Kruse M et al. (2019), Reinterpretation of the substrate specificity o...

XB-ART-55604

J Gen Physiol 2019 Feb 04;1512:258-263. doi: 10.1085/jgp.201812260.

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Reinterpretation of the substrate specificity of the voltage-sensing phosphatase during dimerization.

Kruse M , Kohout SC , Hille B .

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Voltage-sensing phosphatases (VSPs) cleave both 3- and 5-phosphates from inositol phospholipids in response to membrane depolarization. When low concentrations of Ciona intestinalis VSP are expressed in Xenopus laevis oocytes, the 5-phosphatase reaction can be observed during large membrane depolarizations. When higher concentrations are expressed, the 5-phosphatase activity is observed with smaller depolarizations, and the 3-phosphatase activity is revealed with strong depolarization. Here we ask whether this apparent induction of 3-phosphatase activity is attributable to the dimerization that has been reported when VSP is expressed at higher concentrations. Using a simple kinetic model, we show that these enzymatic phenomena can be understood as an emergent property of a voltage-dependent enzyme with invariant substrate selectivity operating in the context of endogenous lipid-metabolizing enzymes present in oocytes. Thus, a switch of substrate specificity with dimerization need not be invoked to explain the appearance of 3-phosphatase activity at high VSP concentrations.

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Species referenced: Xenopus laevis
Genes referenced: nrcam

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Figure 1. Reaction scheme of the Keum et al. (2016) model. Background endogenous kinase and phosphatase reactions in black arrows, and four phosphatase reactions catalyzed by VSP in red arrows. Fig. 2 concerns changes in PI(3,4)P2 as measured by the fTAPP-1 indicator.

Figure 2. Comparison of observed and modeled voltage dependence of fTAPP FRET with different expression levels of Ci-VSP in Xenopus oocytes. (A) Reaction schematic of fTAPP assay where the fTAPP-1-PH FRET sensor shows an increase in FRET when PI(3,4)P2 is produced and a decrease in FRET when PI(3,4)P2 is depleted. (B) Normalized change of fTAPP-1 FRET (ΔFRET/FRETfTAPP-1) versus voltage relationships for six different cRNA injection amounts: 1 ng (light blue), 2.5 ng (blue), 5 ng (yellow), 10 ng (orange), 40 ng (gray), and 80 ng (black). A dashed vertical line is drawn at +56 mV. At high levels of Ci-VSP expression (40 and 80 ng cRNA injections), a FRET increase is observed with small depolarizations, changing to a FRET decrease with the largest depolarizations. At lower levels of Ci-VSP expression (1–5 ng cRNA injections), the FRET increase shifts to higher depolarizations and the FRET decrease is no longer detectable. Error bars are ±SEM, n ≥ 11. Data are from Rayaprolu et al., 2018. (C and D) Simulations of curves in B using two models derived from Keum et al. (2016) as described in the text and Table 1. (C) Calculations using Boltzmann parameters taken from sensing-current measurements on Ci-VSP expressed in oocytes (V0.5 = 57 mV, z = 1.56 electronic charges; Villalba-Galea et al., 2013). (D) Calculations with revised Boltzmann parameters: V0.5 = 95 mV and z = 1.40 electronic charges. Simulations were done with Virtual Cell (http://www.nrcam.uchc.edu; University of Connecticut Health Center).

References [+] :

Campbell, Allosteric activation of PTEN phosphatase by phosphatidylinositol 4,5-bisphosphate. 2003, Pubmed