Editorial: Phosphoinositides and their phosphatases: Linking electrical and chemical signals in biological processes

The voltage-sensing phosphatase (VSP) has changed the way we think about both cellular electrical activity and PIPs (phosphatidylinositol phosphates). Originally discovered in 1999 (Chen et al., 1999), these proteins were not recognized as electrically-controlled enzymes until 2005 (Murata et al., 2005). They constitute the first, and so far the only, example of an enzyme linking electrical signals at the plasma membrane to the catalysis of PIPs (Murata et al., 2005), a ubiquitous family of intracellular signaling molecules (Di Paolo and De Camilli, 2006; Balla, 2013). Before the discovery of VSP, there were no known direct links between the two. Textbook examples would represent this connection with arrows, alluding to indirect or “yet-to-be-defined” signaling pathways. Now we know that VSP serves as a direct connection between the electrical nature of the cell and PIPs, lipid second messengers that are critical for cell survival. However, many questions remain unanswered regarding VSP and its electrical regulation of cellular processes. With the discovery of VSP, the membrane potential must now be considered when studying PIP regulators. PIPs are involved in almost all aspects of cell physiology from survival, proliferation, and migration to pre-programed cell death (Di Paolo and De Camilli, 2006; Logothetis et al., 2010; Koch and Holt, 2012; Balla, 2013). For example, PIP concentrations are actively polarized in migrating cells with phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3) on the leading edge and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) on the lagging edge (Leslie et al., 2008). These gradients in the concentration of PIPs are necessary for activation of Rac and Rho leading to cell motion. PIPs are also crucial for cell growth: PI(3,4,5)P3 activates the mTor cascade leading to increased protein, membrane, and nucleic acid production (Dibble and Manning,2013). Many human diseases have been associated with altered homeostasis of PIPs, including cancer, developmental disorders, and Alzheimer\u27s disease (Simpson and Parsons, 2001; McCrea and De Camilli, 2009; Hakim et al., 2012). Though the physiological relevance of VSP is not yet defined, it is still crucial to human health to understand how PIPs are regulated and that now includes VSP. All cells have an asymmetric composition of ions across their plasma membrane, which, combined with selective permeabilities for these ions, results in a difference in the electrical potential across their plasma membrane. This difference, called the membrane potential, constitutes a form of cell signaling and a source of energy, both driving many biological processes. This electrical potential difference powers neuronal excitability as well as more general processes like proliferation, migration, and development (Levin, 2007; Sundelacruz et al., 2009; Yao et al., 2011). Regulation by the membrane potential has long been the sole purview of ion channels and transporters and that has influenced what questions are asked regarding the changing potential. With our new knowledge of VSP, the changing membrane potential can directly signal the cell by modulating mTor and cell growth pathways, leading to abnormal growth or the M-current in sympathetic ganglion, leading to hyperexcitability. The articles in this Special Topic highlight several features of VSP including its unique activation, its similarities to other enzymes and its use as a versatile tool to study other proteins. In the review article by Hobiger and Friedrich (2015, p. 20), the authors compare the structural similarities and differences between the broader family of protein tyrosine phosphatases and one of its newest members, VSP. They suggest a catalytic mechanism based on this comparison. Castle et al. (2015, p. 63) investigate the activation mechanism of VSP by probing the C2 domain, the C-terminal domain of VSP that has been largely unrecognized before the recent crystal structures showed a direct contribution of the C2 residue Y522 into the active site. The work by Mavrantoni et al. (2015, p. 68) explores the techniques that are used to test VSP and address some of their limitations including the need for expensive electrophysiology equipment as well as the limitations of using channels as functional reporters. They take their methods and apply them to a chimera between the Ciona intestinalis VSP and human PTEN and show how the chimera allows for the investigation of PTEN using standard techniques but with the advantage of regulated activation, voltage. Beyond the molecular mechanism underlying VSP activity, Mori et al. (2015, p. 22) review the use of VSP as a relatively simple tool for manipulating PI(4,5)P2 concentrations in cells. They have used VSP to study the PI(4,5)P2 regulation of transient receptor potential canonical channels involved in receptor-operated calcium currents. Along the same lines, Rjasanow et al. (2015, p. 127) use VSP as a tool that gives them precise control over the PI(4,5)P2 concentrations in the membrane. These authors compared the relative PIP affinities between several ion channels. They also point out an important limitation that the channels must already have a known specificity for a particular PIP because VSP does not destroy PIPs in contrast to phospholipase C; instead, it generates multiple PIPs. All together, these articles underscore the features of VSP and expand our understanding of its function and utility. Though VSP remains relatively unknown to many, this nascent field has shown fast initial growth. The unique nature of these enzymes has inspired many to investigate their properties as well as take advantage of them. Many questions remain unanswered regarding VSP such as how the voltage sensor couples to the enzyme and whether the phosphatase domain is brought to the membrane for activation or whether a conformational change within the active site determines activation. We look forward to the studies that will address these and the many other questions that persist in this exciting field

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

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

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

Multimerization is a key characteristic of most voltage-sensing proteins. The main exception was thought to be the Ciona intestinalis voltage-sensing phosphatase (Ci-VSP). In this study, we show that multimerization is also critical for Ci-VSP function. Using coimmunoprecipitation and single-molecule pull-down, we find that Ci-VSP stoichiometry is flexible. It exists as both monomers and dimers, with dimers favored at higher concentrations. We show strong dimerization via the voltage-sensing domain (VSD) and weak dimerization via the phosphatase domain. Using voltage-clamp fluorometry, we also find that VSDs cooperate to lower the voltage dependence of activation, thus favoring the activation of Ci-VSP. Finally, using activity assays, we find that dimerization alters Ci-VSP substrate specificity such that only dimeric Ci-VSP is able to dephosphorylate the 3-phosphate from PI(3,4,5)P3 or PI(3,4)P2 Our results indicate that dimerization plays a significant role in Ci-VSP function

Voltage-sensing phosphatase modulation by a C2 domain

The voltage-sensing phosphatase (VSP) is the first example of an enzyme controlled by changes in membrane potential. VSP has four distinct regions: the transmembrane voltage-sensing domain (VSD), the inter-domain linker, the cytosolic catalytic domain and the C2 domain. The VSD transmits the changes in membrane potential through the inter-domain linker activating the catalytic domain which then dephosphorylates phosphatidylinositol phosphate lipids. The role of the C2, however, has not been established. In this study, we explore two possible roles for the C2: catalysis and membrane-binding. The Ci-VSP crystal structures show that the C2 residue Y522 lines the active site suggesting a contribution to catalysis. When we mutated Y522 to phenylalanine, we found a shift in the voltage dependence of activity. This suggests hydrogen bonding as a mechanism of action. Going one step further, when we deleted the entire C2 domain, we found voltage-dependent enzyme activity was no longer detectable. This result clearly indicates the entire C2 is necessary for catalysis as well as for modulating activity. As C2s are known membrane-binding domains, we tested whether the VSP C2 interacts with the membrane. We probed a cluster of four positively charged residues lining the top of the C2 and suggested by previous studies to interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) (Kalli et al., 2014). Neutralizing those positive charges significantly shifted the voltage dependence of activity to higher voltages. We tested membrane binding by depleting PI(4,5)P2 from the membrane using the 5HT2C receptor and found that the VSD motions as measured by voltage clamp fluorometry were not changed. These results suggest that if the C2 domain interacts with the membrane to influence VSP function it may not occur exclusively through PI(4,5)P2. Together, this data advances our understanding of the VSP C2 by demonstrating a necessary and critical role for the C2 domain in VSP function