Hydrophobic residues in S1 modulate enzymatic function and voltage sensing in voltage-sensing phosphatase

Abstract

The voltage-sensing domain (VSD) is a four-helix modular protein domain that converts electrical signals into conformational changes, leading to open pores and active enzymes. In most voltage-sensing proteins, the VSDs do not interact with one another, and the S1-S3 helices are considered mainly scaffolding, except in the voltage-sensing phosphatase (VSP) and the proton channel (Hv). To investigate its contribution to VSP function, we mutated four hydrophobic amino acids in S1 to alanine (F127, I131, I134, and L137), individually or in combination. Most of these mutations shifted the voltage dependence of activity to higher voltages; however, not all substrate reactions were the same. The kinetics of enzymatic activity were also altered, with some mutations significantly slowing down dephosphorylation. The voltage dependence of VSD motions was consistently shifted to lower voltages and indicated a second voltage-dependent motion. Additionally, none of the mutations broke the VSP dimer, indicating that the S1 impact could stem from intra- and/or intersubunit interactions. Lastly, when the same mutations were introduced into a genetically encoded voltage indicator, they dramatically altered the optical readings, making some of the kinetics faster and shifting the voltage dependence. These results indicate that the S1 helix in VSP plays a critical role in tuning the enzyme's conformational response to membrane potential transients and influencing the function of the VSD.

Figure 1.

Conserved S1 helices across VSPs from different species. (A) Cartoon representation of a single VSP subunit with the four transmembrane helices of the VSD in purple (S1–S4) and the phosphatase domain in peach. (B) Reactions catalyzed by VSP. Four different reactions with three different substrates. (C) Cartoon representation of a possible VSP dimer organization based on previous results showing both the VSD and phosphatase domains involved in dimerization. (D) PDB ID 4G80 structure of Ci-VSD, biological unit, rendered using ChimeraX (Pettersen et al., 2021). The structure suggests a specific dimer interface. Ribbon representation of S1–S4 with S1s in orange and S4s in green. Position F127, I131, I134, and L137 shown in space-filling cartoon. (E) Alignment of the VSP S1 helices from several species. Arrows indicate the positions to be mutated to alanine. Sequences for alignments: laevisVSP2, gi|325976472|gb|ADZ48071.1; laevisVSP1, gi|148230800|ref|NP_001090072.1; tropicalisVSP, gi|62859843|ref|NP_001015951.1; macaqueVSP, gi|75076430|sp|Q4R6N0.1; chickenVSP, gi|118084924|ref|XP_417079.2; mouseVSP, gi|76827498|gb|AAI07330.1; ratVSP, gi|157820295|ref|NP_001406113.1; humanVSP2, gi|37788781|gb|AAP45144.1; humanVSP1, gi|213972591|ref|NP_954863.2; zebrafishVSP, gi|70887553|ref|NP_001020629.1; cionaVSP, gi|76253898|ref|NP_001028998.1.

Figure 2.

S1 helix modulates kinetics and voltage dependence of PI(3,4,5)P₃ and PI(3,4)P₂ dephosphorylation. (A) Cartoon depicting the biosensor fTAPP’s expected response to VSP 5-phosphate dephosphorylation from PI(3,4,5)P₃ (increase in FRET) and 3-phosphate dephosphorylation from PI(3,4)P₂ (decrease in FRET). (B) Averaged fTAPP data for a voltage step from −100 mV holding the potential to 160 mV for 2 s for the single S1 mutations (F127A, I131A, I134A, and L137A). L137A appears to only show 5-phosphatase activity while the others closely resemble WT kinetics of activity. (C) Voltage-dependent fTAPP activity for the single S1 mutations. The up-FRET component was separated from the down-FRET component (see Materials and methods). While F127A and I134A are very similar to WT, the I131A mutation shifts the voltage dependence of both reactions to higher voltages and the L137A mutation only displays 5-phosphatase activity with the short 2-s steps. (D) Averaged data for a longer voltage step (5 s) from −100 to 160 mV for L137A and WT with the CS control subtracted from each (data without subtraction in Fig. S1 B). A clear FRET decrease is visible for L137A at the longer step, indicating that while the mutation does not eliminate 3-phosphatase activity, it slows it down relative to WT. (E) L137A voltage-dependent fTAPP activity for the 5-s voltage step. Both up and down FRET components are visible though the upward component appears more linear. (F) Averaged data for a longer voltage step (10 s) from −100 to 160 mV for S1-Q and WT with the CS control subtracted from each (data without subtraction in Fig. S1 E). A clear FRET decrease is visible for S1-Q at the longer step, indicating that while the mutation does not eliminate 3-phosphatase activity, it slows it down relative to WT. (G) S1-Q and WT voltage-dependent fTAPP activity for the 10-s voltage step. Both up and down FRET components are visible though the upward component appears more linear. All error bars are ± SEM. Data fit with single Boltzmann sigmoid equations.

Figure S1.

Impact of oocyte endogenous VSP on heterologous expression of Ci-VSP. (A) Voltage-dependent fTAPP activity in X. laevis oocytes. Cells coexpressing catalytically inactive Ci-VSP (C363S, CS) with fTAPP or fTAPP alone. Significantly more background activity from X. laevis VSP2 (Xl-VSP2) is observed in cells expressing inactive Ci-VSP. This result suggested that Xl-VSP2 may be more efficiently trafficked to the plasma membrane in the presence of Ci-VSP. (B) Averaged data for a long voltage step (5 s) from −100 to 160 mV for L137A, WT, and CS with the fTAPP biosensor. Unsubtracted data from Fig. 3 D. While the CS protein is inactive, the resulting fTAPP increase is significant, indicating a substantial amount of Xl-VSP2 activity for the PI(3,4,5)P₃ to PI(3,4)P₂ reaction. (C) Averaged data for a voltage step from −100 to 160 mV (2 s) for S1-Q. 5-phosphatase activity is dramatically reduced while 3-phosphatase activity appears eliminated. (D) Voltage-dependent fTAPP activity for S1-Q, WT, and CS from the 2-s data. While S1-Q is still active, the activity is almost linear and barely above the CS control. (E) Averaged data for a long voltage step (10 s) from −100 to 160 mV for S1-Q, WT, and CS with the fTAPP biosensor. The CS protein shows a significant degree of Xl-VSP activity at the longer time scale. Unsubtracted data from Fig. 3 F. (F) Averaged data for a 2-s voltage step from −100 to 160 mV for the S1-Q mutation with the fPLC biosensor. S1-Q activity above background (CS) was not observed.

Figure 3.

S1 helix impacts the kinetics of PI(4,5)P₂ 5-phosphate dephosphorylation. (A) Cartoon depicting the biosensor fPLC’s expected response to VSP 5-phosphate dephosphorylation from PI(4,5)P₂ (decrease in FRET). (B) Averaged fPLC data for a 2-s voltage step from −100 to 160 mV for the single S1 mutations (F127A, I131A, I134A, and L137A). L137A significantly slowed the 5-phosphatase activity while the other mutations more closely resemble WT kinetics. (C) Voltage-dependent fPLC activity for the single S1 mutations. The I131A mutation shifts the voltage dependence of activity to higher voltages. The L137A mutation displays reduced and right-shifted 5-phosphatase activity. (D) Averaged data for a long voltage step (20 s) from −100 to 160 mV for S1-Q and controls. A FRET decrease is visible for S1-Q at the longer step, indicating that while the mutation does not eliminate 5-phosphatase activity, it significantly slows it down. The CS constructs indicates minimal endogenous Xl-VSP2 activity even with such a long pulse. (E) Voltage-dependent fPLC activity for S1-Q, WT, and CS using the longer (20-s) pulses. While S1-Q is still active, the activity is almost linear with voltage dependence. All error bars are ± SEM. Data fit with single Boltzmann sigmoid equations.

Figure 4.

S1 helix modulates the kinetics of PI(3,4,5)P₃ 3-phosphate dephosphorylation. (A) Cartoon depicting the biosensor gPLC’s expected response to VSP 3-phosphate dephosphorylation from PI(3,4,5)P₃ to PI(4,5)P₂ (increased membrane fluorescence). (B) Averaged gPLC data for a voltage step from −100 to 160 mV for the single S1 mutations (F127A, I131A, I134A, and L137A). L137A significantly slowed the 3-phosphatase activity requiring a 3-s pulse. (C) Voltage dependent gPLC activity for the single S1 mutations. While I134A is similar to WT voltage dependence, it shows an enhanced overall activity. Both the F127A and I131A mutations shift the voltage dependence of activity to higher voltages while L137A displays reduced 3-phosphatase activity and is slightly left shifted though the slope is significantly reduced. (D) Averaged data for a voltage step from −100 to 160 mV for WT and S1-Q. Slower activation was observed for S1-Q so a 3-s pulse was used. (E) Voltage-dependent gPLC activity for WT and S1-Q. The S1-Q significantly reduced the activity and shifted the voltage dependence to higher voltages, more than just the sum of the individual mutations. All error bars are ± SEM. Data fit with single Boltzmann sigmoid equations.

Figure S2.

Quantitation of VSP expression using VCF and sensing currents. (A) VCF expression data for all the cells used in the activity assays. No statistically significant difference was found between WT and the mutations. WT n = 97, CS n = 95, F127A n = 24, I131A n = 25, I134A n = 31, L137A n = 38, and S1Q n = 27. (B) Maximum off-sensing charge from a 150-mV step. Lower current was found for F127A, I134A, and S1-Q. WT n = 15, CS n = 13, F127A n = 10, I131A n = 12, I134A n = 12, L137A n = 13, and S1Q n = 10. (C) VCF data for the same cells in B. Larger fluorescence was found for CS and S1-Q. Statistics of mutations versus WT were determined using the Welch’s t test with a two-tailed distribution. *** P < 0.001; ** P < 0.01; * P < 0.05.

Figure 5.

S1 helix tunes voltage dependence of S4 motion. (A) Representative VCF traces for a voltage step from −80 to 200 mV for all individual S1 mutations. Activation and repolarization kinetics are similar to WT except for L137A which has a second, slower repolarization motion. (B) Voltage dependence of the VSD motions. Mutations in S1 of VSP shift the voltage dependence of VSD motions to lower voltages and introduce a second component in the motion. WT fit by single Boltzmann sigmoid while mutants are fit by double Boltzmann sigmoid. (C) Representative VCF trace for a voltage step from −80 to 200 mV for S1-Q and WT. Repolarization kinetics show an additional slower component for S1-Q. (D) The voltage dependent motions of S1-Q significantly shifted the voltage dependence of activation by almost 90 mV compared to WT. It also introduced a second component. WT fit by single Boltzmann sigmoid, and S1-Q fit by double Boltzmann sigmoid. All constructs have an N-terminal His-tag to compare with co-IP data. All errors are ± SEM.

Figure S3.

Analysis of VCF kinetic and steady-state data. (A) Activation kinetic analysis for WT, F127A, L137A, and S1-Q VCF. Data from Fig. 6, A and C, were fit with double exponential fits, and the resulting τ_a1 and τ_a2 values were plotted versus the voltage measured for each trace. Student’s t tests *P = 0.001–0.049 are shown against WT in the corresponding color for each mutation. (B) Repolarization kinetics were fit with a single exponential (WT) or a double exponential (L137A, S1-Q). The resulting τ_r1 and τ_r2 values were plotted versus the corresponding voltage. Student’s t tests *P = 0.0025–0.034 are shown against WT in the corresponding color for each mutation, except for τ_r2 values which are shown in cyan and are L137A versus S1-Q. (C) Residual for each of the individual mutations and S1-Q, comparing the single and double fits. Significant discrepancies in the single Boltzmann sigmoid are circled in red. The double Boltzmann sigmoid consistently fits all the mutant VCF data better (fits from Fig. 5, B and D).

Figure 6.

Co-IP pull downs show S1 mutations do not disrupt VSP dimers. (A) Cartoon representation of VSP dimers with the His and FLAG tags on the N-terminus. (B) Immunoprecipitation assay using individual S1 mutations F127A, I131A, I134A, L137A. Inputs were checked for both FLAG and His to ensure expression. Each blot is representative of at least three pull-down blots and shows the FLAG-tagged subunit is able to pull down the His-tagged subunit. (C) Same experiment with all four mutations on a single subunit, called S1-Q for quad mutation on S1. The top blot shows pull down (representative of four separate experiments) while the bottom blot does not (one blot) even though protein levels are similar between the two. Source data are available for this figure: SourceData F6.

Figure 7.

Impact of S1 mutations on a GEVI. (A) Cartoon representation of the GEVI CC1 with the VSD from Ci-VSP attached an FP. (B) Averaged series of voltage steps showing the kinetics of fluorescence change for CC1 WT and S1 mutants expressed in HEK293 cells. Faster activation and repolarization kinetics observed for F127A, I134A, and L137A. (C) Voltage-dependent fluorescence change for WT and S1 mutations. F127A and I134A shift the voltage dependence to higher voltages while I131A and L137A shift to lower voltages. (D) Averaged data for a series of voltage steps for WT and S1-Q. S1-Q shows consistently faster kinetics. (E) Voltage-dependent fluorescence change for WT and S1-Q. Strong leftward shift consistent with leftward shift from VCF data. All error bars are ± SEM. Data fit with single Boltzmann sigmoid equations.

Figure 8.

Model for S1 modulation of S4. PDB ID 4G80 ribbon rendering of Ci-VSD dimers using ChimeraX (Pettersen et al., 2021). L137 is depicted in red space-filling, F127, I131, and I134 are depicted in blue-space filling, S1 in orange, and S4 in green. Left: View facing into the membrane. S1 from subunit A could be interacting with S4 from subunit B (black arrow) and/or S1 from subunit B (cyan arrow). Middle: Turned-in membrane view. Arrows show the same possible interactions. Right: Top-down view shows how the S1 from one subunit can interact with the S4 from the same subunit (purple arrow) as well as the S4 from the adjacent subunit (black arrow). Cyan arrows indicate possible intermolecular interactions through the S1s at the dimer interface between subunits A and B. Black arrows indicate possible intermolecular interactions between the S1 from subunit B to S4 from subunit A. Purple arrows indicate possible intramolecular interactions between the S1 and S4 of the same subunit.