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Figure 2. PIP3→PIP2 activity occurs soon after VSD rearrangement associated with gating charge displacementa) Schematic of voltage clamp fluorometry with the environmentally-sensitive fluorophore, TMRM, attached to the G214C (G214C*) at the outer end of S4. b–d) Depolarizing voltage steps dim the fluorescence of G214C*. b) Fluorescence traces evoked by every fifth voltage steps (50mV increments from −150 to +200 mV, from VH = −100 mV). c) F-V relation from measurement at grey bar in (b) fit with a single Boltzmann relation (V1/2 = 67.7 mV ± 0.4; n=11). d) The fluorescence trace evoked by a step to +150 mV for G214C* labeled, catalytically inactive VSP (C363S) is well fit by a double exponential (τfast = 36.5 ± 7.5 s, τslow = 278.2 ± 12.8 s; n=8). e) The rapid increase in PI(3,4)P2 reported by F-TAPP (black) rapidly follows the τfast gating charge motion in the VSD (from (d)).
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Figure 3. Wildtype Ci-VSP appears to have two active enzymatic statesa) ΔFRET (ΔYFP/ΔCFP) for F-PLC (left) and F-TAPP (right) in response to 2 sec depolarizing steps from VH = −100 mV. Left inset) Blowup of early phase of ΔFRET for F-PLC. b) Normalized ΔFRET (mean ± s.e.m.; F-PLC: n=12; F-TAPP: n=17) at the end of a 2s voltage step, plotted against step voltage for F-PLC (closed squares) and F-TAPP (open circles). Also plotted is peak ΔFRET for F-PLC (closed triangles; see inset in (a)) and F-TAPP (open triangles). c) Schematic of working model of VSP enzyme with three sequential states; OFF/inactive (negative potentials), A1 selective for PIP3 (low depolarizing voltages), and A2 selective for PIP2 (high depolarizing voltages).
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Figure 4. VSD mutants stabilize discrete VSD conformationsa) Schematic of voltage clamp fluorometry with TMRM at Q208C (208*) in S3–S4 loop. b,c,e–j) Deplarization-induced ΔF traces (b,e,g,i) and corresponding average F-V relations (c,f,h,j) calculated from fluorescence measured at end of each 500ms step (mean + s.e.m.). b,c) WT (Q208C*) (n=7) has three fluorescence components: F1, F2 and F3. d) Crystal structures of VSD from WT Ci-VSP (resting at zero voltage; S4 “down”; PDB: 4G80) and the R217E mutant (activated at zero voltage; S4 “up”; PDB: 4G7V)43. Deduced activation transition moves R1–R4 (blue) outward (up), with R2 crossing hydrophobic plug (I126, I190 and F161) and R4 crossing hydrophobic residue W182. e,f) F161W/R3K (n=6) shifts F2 to more positive voltage (i.e. stabilizes conformation between F1 and F2). g,h) F161W/R4K (n=5) has relatively unperturbed F1 and F2 components, but F3 is suppressed (i.e. stabilizes the conformation between F2 and F3. i,j) W182A (n=7) shifts F2 and F3 to more negative voltages. Amplitudes of mutant F-Vs (f,h,j) normalized to WT using the F1 component.
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Figure 5. VSD mutants stabilize discrete enzyme activity statesa,b) F161W/R4K mutant favors the A1 (PIP3→PIP2) enzyme activity state. F-PLC detects that F161W/R4K (a, traces; b, solid squares, n=14) augments the accumulation of PI(4,5)P2 (increased FRET due to dephosphorylation of the 3-position phosphate of PI(3,4,5)P3) and suppresses the depletion of PI(4,5)P2 (decreased FRET due to 5-position dephosphorylation of PI(4,5)P2) that are characteristic of WT Ci-VSP (solid grey line). F-TAPP detects that F161W/R4K (a, traces; b, open circles, n=12) augments the accumulation of PI(3,4)P2 (increased FRET due to dephosphorylation of the 5-position phosphate of PI(3,4,5)P3) and suppresses the depletion of PI(3,4)P2 (decreased FRET due to 5-position dephosphorylation of PI(3,4,5)P3) that are characteristic of WT Ci-VSP (dashed grey line). c,d) W182A shifts to more negative voltage the transition from the inactive state to the A1 active state that produces accumulation of PIP2 as well as between A1 and the A2 state that depletes of PIP2, as seen in both F-PLC (c, traces; d, solid squares, n=12) and F-TAPP (c, traces; d, open circles, n=14).
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Figure 6. Two-step VSD conformational control over VSP phosphatase with two active statesa) S4 sequence of with arginines (blue), including R217, whose mutation to glutamate stabilizes an activated conformation of the VSD for crystallography43,67. b,c) F-TAPP traces (b) and F-V (c) shows that the R217E mutant is at peak A1 PI(3,4,5)P3→PI(3,4)P2 activity at zero voltage, suggesting that “up” VSD structure in Fig. 4d corresponds to A1 enzyme state. d) Model of sequential depolarization-driven transitions in VSD that sequentially transition the phosphatase domain from inactive to the PIP3-preferring A1 active state and then to the PIP2-prefering A2 active state.
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