Abstract
Positively charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. We optically tracked the movement of the two most extracellular charged residues (R1 and R2) in the Shaker potassium channel voltage sensor using a fluorescent positively charged bimane derivative (qBBr) that is strongly quenched by tryptophan. By individually mutating residues to tryptophan within the putative pathway of gating charges, we observed that the charge motion during activation is a rotation and a tilted translation that differs between R1 and R2. Tryptophan-induced quenching of qBBr also indicates that a crucial residue of the hydrophobic plug is linked to the Cole-Moore shift through its interaction with R1. Finally, we show that this approach extends to additional voltage-sensing membrane proteins using the Ciona intestinalis voltage-sensitive phosphatase (CiVSP).
Highlights
One would like to follow the movement of individual gating charges in real time as they respond to changes in the electric field
The idea of the present approach is to study the translocation of the gating charge as the membrane potential is changed using the specific quenching of qBBr by a tryptophan (W) that is positioned nearby or in the path of qBBr
In this paper we have demonstrated a technique that allows tracking of a gating charge surrogate in the pathway of a voltage sensor using qBBr
Summary
Positively-charged amino acids respond to membrane potential changes to drive voltage sensor movement in voltage-gated ion channels, but determining the displacements of voltage sensor gating charges has proven difficult. Site directed fluorimetric approaches typically replace a residue with a cysteine and attach a fluorescent dye, providing the additional advantage of being able to monitor conformational changes in real-time, but do not directly follow the movement of the gating charges (Cha and Bezanilla, 1997; Mannuzzu et al, 1996; Priest and Bezanilla, 2015) Charged adducts such as methanethiosulfonate-ethyltrimethylammonium (MTSET) linked to cysteines placed at gating charges can be used to replace the positive charge and characterize discrete gating charges (Ahern and Horn, 2004, 2005; Baker et al, 1998; Larsson et al, 1996). The qBBr-tagging technique described here should be transferrable to other voltage-sensing membrane proteins; as a proof of principle, we demonstrate its use in the voltage-sensitive phosphatase CiVSP
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