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Bio Protoc
2025 Feb 20;154:e5212. doi: 10.21769/BioProtoc.5212.
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Voltage clamp fluorometry in Xenopus laevis oocytes to study the voltage-sensing phosphatase.
Young VC
,
Rayaprolu V
,
Kohout SC
.
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Voltage clamp fluorometry (VCF) is a powerful technique in which the voltage of a cell's membrane is clamped to control voltage-sensitive membrane proteins while simultaneously measuring fluorescent signals from a protein of interest. By combining fluorescence measurements with electrophysiology, VCF provides real-time measurement of a protein's motions, which gives insight into its function. This protocol describes the use of VCF to study a membrane protein, the voltage-sensing phosphatase (VSP). VSP is a 3 and 5 phosphatidylinositol phosphate (PIP) phosphatase coupled to a voltage sensing domain (VSD). The VSD of VSP is homologous to the VSD of ion channels, with four transmembrane helices (S1-S4). The S4 contains the gating charge arginine residues that sense the membrane's electric field. Membrane depolarization moves the S4 into a state that activates the cytosolic phosphatase domain. To monitor the movement of S4, the environmentally sensitive fluorophore tetramethylrhodamine-6-maleimide (TMRM) is attached extracellularly to the S3-S4 loop. Using VCF, the resulting fluorescence signals from the S4 movement measure the kinetics of activation and repolarization, as well as the voltage dependence of the VSD. This protocol details the steps to express VSP in Xenopus laevis oocytes and then acquire and analyze the resulting VCF data. VCF is advantageous as it provides voltage control of VSP in a native membrane while quantitatively assessing the functional properties of the VSD. Key features • Voltage clamp fluorometry using Xenopus laevis oocytes expressing the voltage-sensing phosphatase of Ciona intestinalis. • This protocol uses the fluorophore tetramethylrhodamine-6-maleimide (TMRM). • This protocol details the procedure for a two-electrode voltage clamp using the Dagan CA-1B amplifier.
Figure 1. Map of pSD64TF Ci-VSP plasmid.Vector of pSD64TF with ampicillin selection and an SP6 promoter. The VSD, catalytic domain, and C2 domain of Ci-VSP are noted in the gray regions.
Figure 2.
X. laevis oocytes pre-digestion of follicular membrane.A. Lobe of X. laevis ovary. B. Magnified lobe of X. laevis ovary; black arrow highlights the follicular membrane. C. Morselized ovary pieces.
Figure 3.
X. laevis oocytes post-digestion of the follicular membrane.A. Mixture in the appearance of X. laevis oocytes post-digestion. B. Various qualities of X. laevis oocytes. From left to right: small immature cells, poor-quality stage-V cells, and good-quality stage-V cells. C. Magnified image of a good-quality stage-V cell with part of the follicular membrane attached.
Figure 4. Glass Pasteur pipettes for X. laevis oocyte manipulation.A. Pasteur pipettes for moving oocytes. Top: some pipette taper remaining and a small opening; bottom: pipette taper removed and a larger opening. B. Pasteur pipettes for manual defolliculation. Top: more taper and longer length; bottom: less taper and shorter length.
Video 1. Manual defolliculation of an oocyte. The glass probe approaches from the left and gently presses the oocyte into the Falcon dish until the follicular membrane sticks. The probe is then used to gently roll the oocyte away.
Figure 5. DNA and RNA gels.A. Lanes from left to right: GeneRuler DNA ladder, Ci-VSP circular plasmid, Ci-VSP linearized DNA. B. Left lane: MillenniumTM Marker RNA ladder; right lane: Ci-VSP mRNA product.
Figure 6. Supplies and setup for X. laevis injection.A. Glass injection needles. Top: directly from the glass pipette puller; bottom: after being trimmed. B. Microscope view of the needle aligned with the parafilm corner and the mineral oil being ejected out. C. View of the injector and injection needle with mineral oil being ejected out. The metal plunger is halfway down the needle. D. Oocytes resting in the etched Petri dish.
Video 2. RNA injection of an oocyte. The video shows the various steps of RNA injection in an oocyte as outlined in section C.
Figure 7. VCF setup.A. An oocyte under voltage clamp showing the bath and headstage setup. The black arrows point to the wells containing the silver-chloride wires and agarose bridges. The yellow wire connects to the P1 and V2 headstages, and the white wire connects to the P2 headstage. B. Silver-chloride wires. Top: properly chlorinated; bottom: needs to be chlorinated as shiny metal patches are showing. C. Oocyte in the bath chamber with glass microelectrodes inserted; the surrounding gray mesh prevents the oocyte from rolling. D. Front of Dagan CA-1B amplifier in the TEV setting. The digital monitor is set to V2 showing the bath clamp; note that the amplifier internally inverts the command (i.e., a -80 mV holding shows +80 mV). To the right, the CLAMP is in the ON position and BATH/GUARD switch to ACTIVE.
Figure 8. Making of agarose bridges.A. 3.5” glass capillary marked at 1.5”. B. The glass capillary from A cut with bend points marked 0.25” from either end. C. Top: palladium wire cut the length of the glass capillary. Bottom: the glass capillary bent at the 0.25” mark. D. Palladium wire threaded through the bent glass capillary. E. Bent and palladium-filled glass capillary attached to the suction tube, which is attached to a syringe. F. Close view of the bent and palladium-filled glass capillary attached to the suction tube. G. The final bridge made of a bent glass capillary filled with the bridge agarose solution and a palladium wire.
Figure 9. Dish for holding glass microelectrodes.The holder is a 150 mm Petri dish with modeling clay pressed into the bottom.
