Hv1 Proton Channel Opening Is Preceded by a Voltage-independent Transition

AbstractThe voltage sensing domain (VSD) of the voltage-gated proton channel Hv1 mediates a H+-selective conductance that is coordinately controlled by the membrane potential (V) and the transmembrane pH gradient (ΔpH). Allosteric control of Hv1 channel opening by ΔpH (V-ΔpH coupling) is manifested by a characteristic shift of approximately 40 mV per ΔpH unit in the activation. To further understand the mechanism for V-ΔpH coupling in Hv1, H+ current kinetics of activation and deactivation in excised membrane patches were analyzed as a function of the membrane potential and the pH in the intracellular side of the membrane (pHI). In this study, it is shown for the first time to our knowledge that the opening of Hv1 is preceded by a voltage-independent transition. A similar process has been proposed to constitute the step involving coupling between the voltage-sensing and pore domains in tetrameric voltage-gated channels. However, for Hv1, the VSD functions as both the voltage sensor and the conduction pathway, suggesting that the voltage independent transition is intrinsic to the voltage-sensing domain. Therefore, this article proposes that the underlying mechanism for the activation of Hv1 involves a process similar to VSD relaxation, a process previously described for voltage-gated channels and voltage-controlled enzymes. Finally, deactivation seemingly occurs as a strictly voltage dependent process, implying that the kinetic event leading to opening of the proton conductance are different than those involved in the closing. Thus, from this work it is proposed that Hv1 activity displays hysteresis

Voltage-Controlled Enzymes: The New Janus Bifrons

The Ciona intestinalis voltage-sensitive phosphatase, Ci-VSP, was the first Voltage-controlled Enzyme (VEnz) proven to be under direct command of the membrane potential. The discovery of Ci-VSP conjugated voltage sensitivity and enzymatic activity in a single protein. These two facets of Ci-VSP activity have provided a unique model for studying how membrane potential is sensed by proteins and a novel mechanism for control of enzymatic activity. These facets make Ci-VSP a fascinating and versatile enzyme. Ci-VSP has a voltage sensing domain (VSD) that resembles those found in voltage-gated channels (VGC). The VSD resides in the N-terminus and is formed by four putative transmembrane segments. The fourth segment contains charged residues which are likely involved in voltage sensing. Ci-VSP produces sensing currents in response to changes in potential, within a defined range of voltages. Sensing currents are analogous to gating currents in VGC. As known, these latter proteins contain four VSDs which are entangled in a complex interaction with the pore domain - the effector domain in VGC. This complexity makes studying the basis of voltage sensing in VGC a difficult enterprise. In contrast, Ci-VSP is thought to be monomeric and its catalytic domain - the VSP\u27s effector domain - can be cleaved off without disrupting the basic electrical functioning of the VSD. For these reasons, VSPs are considered a great model for studying the activity of a VSD in isolation. Finally, VSPs are also phosphoinositide phosphatases. Phosphoinositides are signaling lipids found in eukaryotes and are involved in many processes, including modulation of VGC activity and regulation of cell proliferation. Understanding VSPs as enzymes has been the center of attention in recent years and several reviews has been dedicated to this area. Thus, this review will be focused instead on the other face of this true JanusBifrons and recapitulate what is known about VSPs as electrically active proteins

The gating charge should not be estimated by fitting a two-state model to a Q-V curve

The voltage dependence of charges in voltage-sensitive proteins, typically displayed as charge versus voltage (Q-V) curves, is often quantified by fitting it to a simple two-state Boltzmann function. This procedure overlooks the fact that the fitted parameters, including the total charge, may be incorrect if the charge is moving in multiple steps. We present here the derivation of a general formulation for Q-V curves from multistate sequential models, including the case of infinite number of states. We demonstrate that the commonly used method to estimate the charge per molecule using a simple Boltzmann fit is not only inadequate, but in most cases, it underestimates the moving charge times the fraction of the field

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

Hysteretic Behavior in Voltage-Gated Channels

An ever-growing body of evidence has shown that voltage-gated ion channels are likely molecular systems that display hysteresis in their activity. This phenomenon manifests in the form of dynamic changes in both their voltage dependence of activity and their deactivation kinetics. The goal of this review is to provide a clear definition of hysteresis in terms of the behavior of voltage-gated channels. This review will discuss the basic behavior of voltage-gated channel activity and how they make these proteins into systems displaying hysteresis. It will also provide a perspective on putative mechanisms underlying hysteresis and explain its potential physiological relevance. It is uncertain whether all channels display hysteresis in their behavior. However, the suggested notion that ion channels are hysteretic systems directly collides with the well-accepted notion that ion channel activity is stochastic. This is because hysteretic systems are regarded to have “memory” of previous events while stochastic processes are regarded as “memoryless.” This review will address this apparent contradiction, providing arguments for the existence of processes that can be simultaneously hysteretic and stochastic

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing

Sensing Charges of Ci-VSP

The Ciona intestinalis Voltage Sensor-containing Phosphatase (Ci-VSP) is a member of the Voltage Sensitive Phosphatase family that exhibits membrane potential-controlled enzymatic activity. Alignments of the amino acid sequence of Ci-VSP against Voltage Gated Channels (VGC) indicate that its Voltage Sensing Domain (VSD) is formed by four transmembrane segments. The putative fourth segment (S4) of Ci-VSP extends between the arginine (R) in position 217 and the glutamine (Q) in position 239, containing five arginines, which might be the voltage sensing charges. Although it has been shown that R229 and R232 are critical for voltage sensing in Ci-VSP (Murata et al., 2005), the role of the remaining charges is still unclear. To address this issue, we have performed a partial Histidine Scanning of the S4 of Ci-VSP, following the paradigm established for the VGC Shaker (Starace and Bezanilla, 2004). The voltage dependence of the sensing current of the R217H mutant was modulated by pH. Decreasing the external pH shifted the Q-V curve towards positive potentials, while a pH increase had the converse effect, consistent with the finding that neutralizing R217 (R217Q) produces a negative shift of the voltage dependence of Ci-VSP (Kohout et al., 2008). However, the total net charge of R217H did not change with pH, indicating that R217 does not participate in sensing the membrane potential. When the second arginine is replaced by histidine, the resultant mutant (R223H) exhibits a voltage dependent proton current which closes at positive potentials, resembling the current recorded from Shaker-W434F with its first gating charge replaced by histidine (R362H). This result strongly suggests that R223 has access to both the intra- and the extracellular media depending on voltage. Taken together, our results indicate that R223 is the most extracellularly located sensing charge of the Ci-VSP S4 segment. (Support: NIHGM030376