Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence

Keum, Dongil; Kruse, Martin; Kim, Dong-Il; Hille, Bertil; Suh, Byung‐Chang

doi:10.1073/pnas.1606472113

Cited by 37 publications

(58 citation statements)

References 80 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…After the paper was accepted, a paper characterizing voltage dependence of phosphatase activity of VSP shared among four subreactions toward distinct phosphoinositides was published (34).…”

Section: Methodsmentioning

confidence: 99%

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Sakata

Jinno

Kawanabe

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation. FRET analysis showed that the catalytic region remains situated beneath the plasma membrane, irrespective of the voltage level. Moreover, Anap fluorescence from a membrane-facing loop in the C2 domain showed a pattern reflecting substrate turnover. These results indicate that the voltage sensor regulates Ci-VSP catalytic activity by causing conformational changes in the entire catalytic region, without changing their distance from the plasma membrane.

show abstract

“…After the paper was accepted, a paper characterizing voltage dependence of phosphatase activity of VSP shared among four subreactions toward distinct phosphoinositides was published (34).…”

Section: Methodsmentioning

confidence: 99%

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Sakata

Jinno

Kawanabe

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…Together these reactions lead to the formation of PI(4)P via one or two steps. However, PI(4,5)P 2 is the most abundant phosphoinositide in the membrane and the phosphatase action at position 5 of PI(4,5)P 2 occurs at a much higher rate than the other reactions, therefore resulting in the predominant Dr-VSP activity (Keum et al 2016). Heteromeric Kv7.2/7.3 channels are activated by PI(3,4,5)P 3 as well as by PI(4,5)P 2 with similar potency.…”

Section: Discussionmentioning

confidence: 99%

“…PIP 2 sensitivity is decreased in the dephosphomimetic mutant of Kv7.2 PIP 2 is generally required for Kv7 channel opening, and Kv7.2 is particularly sensitive to changes in membrane PIP 2 levels (Suh et al 2006;Gamper & Shapiro, 2007). To test Kv7.2 for its PIP 2 sensitivity, channels were coexpressed with the Danio rerio voltage-sensitive phosphatase (Dr-VSP) that, upon depolarization, predominantly removes the phosphate at position 5 of the inositol ring of PI(4,5)P 2 Keum et al 2016) thereby reducing currents through Kv7.2 (Zhang et al 2013;Rjasanow et al 2015). Currents through Kv7 were elicited by switching the membrane potential from −80 to −30 mV, and Dr-VSP was activated by intermittent depolarizations to +100 mV for 0.01-4.8 s. The ratio of current amplitudes at −30 mV just before and right after these intermittent depolarizations was determined ( Fig.…”

Section: Identification Of In Vivo Phosphorylation Sites In Kv72mentioning

confidence: 99%

Phosphorylation regulates the sensitivity of voltage‐gated Kv7.2 channels towards phosphatidylinositol‐4,5‐bisphosphate

Salzer

Erdem

Chen

et al. 2016

The Journal of Physiology

View full text Add to dashboard Cite

Key points Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is a key regulator of many membrane proteins, including voltage‐gated Kv7.2 channels.In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2‐binding domains in Kv7.2.Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2.Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors.Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channel via G protein‐coupled receptors. AbstractThe function of numerous ion channels is tightly controlled by G protein‐coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol‐4,5‐bisphosphate (PIP2). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography‐coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2‐binding domains. To evaluate the effect of phosphorylation on PIP2‐mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage‐sensitive phosphatase Dr‐VSP than were wild‐type channels. In vitro phosphorylation assays with the purified C‐terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild‐type but not mutant Kv7.2 channels towards PIP2 depletion via Dr‐VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2‐binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR‐mediated channel regulation.

show abstract

“…In such an intermediate state, is the rate of enzymatic activity different from the rate of the fully activated state, but with constant substrate specificity? The results of Keum et al (5) do not exclude the presence of an intermediate state. A three-state model could better explain the actual PI profiles, especially because PI composition can change much more rapidly than can be detected with FRET sensors.…”

mentioning

confidence: 84%

Simple scheme of lipid enzyme can explain complex lives of phosphoinositides

Okamura

2016

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Phospholipids in biological membranes play multiple roles: They not only allow electrical signaling but also serve as precursors of second messengers in morphogenesis and cancer progression. Voltage-sensing phosphatases (VSPs) (1, 2) are unique membrane proteins that link these two functions. The VSP contains both a cytoplasmic lipid phosphatase and a transmembrane voltage sensor that is similar to the voltage sensor of voltage-gated ion channels. The voltage sensor domain is connected to a downstream cytoplasmic region with remarkable similarity to a tumor suppressor enzyme, phosphatase and tensin homolog (PTEN). PTEN dephosphorylates two species of phosphoinositides (PIs), PI(3,4,5)P 3 and PI(3,4)P 2 , in plasma membranes, antagonizing PI-3 kinase activity. Changes of PTEN's phosphatase activities are known to be associated with many human diseases including cancer, diabetes, and autism spectrum disorders (3). In the VSP, membrane depolarization activates the voltage sensor domain, leading to activation of PI phosphatase activity (4). Despite remarkable structural similarity to PTEN, VSP substrate preference toward PIs differs from substrate preference of PTEN; in particular, VSP dephosphorylates PI(4,5)P 2 , whereas PTEN does not. To understand the physiological roles of VSP, it is important to know the kinetic profiles of the various PIs that can be dephosphorylated by VSP upon membrane potential change. In PNAS, Keum et al. (5) precisely characterize and model the kinetics and voltage dependence of VSP activity toward different PI substrates.Detailed properties of enzymatic activities toward diverse PIs have previously been studied by in vitro enzyme assays using color detection of released phosphate with malachite green or separation of radiolabeled PIs by TLC (6). However, these methods have some limitations because they are difficult to apply to the full-length protein of VSP harboring the transmembrane segments. Several PI-binding motifs, such as the plextrin homology domain, can selectively recognize the headgroup of each species of PI, and fusions of such motifs with fluorescent polypeptides can report dynamic change of each species of PI in living cells. These probes have been used by many laboratories to study multiple types of PIs [PI(3,4,5)P 3 , PI(4,5)P 2 , and PI(3,4)P 2 ] in VSP-expressing cells under different membrane potentials (4, 6-9). One puzzling phenomenon in some of these detailed studies is that upon membrane depolarization, PI(3,4)P 2 initially increases, but it later decreases. These changes depend on the dual-enzyme activity of VSP toward PI(3,4,5)P 3 and PI(3,4)P 2 : PI(3,4,5)P 3 is turned into PI(3,4)P 2 through 5′-phosphatase activity, and PI(3,4)P 2 is then dephosphorylated by 3′-phosphatase activity. Such dynamic regulation of PI(3,4)P 2 by VSP activity might be physiologically relevant, because chick fibroblasts overexpressing VSP form remarkable fine processes that depend on increased PI(3,4)P 2 (10). Interestingly, an increase of PI(3,4)P 2 is observed at a lower voltage th...

show abstract

Phosphoinositide 5- and 3-phosphatase activities of a voltage-sensing phosphatase in living cells show identical voltage dependence

Cited by 37 publications

References 80 publications

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid

Phosphorylation regulates the sensitivity of voltage‐gated Kv7.2 channels towards phosphatidylinositol‐4,5‐bisphosphate

Simple scheme of lipid enzyme can explain complex lives of phosphoinositides

Contact Info

Product

Resources

About