Optically Mapping the Movement of Discrete Gating Charges in Shaker

Abstract

The nature of the movement of the voltage sensor in voltage-gated ion channels has been a subject of intense study. In the canonical voltage-gated potassium channel Shaker, one major branch of experimentation has been to examine discrete gating charges in the voltage sensor, such as R362 (R1) or R365 (R2), to determine, via site-directed mutagenesis or cross-linking, the residues that they interact with, such as F290 or F416. However, site-directed mutagenesis may uncover indirect interactions rather than direct ones, while cross-linking may uncover rare states. We have aimed to track the movement of the charged residues directly, using a positively charged bimane derivative (qBBr) conjugated to a cysteine replacing either R362 or R365. Although bulkier than arginine, the distance of the positive charge from the protein backbone is similar, and the introduction of the dye is sufficient to restore typical gating current properties to these cysteine mutant constructs, strongly suggesting that qBBr is mimicking the natural function of the endogenous arginine. We have taken advantage of the properties of the bimane class of fluorescent dyes which are highly quenched by proximity to tyrosine and tryptophan but not other amino acids. Using qBBr attached to a chosen gating charge of Shaker proteins expressed in Xenopus laevis oocytes under voltage-clamp, we can optically measure the motion of a specific gating charge in relationship to a specific quenching site in response to changes in membrane potential. The quenching site can be either naturally occurring or artificially substituted into the protein. This method for optically mapping the movement of discrete gating charges in Shaker should be transferable to a wide variety of voltage-gated membrane proteins. Support: NIH GM030376.