Engineering of a genetically encodable fluorescent voltage sensor exploiting fast Ci-VSP voltage-sensing movements

Abstract

Ci-VSP contains a voltage-sensing domain (VSD) homologous to that of voltage-gated potassium channels. Using charge displacement ('gating' current) measurements we show that voltage-sensing movements of this VSD can occur within 1 ms in mammalian membranes. Our analysis lead to development of a genetically encodable fluorescent protein voltage sensor (VSFP) in which the fast, voltage-dependent conformational changes of the Ci-VSP voltage sensor are transduced to similarly fast fluorescence read-outs.

Figure 1. Fast voltage-dependent VSD movements and slow fluorescence signals in VSFP2.3.

(a) Membrane topology of single Kv channel subunit, Ci-VSP and VSFP2.3. VSDs are shown in blue. (b) Change in yellow fluorescence induced by depolarizing voltage steps recorded from a PC12 cell stably expressing VSFP2.3. Red traces are single exponential fits. (c) On- and off ‘gating’ currents induced by the same voltage steps. The On-‘gating’ decay is fitted by single exponential functions (red traces). (d) Fluorescence-voltage (F-V) (n = 11, blue) and charge-voltage (Q-V) relations (n = 10, black) of VSFP2.3. (e) Voltage-dependency of time-constants for VSFP2.3 fluorescence activation (blue) and the decay of on-‘gating’ currents (black). Note broken time scale.

Figure 2. Development of a fast reporting VSFP.

The membrane topology of VSFP2A(R217Q) (a), VSFP2A(R217Q) after photobleaching YFP (b) and VSFP3.1 (c) are shown in the top panel. Underneath are emission spectra recorded from each construct using 440 nm excitation light. The lower panel shows the fluorescence signals recorded in the yellow and cyan channels. For VSFP2A(R217Q) a scaled mirror-image of the cyan signal is shown aligned with the yellow signal; note the fast CFP component. For VSFP3.1 the onset of the fluorescence signal is shown on an expanded time scale; note the dramatically faster response of VSFP3.1.

Figure 3. Characterization of VSFP3.1 ‘gating’ currents and fluorescence response.

Representative ‘gating’ currents (a) and fluorescence responses (b) of VSFP3.1 elicited from single cells upon a family of voltage steps. (c) Voltage-dependency of the fast component of the fluorescence activation time-constant (blue) and the time-constant for the decay of on-‘gating’ currents (black). The inset shows single exponential fits to the fast fluorescence time-constant component from averaged fluorescence traces (n = 8) obtained with voltage steps to −30 mV and +70 mV.