Gada KD et al. (2023), Optogenetic dephosphorylation of phosphatidylin...

XB-ART-59646

STAR Protoc 2023 Mar 17;41:102003. doi: 10.1016/j.xpro.2022.102003.

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Optogenetic dephosphorylation of phosphatidylinositol 4,5 bisphosphate in Xenopus laevis oocytes.

Gada KD , Xu Y , Winn BT , Masotti M , Kawano T , Vaananen H , Plant LD .

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Here, we present a protocol for optogenetic dephosphorylation of the phosphoinositide PI(4,5)P2 at the plasma membrane of Xenopus laevis oocytes. We first describe the co-injection of oocytes with cRNAs encoding (1) a light-activated PI(4,5)P2 5-phosphatase fusion protein, (2) its dimerization partner fused to the plasma membrane, and (3) the potassium channel reporter for PI(4,5)P2 dephosphorylation. We then detail blue light illumination to induce PI(4,5)P2 dephosphorylation, combined with simultaneous two-electrode voltage clamp electrophysiological recording to assess potassium channel current responses. For complete details on the use and execution of this protocol, please refer to Xu et al. (2022).1.

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Species referenced: Xenopus laevis
Genes referenced: dtl
GO keywords: ion channel activity [+]

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References [+] :

Gada, PKC regulation of ion channels: The involvement of PIP2. 2022, Pubmed

	Graphical abstract
	Figure 1. WinWCP screenshots to create a voltage-ramp recording protocol (A) Recording details entered in each field. (B) Waveform details entered by dragging hold and ramp waveforms into relevant boxes. Blue arrows indicate the box that needs to be clicked to enter details of that waveform to the right of the window.
	Figure 2. Expected time-course of Kir2.1 current obtained at the -80 mV phase of consecutive voltage ramps Upon illumination with blue light, a contemporaneous decrease in Kir2.1 channel activity is initiated that corresponds to an inhibition of the current caused by dephosphorylation of PI(4,5)P2. The inhibition reaches a plateau and leaves behind residual current that is blocked by 10 mM barium.
	Figure 3. Raw current data from Kir2.1 channels (A and B) Example of raw current data from Kir2.1 channels shown as the overall of all sweeps from a single time course study in WinWCP software, in (A), and after transferring the data values from the - 80 mV portion of each consecutive sweep to Graphpad Prism (B).