Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity

Rayaprolu, Vamseedhar; Royal, Perrine; Stengel, Karen; Sandoz, Guillaume; Kohout, Susy C.

doi:10.1085/jgp.201812064

Cited by 20 publications

(58 citation statements)

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“…However, the apparent enzymatic activity of VSP will also depend on the level of its surface expression ( Murata and Okamura, 2007 ). In Xenopus oocytes, the expression level can be controlled by regulating the amount of injected RNA ( Rayaprolu et al, 2018 ). On the other hand, the protein expression level in transfected cultured cells often varies from cell to cell dependent on the copy number of introduced plasmids.…”

Section: Resultsmentioning

confidence: 99%

Engineering an enhanced voltage-sensing phosphatase

Kawanabe

Mizutani

Polat

et al. 2020

Journal of General Physiology

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Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (PIs). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding to ion channels and transporters. For example, by applying simple changes in the membrane potential, Danio rerio VSP (Dr-VSP) has been used effectively to manipulate PI(4,5)P2 in mammalian cells with few, if any, side effects. In the present study, we report an enhanced version of Dr-VSP as an improved molecular tool for depleting PI(4,5)P2 from cultured mammalian cells. We modified Dr-VSP in two ways. Its voltage-dependent phosphatase activity was enhanced by introducing an aromatic residue at the position of Leu-223 within a membrane-interacting region of the phosphatase domain called the hydrophobic spine. In addition, selective plasma membrane targeting of Dr-VSP was facilitated by fusion with the N-terminal region of Ciona intestinalis VSP. This modified Dr-VSP (CiDr-VSPmChe L223F, or what we call eVSP) induced more drastic voltage-evoked changes in PI(4,5)P2 levels, using the activities of Kir2.1, KCNQ2/3, and TRPC6 channels as functional readouts. eVSP is thus an improved molecular tool for evaluating the PI(4,5)P2 sensitivity of ion channels in living cells.

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Section: Resultsmentioning

confidence: 99%

Engineering an enhanced voltage-sensing phosphatase

Kawanabe

Mizutani

Polat

et al. 2020

Journal of General Physiology

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“…Second, a single VSD regulates a single CCR, in contrast with VGICs, in which four homologous units (repeat or subunit) assemble to regulate a central pore gate in a complex manner. Although a recent study suggested that VSPs can form dimers with high density expression and that dimeric VSPs exhibit slightly different molecular properties than those of monomers, the basic nature of voltage-dependent enzyme activity is innate to monomers (Rayaprolu et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

The hydrophobic nature of a novel membrane interface regulates the enzyme activity of a voltage-sensing phosphatase

Kawanabe

Hashimoto

Nishizawa

et al. 2018

eLife

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Voltage-sensing phosphatases (VSP) contain a voltage sensor domain (VSD) similar to that of voltage-gated ion channels but lack a pore-gate domain. A VSD in a VSP regulates the cytoplasmic catalytic region (CCR). However, the mechanisms by which the VSD couples to the CCR remain elusive. Here we report a membrane interface (named ‘the hydrophobic spine’), which is essential for the coupling of the VSD and CCR. Our molecular dynamics simulations suggest that the hydrophobic spine of Ciona intestinalis VSP (Ci-VSP) provides a hinge-like motion for the CCR through the loose membrane association of the phosphatase domain. Electrophysiological experiments indicate that the voltage-dependent phosphatase activity of Ci-VSP depends on the hydrophobicity and presence of an aromatic ring in the hydrophobic spine. Analysis of conformational changes in the VSD and CCR suggests that the VSP has two states with distinct enzyme activities and that the second transition depends on the hydrophobic spine.

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“…PH domains are well known proteins that bind specifically to certain PIPs (38-40). We chose PH domains from tandem PH domain containing protein 1 (TAPP) and phospholipase C (PLC) because they bind specifically to phosphatidylinositol-3,4-bisphosphate [PI(3,4)P 2 ] and PI(4,5)P 2 , respectively, allowing us to monitor both 5- and 3-phosphatase activities (12,14,41,42). To follow PI(3,4)P 2 , we used the biosensor fTAPP that utilizes the PH domain from TAPP flanked by an N-terminal CFP and C-terminal YFP, while the whole sensor is anchored to the membrane through a prenylation site at the C-terminus (Fig 6B) (41-43).…”

Section: Resultsmentioning

confidence: 99%

“…This small signal is expected because it has been previously shown that a low level of endogenous Xl-VSP activity is present in X . laevis oocytes (14,22,41,42,44). Interestingly, the same fTAPP experiment with Xl-VSP2 only showed a FRET increase (Fig 6C, green) even when we extended the voltage pulse to 60 seconds (data not shown).…”

Section: Resultsmentioning

confidence: 99%

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The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells

Ratzan

Rayaprolu

Killian

et al. 2018

Preprint

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Voltage-sensing phosphatases (VSPs) are transmembrane proteins that couple changes in membrane potential to hydrolysis of inositol signaling lipids. VSPs catalyze the dephosphorylation of phosphatidylinositol phosphates (PIPs) that regulate diverse aspects of cell membrane physiology including cell division, growth and migration. VSPs are highly conserved among chordates, and their RNA transcripts have been detected in the adult and embryonic stages of frogs, fish, chickens, mice and humans. However, the subcellular localization and biological function of VSP remains unknown. Using reverse transcriptase-PCR (RT-PCR), we show that both Xenopus laevis VSP (Xl-VSP1 and Xl-VSP2) mRNAs are expressed in early embryos, suggesting that both Xl-VSPs are involved in early tadpole development. To understand which embryonic tissues express Xl-VSP mRNA, we used in situ hybridization (ISH) and found Xl-VSP mRNA in both the brain and kidney of NF stage 32-36 embryos. By Western blot analysis with a VSP antibody, we show increasing levels of Xl-VSP protein in the developing embryo, and by immunohistochemistry (IHC), we demonstrate that Xl-VSP protein is specifically localized to the apical membrane of both embryonic and adult kidney tubules. We further characterized the catalytic activity of both Xl-VSP homologs and found that while Xl-VSP1 catalyzes 3- and 5-phosphate removal, Xl-VSP2 is a less efficient 3-phosphatase with different substrate specificity. Our results suggest that Xl-VSP1 and Xl-VSP2 serve different functional roles and that VSPs are an integral component of voltage-dependent PIP signaling pathways during vertebrate kidney tubule development and function.

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Dimerization of the voltage-sensing phosphatase controls its voltage-sensing and catalytic activity

Abstract: The Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) was not thought to multimerize. Rayaprolu et al. show that Ci-VSP exists as a dimer and that this interaction lowers the voltage dependence of activation and alters substrate specificity.

Cited by 20 publications

References 50 publications

Engineering an enhanced voltage-sensing phosphatase

Engineering an enhanced voltage-sensing phosphatase

The hydrophobic nature of a novel membrane interface regulates the enzyme activity of a voltage-sensing phosphatase

The voltage sensing phosphatase (VSP) localizes to the apical membrane of kidney tubule epithelial cells

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