction of MD simulation, which blocks the exchange of water molecules between extracellular bulk and the water binding pocket behind selectivity filter
Summary
C-type inactivation, termed ‘slow-inactivation’ due to its delayed kinetics, in voltage-gated potassium channels describes a phenomenon by which ionic conductance through the channel pore is decreased in the presence of continued depolarizing stimulus (Choi et al, 1991; Hoshi et al, 1991). In the context of the Xenopus laevis oocyte, the cRNA of the target protein contains an introduced stop codon (often amber, TAG) that is subsequently suppressed by a co-injected orthogonal tRNA that has been misacylated in vitro with the ncAA. This approach was used to encode a tryptophan homologue, 2-amino-3-indol-1-yl-propionic acid
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