Tsutsui H et al. (2024), Structural change of the cytoplasmic N-terminus...

XB-ART-60734

Acta Physiol (Oxf) 2024 May 01;2405:e14137. doi: 10.1111/apha.14137.

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Structural change of the cytoplasmic N-terminus and S1 segment of voltage-sensing phosphatase reported by Anap.

Tsutsui H , Jinno Y , Mizutani N , Okamura Y .

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BACKGROUND: Voltage-sensing phosphatase contains a structurally conserved S1-S4-based voltage-sensor domain, which undergoes a conformational transition in response to membrane potential change. Unlike that of channels, it is functional even in isolation and is therefore advantageous for studying the transition mechanism, but its nature has not yet been fully elucidated. This study aimed to address whether the cytoplasmic N-terminus and S1 exhibit structural change. METHODS: Anap, an environment-sensitive unnatural fluorescent amino acid, was site-specifically introduced to the voltage sensor domain to probe local structural changes by using oocyte voltage clamp and photometry. Tetramethylrhodamine was also used to probe some extracellularly accessible positions. In total, 51 positions were investigated. RESULTS: We detected robust voltage-dependent signals from widely distributed positions including N-terminus and S1. In addition, response to hyperpolarization was observed at the extracellular end of S1, reflecting the local structure flexibility of the voltage-sensor domain in the down-state. We also found that the mechanical coupling between the voltage-sensor and phosphatase domains affects the depolarization-induced optical signals but not the hyperpolarization-induced signals. CONCLUSIONS: These results fill a gap between the previous interpretations from the structural and biophysical approaches and should provide important insights into the mechanisms of the voltage-sensor domain transition as well as its coupling with the effector.

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Species referenced: Xenopus laevis

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	FIGURE 1 Anap photometry at 42 different positions in the Ci-VSP-derived VSD. The traces show representative Anap signals in response to the depolarization step from −100 to 100 mV in the construct S1-S4(C159F/R217Q)-HaloTag.
	FIGURE 2 Mapping of Anap signal directions. Positions showing positive (i.e., fluorescence increase) and negative (i.e., fluorescence decrease) responses are highlighted in red and blue of the ball-and-stick model, respectively. The drawing is based on PDB# 4G7V.
	FIGURE 3 Anap responses during the down-to-up transition in the N-terminal region of Ci-VSP (C363S). (A) A cartoon showing Ci-VSP and the seven positions examined. (B) Representative Anap signals detected through 440/40 nm band-pass filter in response to depolarization to +100 mV from a holding potential of −60 mV in the twelve positions indicated.
	FIGURE 4 Effect of VSD-PD coupling on Anap responses at seven positions during the transition. (A) A cartoon showing Ci-VSP and the seven positions examined. (B) Representative Anap signals detected through 440/40 nm band-pass filter in response to depolarization to +100 mV from a holding potential of −60 mV at the seven positions. Data in the presence of VSD-PD coupling (i.e., linker: RR) or absence (i.e., linker: AA) are shown in black and red traces, respectively.
	FIGURE 5 Effect of VSD-PD coupling on the N-terminal motion during the transition. (A−D) Anap signals in response to the depolarizing voltage steps from holding voltage of −60 mV at position 93 (A, B) and 94 (C, D). The VSD and PD are uncoupled in (B) and (D) due to R235A/R254A mutations. (E, F) Analysis of steady-state Anap signals (ΔF/F) at the position 93. The plots in E and F show data at 440 and 485 nm, respectively. Data for Ci-VSP(C363S) (n = 5 cells) and Ci-VSP(C363S/R235A/R254A) (n = 6) are plotted in black and red, respectively. (G−I) The plots show analysis of optical signals at position 94 for the initial rise at 440 nm (G), steady-state responses at 440 (H), and steady-state responses at 485 nm (I). Data for Ci-VSP(C363S) (n = 3) and Ci-VSP(C363S/R235A/R254A) (n = 5) are plotted in black and red, respectively.
	FIGURE 6 Anap responses to depolarization and hyperpolarization at the two positions from the S1-S2 loop. (A) The Anap signals in response to depolarizing and hyperpolarizing voltage steps from holding voltage of −60 mV at position 137. (B) Plots of Anap response measured as average of ΔF/F for the last 50 ms of voltage pulse versus membrane potential (n = 8 cells). The open circle and filled square represent signals at 440 and 485 nm, respectively. The open triangle and solid line indicate normalized gating charge and its Boltzmann fit, respectively. (C) Anap responses at position E144. (D) A plot of responses in E144Anap (n = 5 cells) and gating charge. The same symbols as in B are applied. (E) Representative Anap responses at position 137 in Ci-VSP(D129R/C363S). (F) Representative Anap responses at position 144 in Ci-VSP(D129R/C363S).
	FIGURE 7 TMR photometry from position 144 in S1-S2 loop. (A, B) Representative TMR signals in response to voltage steps from holding voltage of −60 mV in Ci-VSP(C363S) (A) and Ci-VSP(C363S/R253A/R254A) (B). (C, D) Analysis of TMR signals in Ci-VSP(C363S) (n = 5 cells, C) and Ci-VSP(C363S/R253A/R254A) (n = 8 cells, D). The filled square and open circles show the transient and persistent responses measured at the first and last 100 ms time windows after the onset of the voltage pulse, respectively.
	Supplementary Figure S1. The plots show Q-V curves of the oocytes expressing Anap mutants of Ci-VSP(C363S) used in this study. Solid lines indicate curve fit with a single Boltzmann function. Gating parameters and number of cells are also shown.
	Supplementary Figure S2. The panels show the positions of the amino acids examined in this study except for those in the N-terminal region onto the crystal structure model (PDB #4G7V).
	Supplementary Figure S3. The Anap signals in response to depolarizing and hyperpolarizing voltage steps from holding voltage of -60 mV at position 133. Onsets for some of the hyperpolarization-induced responses are indicated by arrows.