Engineering an enhanced voltage-sensing phosphatase.
Journal
The Journal of general physiology
ISSN: 1540-7748
Titre abrégé: J Gen Physiol
Pays: United States
ID NLM: 2985110R
Informations de publication
Date de publication:
04 05 2020
04 05 2020
Historique:
received:
17
09
2019
revised:
05
12
2019
accepted:
16
02
2020
entrez:
14
3
2020
pubmed:
14
3
2020
medline:
12
6
2021
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 32167537
pii: 133870
doi: 10.1085/jgp.201912491
pmc: PMC7201886
pii:
doi:
Substances chimiques
Phosphatidylinositol 4,5-Diphosphate
0
Potassium Channels, Voltage-Gated
0
TRPC6 Cation Channel
0
Phosphoric Monoester Hydrolases
EC 3.1.3.2
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Informations de copyright
© 2020 Kawanabe et al.
Références
J Physiol. 2007 Sep 15;583(Pt 3):875-89
pubmed: 17615106
J Physiol. 2012 Mar 1;590(5):1101-19
pubmed: 22183723
Channels (Austin). 2012 May-Jun;6(3):206-9
pubmed: 22760061
J Gen Physiol. 2010 Feb;135(2):99-114
pubmed: 20100891
Physiol Rev. 2013 Jul;93(3):1019-137
pubmed: 23899561
J Gen Physiol. 2019 Aug 5;151(8):986-1006
pubmed: 31182502
Biochim Biophys Acta. 2015 Jun;1851(6):844-56
pubmed: 25241941
Proc Natl Acad Sci U S A. 2016 Jun 28;113(26):E3686-95
pubmed: 27222577
Dev Cell. 2013 Sep 16;26(5):511-24
pubmed: 23993788
J Biol Chem. 2015 Jul 3;290(27):16517-29
pubmed: 25957411
J Physiol. 2014 Mar 1;592(5):899-914
pubmed: 24277865
J Physiol. 2015 Feb 1;593(3):541-58
pubmed: 25398525
J Gen Physiol. 2012 Aug;140(2):189-205
pubmed: 22851677
Front Pharmacol. 2015 Jun 19;6:127
pubmed: 26150791
Curr Opin Neurobiol. 2005 Jun;15(3):370-8
pubmed: 15922587
J Physiol. 2013 Sep 15;591(18):4427-37
pubmed: 23836686
Elife. 2019 Mar 12;8:
pubmed: 30860481
Physiol Rev. 2018 Oct 1;98(4):2097-2131
pubmed: 30067160
J Physiol. 2009 Feb 1;587(3):513-20
pubmed: 19074969
J Biol Chem. 2008 Jun 27;283(26):18248-59
pubmed: 18375390
Nature. 2019 Jan;565(7740):516-520
pubmed: 30602789
J Physiol. 2011 Jun 1;589(Pt 11):2687-705
pubmed: 21486809
Physiol Rep. 2019 Jul;7(14):e14156
pubmed: 31342668
Neuron. 2010 Jul 29;67(2):224-38
pubmed: 20670831
Psychopharmacology (Berl). 2014 Sep;231(17):3493-501
pubmed: 24553581
J Gen Physiol. 2018 May 7;150(5):683-696
pubmed: 29695412
Elife. 2018 Nov 28;7:
pubmed: 30484774
Proc Natl Acad Sci U S A. 2012 Aug 28;109(35):E2316-23
pubmed: 22847441