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.
Highlights
Cells use voltage sensor containing proteins to control the membrane potential and for signaling processes. Among these proteins are the extensively studied voltage-gated potassium channels (Kv channels), which are constituted by four homologous subunits each with transmembrane segments S1–S4 forming a voltage-sensing domain (VSD) and S5–S6 contributing to the pore structure (Figure 1a left) [1,2]
A homolog to the VSD of Kv channels was discovered to be coupled to a phosphatase in the ascidian Ciona intestinalis (Ciona intestinalis voltage-sensor containing phosphatase; Ci-VSP) (Figure 1a middle) [3], and unlike Kv channel subunits Ci-VSP can exist in the membrane as a monomer [4]
To address if the slow fluorescence response of VSFP2.3 is due to intrinsically slow operations of its VSD, we measured fluorescence signals along with ‘gating’ currents in a PC12 cell-line stably expressing VSFP2.3 (Figure S2, Supplementary Methods)
Summary
Cells use voltage sensor containing proteins to control the membrane potential and for signaling processes. To address if the slow fluorescence response of VSFP2.3 is due to intrinsically slow operations of its VSD, we measured fluorescence signals along with ‘gating’ currents in a PC12 cell-line stably expressing VSFP2.3 (Figure S2, Supplementary Methods).
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