Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels

Tang, Qiong-Yao; Larry, Trevor C.; Hendra, Kalen; Yamamoto, Erica; Bell, Jessica; Cui, Meng; Logothetis, Diomedes E.; Boland, Linda M.

doi:10.1074/jbc.m115.640409

Cited by 13 publications

(13 citation statements)

References 52 publications

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“…To test whether a similar change takes place upon amino acid substitution in the corresponding region of Dr-VSP, the WT enzyme or a mutant in which Leu-223 or Tyr-224 in the hydrophobic spine was replaced with Phe or Gln were heterologously expressed in HEK293T cells. An inward rectifying K + channel (mouse Kir2.1) was used for the read out of the voltage-dependent phosphatase activity toward PI(4,5)P 2 in the plasma membrane, as previously established ( Murata and Okamura, 2007 ; Sakata and Okamura, 2014 ; Rjasanow et al, 2015 ; Tang et al, 2015 ).…”

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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“…Although it is a potent inhibitor of PI-3-kinase, Wortmannin also inhibits phosphoinositide 4-kinase at concentrations at the micromolar range (Nakanishi and Catt, 1995). Because of this, Wortmannin has been shown to effectively decrease the concentration of PI (4,5)P 2 in the plasma membrane (Suh and Hille, 2002;Zhang et al, 2003;Hirdes et al, 2004;Bernier et al, 2008a;Bernier et al, 2008b;Rodriguez-Menchaca et al, 2012;Dickson et al, 2014;Tang et al, 2015;Kirchner et al, 2017). Oocytes expressing K V 7.2/K V 7.3 channels were incubated in 10-20 μM Wortmannin for 30-60 min; the concentration of Wortmannin varied depending on the sensitivity to the treatment of each oocyte batch.…”

Section: Phosphatase Activity Hampers the Activation-induced Decreasementioning

confidence: 99%

Modulation of KV7 Channel Deactivation by PI(4,5)P2

Villalba-Galea

2020

Front. Pharmacol.

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The activity of K V 7 channels critically contributes to the regulation of cellular electrical excitability in many cell types. In the central nervous system, the heteromeric K V 7.2/K V 7.3 channel is thought to be the chief molecular entity giving rise to M-currents. These K +-currents as so called because they are inhibited by the activation of Gq protein-coupled muscarinic receptors. In general, activation of Gq protein-coupled receptors (GqPCRs) decreases the concentration of the phosphoinositide PI(4,5)P 2 which is required for K V 7 channel activity. It has been recently reported that the deactivation rate of K V 7.2/K V 7.3 channels decreases as a function of activation. This suggests that the activated/open channel stabilizes as activation persists. This property has been regarded as evidence for the existence of modal behavior in the activity of these channels. In particular, it has been proposed that the heteromeric K V 7.2/ K V 7.3 channel has at least two modes of activity that can be distinguished by both their deactivation kinetics and sensitivity to Retigabine. The current study was aimed at understanding the effect of PI(4,5)P 2 depletion on the modal behavior of K V 7.2/K V 7.3 channels. Here, it was hypothesized that depleting the membrane of P(4,5)P 2 would hamper the stabilization of the activated/open channel, resulting in higher rates of deactivation of the heteromeric K V 7.2/K V 7.3 channel. In addressing this question, it was found that the activity-dependent slowdown of the deactivation was not as prominent when channels were co-expressed with the chimeric phosphoinositide-phosphatase Ci-VS-TPIP or when cells were treated with the phosphoinositide kinase inhibitor Wortmannin. Further, it was observed that either of these approaches to deplete PI(4,5)P 2 had a higher impact on the kinetic of deactivation following prolonged activation, while having little or no effect when activation was short-lived. Furthermore, it was observed that the action of either Ci-VS-TPIP or Wortmannin reduced the effect of Retigabine on the kinetics of deactivation, having a higher impact when activation was prolonged. These combined observations led to the conclusion that the deactivation kinetic of K V 7.2/K V 7.3 channels was sensitive to PI(4,5)P 2 depletion in an activation-dependent manner, displaying a stronger effect on deactivation following prolonged activation.

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“…Earlier work on mammalian Kir channels found that PI(4,5)P 2 interacting residues are generally conserved between family members, and the difference between high and low affinity channels could be traced to a subtle conformational change caused by a relatively conservative substitution of Leucin to Isoleucin 19 . Recently it was shown that a Kir channel from a marine sponge Amphimedon queenslandica displays very weak interactions with PI(4,5)P 2 , and lacks two key positively charged PI(4,5)P 2 interacting residues that are highly conserved in mammalian Kir channels 20 . Reintroduction of the two positively charged residues substantially strengthened the interactions of the invertebrate Kir channel with PI(4,5)P 2 20 .…”

mentioning

confidence: 99%

“…Recently it was shown that a Kir channel from a marine sponge Amphimedon queenslandica displays very weak interactions with PI(4,5)P 2 , and lacks two key positively charged PI(4,5)P 2 interacting residues that are highly conserved in mammalian Kir channels 20 . Reintroduction of the two positively charged residues substantially strengthened the interactions of the invertebrate Kir channel with PI(4,5)P 2 20 .…”

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confidence: 99%

A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

Velisetty

Borbíró

Kasimova

et al. 2016

Sci Rep

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Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is an important cofactor for ion channels. Affinity for this lipid is a major determinant of channel inhibition by depletion of PI(4,5)P2 upon phospholipase C (PLC) activation. Little is known about what determines PI(4,5)P2 affinity in mammalian ion channels. Here we report that two members of the Transient Receptor Potential Vanilloid (TRPV) ion channel family, TRPV5 and TRPV6 lack a positively charged residue in the TM4-TM5 loop that was shown to interact with PI(4,5)P2 in TRPV1, which shows high affinity for this lipid. When this positively charged residue was introduced to either TRPV6 or TRPV5, they displayed markedly higher affinities for PI(4,5)P2, and were largely resistant to inhibition by PI(4,5)P2 depletion. Furthermore, Ca2+-induced inactivation of TRPV6 was essentially eliminated in the G488R mutant, showing the importance of PLC-mediated PI(4,5)P2 depletion in this process. Computational modeling shows that the introduced positive charge interacts with PI(4,5)P2 in TRPV6.

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Mutations in Nature Conferred a High Affinity Phosphatidylinositol 4,5-Bisphosphate-binding Site in Vertebrate Inwardly Rectifying Potassium Channels

Cited by 13 publications

References 52 publications

Engineering an enhanced voltage-sensing phosphatase

Engineering an enhanced voltage-sensing phosphatase

Modulation of KV7 Channel Deactivation by PI(4,5)P2

A molecular determinant of phosphoinositide affinity in mammalian TRPV channels

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