Characterization of a Fast Voltage-Sensing Protein using Voltage-Clamp Fluorometry

Abstract

Voltage-sensing domains in voltage-activated ion channels and other voltage-sensing proteins contain well-conserved S4 helices containing basic residues capable of sensing changes in membrane potential to trigger opening or closing of ion-selective pores or phosphatase activity in voltage-sensitive phosphatases. Here we report the discovery of a protein which contains a voltage-sensing domain capable of rapid and slow rearrangements in response to changes in voltage. In place of a pore domain, this voltage sensor has large N- and C-termini predicted to contain structured and disordered domains for interaction with intracellular proteins. Our working hypothesis is that this protein, which we name Coupled Voltage Sensor (CVS), functions as a voltage sensor that couples to intracellular signaling pathways (as yet undefined). Our goal in the present experiments was to demonstrate that CVS undergoes voltage-dependent conformation changes and to characterize those using site-specific voltage-clamp fluorometry. We identified several positions at the external end of S4 where introduced and fluorophore-labeled Cys residues produce changes in fluorescence as a function of membrane potential. Several positions give complex fluorescence responses, starting with a rapid fluorescence increase (dequenching), followed by slower fluorescence decrease (quenching). Interestingly the rapid component is as fast as the clamp speed when using the cut-open voltage clamp technique (tau ∼ 200 μs). To exclude the possibility of Stark effect we examined the effects of a series of soluble quenchers and observed enhanced quenching with membrane depolarization, consistent with an outward movement of the S4 helix. Overall, our results support a model in which CVS undergoes two temporally distinct conformation rearrangements in response to membrane depolarization.