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Zebrafish (Danio rerio) as a Model for the Study of Glial Calcium Signaling in Awake, Behaving Animals
Full text linkGlial biology has been studied for over a century, yet the existence of dynamic calcium signaling in astrocytes was only discovered in 1990. Early studies used cultured astrocytes to investigate this phenomenon; however, studies in cultured cells are highly vulnerable to artifactual results that do not accurately represent normal physiological conditions. More recent studies in vivo have revealed a broad heterogeneity in astrocyte signaling—the mechanisms that trigger a calcium rise, the sources of that calcium rise, and the downstream effects of astrocyte signaling all vary with different brain regions and types of stimulation. In zebrafish (Danio rerio), the primary glial cell type is radial glia that persist into adulthood, which have previously been shown to perform many of the same roles as mammalian astrocytes, including regulating synaptic glutamate uptake and maintaining CNS water balance. This study establishes that zebrafish are an appropriate model for studying glial calcium signaling, which can be triggered by eliciting an acoustic startle response in awake, behaving fish. The glial calcium signal, measured by the genetically encoded calcium indicator GCaMP5, reliably peaks following an escape response and is mediated by the group I metabotropic glutamate receptor mGluR1
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Allosteric Substrate Switching in Novel Voltage Sensing Lipid Phosphatase
Full text linkThe explosion of protein diversity through domain rearrangements and inter-domain coupling supported the evolution of multicellular organisms. To perform the advanced signaling necessary for multicellularity, sometimes unrelated protein domains combined to form novel domain architectures that over time evolved tight mechanisms of allosteric coupling. One such protein, the voltage sensing phosphatase (VSP), developed a sophisticated mechanism of inter-domain coupling, which enabled cells to integrate changes in membrane potential into chemical changes in a class of secondary signaling lipids called phosphatidylinositol-phosphates (PIPs).Phosphoinositol phosphate signaling lipids (PIPs) are important second messengers that regulate ion channels, transporters, cell motility and endo/exocytosis. PIP concentrations are controlled by enzymes, including VSP, which has broad specificity for a diverse class of PIPs. VSP is a novel lipid phosphatase, which contains a voltage sensing domain (VSD) homologous to voltage-gated ion channels, and a lipid phosphatase domain (PD). Until now it was not known what properties of the cytosolic PD were allosterically regulated by the membrane-associated VSD. Using a pair of new PIP sensors to monitor enzyme activity and voltage clamp fluorometry to monitor conformational changes in the VSD, it becomes clear the Ciona intestinalis VSP (Ci-VSP) has two distinct voltage regulated enzyme active states: a faster low-voltage state with substrate preference for PIP3 and a slower high-voltage state with preference for PIP2. This novel 2-step allosteric switch for enzyme specificity enables membrane potential to function as an allosteric effector that dynamically regulates PIP concentration.In this work, it is show that two unrelated domains, a VSD from voltage dependent ion channels and a lipid PD homologous to protein tyrosine phosphatases evolved a tight mechanism of allosteric regulation that transduces fluctuations in membrane potential into changes in the enzyme selectivity of a novel lipid phosphatase. This regulation of active site specificity in the PD by an allosteric effector domain represents a significant advancement in our understanding of allosteric regulation, which has previously been restricted to control of activity on only one type of substrate
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Synaptic heterogeneity and the underlying molecular mechanisms at the Drosophila neuromuscular junction.
Full text linkSynapses are the fundamental units of communication in the nervous system and reliable synaptic transmission is central to key nervous system processes such as learning, memory, and sensory adaptation. Moreover, due to dysfunctions at the synapse, many neurological diseases may develop. While electrophysiological studies of synaptic transmission have been around for a long time, only the recent development of optical quantal analysis (OQA) tools has made possible to correlate morphological and structural elements to transmission properties of individual synapses. Studies using OQA have revealed a much larger diversity in synaptic transmission, even among neighboring synapses, than was previously thought. The advent of higher-resolution OQA, such as the one developed in Chapter 2 of this thesis, called “QuaSOR”, and super-resolution structural imaging methods opens up an exciting frontier of being able to investigate how neural activity shapes (and is shaped by) synaptic diversity, what are the molecular determinants and mechanisms the set this diversity, and how do these molecular determinants shape synaptic diversity. By using OQA, we’ve shown that synaptic diversity, as measured by difference in synaptic strength (i.e., probability of action potential evoked transmission; Pr ) is extremely heterogeneous (Pr: 0.01–0.62) within a single neuron synapsing onto a single target cell. This high degree of heterogeneity leads us to the central hypothesis of my thesis which is that synaptic strength is set by a very precise, local distribution of key proteins. To test this hypothesis I used the model glutamatergic synapse–Drosophila melanogaster larval neuromuscular junction (NMJ)– where I will investigated hundreds of synapses in parallel, in vivo, and addressed synaptic heterogeneity from both functional and structural perspectives at single synapse resolution (50-100nm).
In Chapter 2 of this thesis, using a combination of QuaSOR and super-resolution structural imaging methods, we found that essential active zone (AZ) proteins such as Bruchpilot (Brp) and the Ca2+-channel Cacophony (Cac) vary greatly among synapses and can explain ~31% of the diversity that we observe in basal Pr. Moreover, when investigating complexin, which acts as a break on release, we found that it suppresses both spontaneous and evoked release but in different ways. Taking the functional-structural tools developed in Chapter 2, I then wanted to ask how the neuromodulator octopamine, which is released from type II motor neurons (MNs) impacts synaptic release at type I MNs. In Chapter 3, I was able to show that OA affects release at type I MN in an input-specific manner (increasing release at type Ib MNs but having no effect at type Is MNs). Then, combining the QuaSOR method with structural imaging, I was able to show that the effect of modulation by OA on a single-synapse level is at least partially due to the amount of Unc13A at the synapse and interestingly, is dependent on the PLC pathway
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Optical quantal analysis of synaptic diversity and plasticity at the Drosophila neuromuscular junction
Full text linkSynapses are the cellular structures that allow neurons throughout the nervous system to communicate, perform calculations, and store information. Due in part to their small size, complex spatial organization, heterogeneity, stochastic behavior, and plastic nature, monitoring the behavior of networks of synapses in intact neural circuits remains an enormous challenge. Yet, this level of understanding will be critical for identifying the central principles and the specific molecular mechanisms that govern neural function, orchestrate activity in networks of neurons and ultimately direct organismal behavior.To begin to answer some of these fundamental questions regarding the activity of synapses in intact networks, I turned to the Drosophila larval neuromuscular junction (NMJ). The NMJ is a model glutamatergic synapse that directly controls locomotor behavior in the developing larva. A single larval muscle cell receives primary excitatory input from two glutamatergic axons with different morphologies and functions. Each input forms hundreds of individual heterogeneous synaptic contacts with the target muscle cell. Furthermore, each larva only uses a few hundred motor neurons and muscle cells to accomplish all of its behaviors. Thus, this single system can provide diverse information about synaptic function at multiple levels important to the function of the motor system, from the release of a single vesicle of neurotransmitter (quantum) at a single synapse, all the way up to large-scale coordination of muscles during complex locomotive behavior. Through this work, I implemented several generations of optical tools that provide quantal, single-synapse resolution measurements of multiple modes of synaptic transmission and synaptic plasticity. I generated the experimental and analytic methods that allow these measurements, for the first time, to be applied in vivo in the live, behaving animal to monitor quantal synaptic transmission during native activity. I also combined these optical measurements with the precise optical manipulation of intracellular signaling, genetic mutations, and cell-specific alterations in gene expression or neuronal function. Using combinations of these techniques, I was able to uncover several novel homeostatic mechanisms that help to stabilize synaptic and neuronal function. First, I utilized a unique combination of optopharmacology (light-controlled ligands) and optogenetic pharmacology (light-controlled proteins) to manipulate the signaling of native and orthogonal presynaptic metabotropic glutamate receptors (mGluRs), while simultaneously monitoring single-synapse quantal transmission at hundreds of synapses. I was able to demonstrate that it is possible to track changes in synaptic strength at multiple synapses, during several forms of plasticity. This included monitoring the number of active synapses, quantal amplitudes and presynaptic quantal release probability (Pr, the likelihood of quantal release), all important factors that work together to regulate synaptic strength. Global activation of mGluRs throughout the NMJ, using an orthogonal, light-agonized mGluR, LimGluR2, produces a powerful and uniform suppression of Pr. However, the NMJ contains a highly heterogeneous collection of synapses that may introduce spatial variability in presynaptic mGluR activation. To address this, I used either local LimGluR2 photoactivation or local glutamate uncaging to activate the native mGluR, DmGluRA. With these methods, I was able to show that local excesses of glutamate and presynaptic G protein signaling, produce a combination of local and global, autoreceptor-dependent, negative feedback suppression of Pr. This mechanism likely stabilizes and balances synaptic strength throughout the NMJ, while maintaining synapse-to-synapse heterogeneity.Next, I extended these optical measurements to both of the convergent glutamatergic inputs to demonstrate that there are enormous, input-specific differences in spontaneous glutamate release, basal evoked Pr, and short-term plasticity. These differences were found to shape the even more divergent behavior of these two inputs in vivo, during locomotion, in the intact larva. By manipulating the postsynaptic muscle sensitivity to glutamate, I was able to show that there is a long-term, retrograde, homeostatic increase in synaptic strength that only occurs at the physiologically dominant input, while not affecting the neighboring, modulatory input. Mechanistically, this input-specificity was found to be due to differential postsynaptic Ca2+ activity and different activity of postsynaptic Ca2+/calmodulin-dependent kinase II (CaMKII), which likely functions to spatially and temporally integrate postsynaptic activity, shaping the retrograde signals to only one of the presynaptic motor neurons. These signals then appropriately adjust glutamate release in order to maintain effective contraction of the muscle.I then combined this input-specific, synapse-specific quantal framework with multiple mutations that affect presynaptic active zone (AZ) structure and function. I found that these presynaptic mutations are phenotypically heterogeneous, when observed with such high spatial precision and under multiple, interrelated synaptic transmission modes. These mutations produced differential effects on spontaneous transmission, locally and globally. They differentially affected presynaptic Pr, with one mutation increasing the Pr so much, that single-synapse basal transmission switched from univesicular to multivesicular release. I found that short-term plasticity was less affected by basal Pr, as has been suggested by classic models. Rather, plasticity mechanics are more strongly governed by some, as-yet-to-be-identified, input-specific, frequency-dependent factor. I then demonstrated that these alterations in synaptic function are not entirely due to cell autonomous changes at the NMJ specifically, but reflect the systemic, pan-neuronal function of these proteins. Furthermore, neurotransmitter release-defective mutant NMJs have very different behaviors when considered during traditional, semi-intact recording conditions versus when they are observed truly in vivo. In fact, I demonstrated that input-specific synaptic phenotypes are completely inverted when considered under these two recording conditions, suggesting a phenotypic complexity that we are only beginning to uncover.Finally, in response to the conclusion that mutations may not produce cell autonomous, synapse-autonomous phenotypes, as predicted, I have begun to develop a platform for understanding molecular mechanisms, and homeostatic compensation more broadly. This system combines quantal imaging in vivo and under controlled levels of activity, with cell-specific gene knockdown and network-level activity analysis. This system will provide a full phenotypic characterization of the function of any gene within a specific cell type in the nervous system, while simultaneously identifying the synapse-specific, input-specific, and cell-specific compensatory mechanisms induced by neuronal dysfunction.Together, this work demonstrates the exquisite precision and diversity in the regulation of synaptic strength employed by even a relatively simple network of synapses, governing a straightforward process of muscle contraction. In particular, this work uncovers several homeostatic mechanisms that the larva can use to stabilize NMJ function in response to diverse perturbations. These changes alter the activity of individual synapses, individual neurons, and the behavior of the entire motor system. Because many of the molecular mechanisms at work within the Drosophila NMJ are conserved throughout many animals, including the most common mammalian central synapses, the framework provided by this work will help to shape our understanding of the broader mechanisms that regulate synaptic function, and as a consequence regulate neuronal circuit function and behavior
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Short-term and long-term control of synaptic strength by light activatable glutamate receptors at the Drosophila neuromuscular junction
Full text linkDrosophila neuromuscular junctions (NMJs) exhibit structural and physiological homeostasis during larval development in which the number of boutons and the amount of neurotransmitter released increases in coordination with larval muscle size growth. The Bone Morphogenetic Protein (BMP) signaling pathway, including Glass bottom-boat (Gbb), a BMP ligand, and Wishful thinking (Wit), its presynaptic BMP receptor, are important for regulating this homeostatic growth in larvae. Genetic analysis of Gbb suggests it is released as a retrograde signal from the postsynaptic muscle to initiate presynaptic BMP signaling for synaptic growth. However, muscle expression of Gbb fails to rescue synaptic transmission defects in the gbb mutant, which is instead rescued by nervous system expression of Gbb. To resolve this conflicting data and elucidate the role of Gbb at the NMJ, we investigated the expression of Gbb during Drosophila development at the NMJ. We fused EclipiticGFP to Gbb for visualizing its expression pattern at third-instar larval NMJs. Finally, we demonstrate genetic rescue of the gbb mutant with our transgenic line and provide evidence that Gbb released from the muscle may play a role in higher order synapses beyond the NMJ.Development of the larval neuromuscular junction (NMJ) in Drosophila has been well characterized using genetic mutants and advanced imaging methods. However, the time course of activity-dependent changes in synaptic strength at the larval NMJ has not yet been fully investigated. To further understand the time course of synaptic plasticity at the NMJ, we used the Gal4/UAS system to express the Light-Gated Glutamate Receptor (LiGluR) in the muscle to precisely control postsynaptic activity while performing electrophysiological recordings. Our experiments reveal that long-term postsynaptic LiGluR expression during development induces a homeostatic decrease in bouton density and evoked synaptic transmission. With acute activation of LiGluRs, we potentiate synaptic transmission during high frequency stimulation. CamKII activity is required for this enhancement in synaptic strength by rapid LiGluR activation but it is not necessary for the long-term decrease in bouton density. Finally, we provide evidence that suggests the Wit BMP receptor is not required for the rapid potentiation of synaptic transmission but we provide data to possibly implicate cAMP signaling as a downstream mediator of this effect. These results suggest that a transient increase in postsynaptic activity generated by LiGluR activation may produce a rapid retrograde signal that enhances neurotransmitter release
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Vision restoration in animal models of human blindness using natural and engineered light-gated receptors
Full text linkMost inherited forms of human blindness are caused by mutations that lead to photoreceptor cell death, but spare the inner retina, providing an opportunity for treatment. With the help of azobenzene-derived chemicals that we call ‘photoswitches’, I engineered light gated receptors to be applied as therapeutics towards vision restoration in animal models of human blindness. The first part of my thesis describes this work.Next, I targeted expression of natural and engineered light-gated proteins to the remaining neurons of the retina, using viruses as gene delivery vehicles. I asked if these cells would then function as the new photoreceptors and if they would be able to drive visual responses. The quick answer is: yes, they do. In blind mice, I compared the ability of different target cells to act as new photoreceptors. Installing light sensors downstream in retinal ganglion cells lead to robust and uniform responses, whereas expression upstream in bipolar cells lead to more diverse activity patterns in response to light. I characterized mammalian proteins as optical actuators and found that light gated ion channels drive fast responses but require very high light intensities whereas G-protein coupled receptors are about 1000x more sensitive to light but at the cost of slow kinetics. I then further extended our studies to dogs and were able to show that our treatment restored light responses in blind rcd1 dog retinas in vitro and was safe and well tolerated in vivo. My results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation. These findings are summarized in the second part of my thesis.Finally, I explored non-invasive approaches to restore a sense of ‘space’ and enable navigation for the blind. Towards this end, I helped design and prototype a sensory substitution device, the ‘sonic eye’. This device is inspired by bats and human echolocators and it allows users to ‘see with sound’. This work is described in the last part of my thesis
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Generating Diversity and Maintaining Stability in the Nervous System, From Synapses to Behavior
Full text linkA central question in neuroscience is how synapses are assembled within neuronal circuits to regulate and maintain complex behaviors. These fundamental nodes of information transfer must be malleable yet maintain stability, all within a nervous system of a developing, learning, and behaving animal. In my thesis work, I have sought to help bridge some of these divides between the many scales of the nervous system and disentangle the overwhelming intricacy that lies between molecules at the synapse and emergent behavior. Behavioral variability in animal populations is thought to increase fitness and aid adaptation to environmental change, yet the underlying neural mechanisms are poorly understood. We found that variation between individuals in neuromodulatory input contributes to individuality in short-term habituation of the zebrafish (Danio Rerio) acoustic startle response (ASR). From here, I studied heterogeneity on a different scale and began researching the synaptic diversity of the model system, the Drosophila neuromuscular junction (NMJ). Most electrophysiological studies of the NMJ have ignored the functional differences between its two distinct motor neuron inputs, the Ib and Is, both of which are comprised of hundreds of synapses of varying vesicular release properties. We found that the Ib and Is inputs generate distinct forms of excitatory drive during crawling and differ in key transmission properties. Finally, after a desire to connect the synaptic level of the NMJ to the behavioral level of the larval zebrafish, my most recent work investigates how circuits increase motor neuron output to maintain normal behavior in response to a reduction in synaptic strength at the NMJ. Although diverse in nature, these model systems provide a unique insight into the complex interplay between generating diversity and maintaining stability in the nervous system
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From networks to neurons: A multilevel investigation of experience-dependent improvement in prey capture behavior in the larval zebrafish
Full text linkUnderstanding the neural mechanisms that guide behavior is one of the biggest quests in neuroscience. This question is tackled at different levels of analysis, from studying whole-brain blood oxygenation levels in humans, to minuscule receptor movements in cells. In this dissertation, I describe work aiming to explore the neural basis of behavioral improvement in the larval zebrafish from the network level, across brain areas, to the neuron level, looking at specific neuronal ensembles.In chapter one, I introduce the study of experience dependent changes in behavior through history and describe the advantages of using the ethologically relevant prey capture behavior in larval zebrafish as a model. In chapter two, in collaboration with Claire Oldfield, I studied how experience hunting live prey affects prey capture behavior and the underlying neural activity. I show that previous experience with live prey improves hunting performance compared to larvae that have been fed with inert fish flakes. Consequently, looking at whole-plane neural activity, I observed no differences in the neural representations of prey in the visual areas, however, experienced fish showed increased correlations between output neurons of the tectum and the forebrain, and an increased probability for visual activity to evoke motor action. This led to the hypothesis that experience may lower the threshold for visual information transfer to motor areas, via an increase of activity in the forebrain. To test this hypothesis, I specifically ablated cells in the habenula, one of the forebrain structures, and observed a reduction in eye convergences and prey consumption. These findings show the involvement of the forebrain in experience-dependent improvement of prey capture for the first time. In chapter three, I describe our attempts to study experience-dependent changes in forebrain activity and network dynamics between visual areas and the forebrain at a single-cell resolution, using multi-plane two-photon imaging. This project is still at its beginnings, but I describe the adaptation of the behavioral paradigm to the two-photon microscope, and the potential of recording large populations of neurons at cellular level during a naturalistic behavior
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Optical control and measurement of metabotropic glutamate receptors and K2P potassium channels
Full text linkG protein-coupled receptors (GPCRs) are an extremely important class of membrane receptors that convert extracellular stimuli into intracellular signals through interaction with G proteins. These receptors are intimately involved in most physiological processes and for this reason are the largest drug targets in biology. In the nervous system a wide range of GPCRs function in nearly all subcellular locations, including the synapse, where they modulate cellular excitability, neurotransmission, synaptic plasticity, and behavior. An ultimate goal for the understanding of GPCR neurophysiology is to reconstruct a functional map of when and where GPCRs are activated and how this activation affects larger scale network outputs like behavior. However, due to the limitations of classical techniques, such as pharmacology and transgenic approaches, it has been difficult to decipher the role of individual GPCRs in specific cell types with temporal precision. In order to gain a foothold toward understanding the contribution of different GPCRs in physiological functions, I developed a means of optically controlling individual GPCRs. This work focused on the metabotropic glutamate receptors (mGluRs) which are crucial neuronal GPCRs that respond to the major excitatory neurotransmitter, glutamate. There are eight different subtypes of mGluRs that have unique, but overlapping expression profiles and distinct G protein coupling and regulatory properties. Since pharmacological agents often can't distinguish between subtypes, can only be poorly targeted spatially, and are slow to apply and remove, we used a chemical optogenetic approach to individually agonize or antagonize mGluRs with light. This approach was based on a previous body of work from the Isacoff lab and is the basis of chapters 2 and 3. I show the molecular engineering and characterization of these tools, their initial characterization as tools for optical control of neuronal activatity, and validate their in vivo function in zebrafish and mice. Light-activated mGluRs, or "LimGluRs", are both a useful tool that are applicable in many contexts, but also an important test case that should serve as a model for the development of optical control over other GPCRs.Structurally, GPCRs share a common 7 transmembrane domain structure but show divergence in extracellular N-terminal domains and intracellular C-terminal domains. Recent breakthroughs in X-ray crystallography have led to a greater structural understanding of how GPCRs bind ligands, activate, interact with G proteins, and oligomerize. However, functional experiments, which are required to gain a more complete understanding, have been hampered by the lack of high resolution techniques to probe these same processes. mGluRs are particulary interesting in this biophysical context because of their dimeric arrangement and large extracellular ligand binding domains (LBDs) that indirectly couple glutamate binding to G protein activation. In chapters 4 and 5, I use a combination of optical techniques including LimGluRs to measure oligomerization, structural dynamics, and function of mGluRs. While Fӧrster resonance energy transfer (FRET) is an established technique for probing of protein structure, it has recently been greatly enhanced in power by its application at the single molecule level. Single molecule FRET (smFRET) allows for individual receptors to be measured which allows for the observation of distinct states and their transitions, without the obscuring effects of averaging. Using intersubunit smFRET experiments on the extracellular ligand binding domains of mGluRs I found that these receptors visit three distinct conformations that have dynamics which determine receptor activation properties. Using another single molecule fluorescence technique based on counting fluorophore bleaching steps, I was able to show that mGluRs form homo- and heterodimers in living cell membranes. Furthermore, using a variety of perturbations I found that mGluRs have a covalent and non-covalent dimer interface within their LBDs that is complemented by a weak interface at the trans-membrane domains. Using LimGluRs, which allow for individual subunits within a dimer to be liganded, I demonstrate cooperativity that is dependent on receptor subtype and dynamics. All of these studies have given insight into the molecular biophysics of mGluRs and should serve as the basis for future studies on other GPCRs. A final goal of the study of GPCRs is to understand the responses of their downstream signaling targets, including ion channels. One ion channel subfamily of particular interest for this objective are the K2P potassium channels which classically function as leak channels to maintain cellular resting potential. K2P channels have been shown to be highly regulated by different extracellular and intracellular signals, including GPCR activation. Despite the extreme regulation of these channels by a variety of signals, it has been difficult to determine which forms of regulation happen endogenously on individual channel subtypes. This is especially complicated by the fact that there is very limited pharmacology to individually block K2P channels. To overcome this, I developed, using a similar design to LimGluRs, a means of optically blocking the K2P channel, TREK1. Using a conditional expression system, native TREK1 channels were manipulated in hippocampal neurons where it was shown that they are non-canonical targets of GABAB receptor activation. This work is reported in Chapter 6 and provides the basis for future understanding of the physiological contribution of individual K2P channels. Finally, in Chapter 7 using a variety of approaches I show that TREK channels are specifically regulated by the enzyme phospholipase D through direct interaction. Using the photoswitchable conditional subunit system, this regulation was shown to occur in hippocampal neurons where it may explain some of the long term effects of alcohol exposure
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Development of Small Molecule Ligands for Voltage-gated Potassium Channels and Functional Characterization of Voltage-gated Phosphatases
Full text linkMembrane proteins respond to both chemical and electrical stimuli. This work explores the molecular mechanisms by which membrane voltage controls voltage-gated proteins and describes the development of tools to modulate voltage-gated protein function. Voltage-gated potassium (Kv) channels are tetrameric transmembrane proteins that translate changes in the membrane electric field into the controlled permeation of potassium across the plasma membrane. Kv channels mediate the initiation and regulation of action potentials, muscle contraction, hormone secretion, and information processing, rendering them important drug targets. We employed organic synthesis, molecular dynamics and electrophysiology techniques to demonstrate that calix[4]arenes with free phenolic OH groups at the lower rim and positively-charged groups at the upper rim constitute a versatile class of reversible ligands for homotetrameric Kv1.x channels. Synthesis of a panel of calix[4]arenes with variable upper and lower rim substituents enabled the systematic development of Kv1.x channel-compatible ligands. We used molecular modeling to predict calix[4]arene binding to the pore domain, and through electrophysiology experiments, we demonstrated that the calix[4]arene ligands function as reversible blockers of Kv1.x channels. We probed the mechanism of calix[4]arene-channel interactions using voltage clamp fluorometry and found these ligands modify the voltage-dependent motions of the Shaker Kv channel in addition to inhibiting ion current. These calix[4]arene ligands provide a new set of tools to control cell excitability by specifically targeting Kv channels. Until recently, ion channels were the only proteins known to sense changes in membrane potential. This changed with the discovery of Ciona intestinalis voltage-sensor containing phosphatase (Ci-VSP) which has a voltage sensing domain like voltage-gated ion channels and a cytosolic phosphatase domain resembling the phosphoinositide phosphatase PTEN. Ci-VSP is the first member of the voltage dependent family of proteins that is not an ion channel. Instead, Ci-VSP takes an electrical signal in the form of membrane voltage and converts it to a chemical signal through its phosphatase activity. To study the mechanism of voltage-sensing in Ci-VSP, we combined electrophysiology and fluorescence methods in living cells to determine the oligomerization state of Ci-VSP and monitor the functional transitions that result in Ci-VSP mediated changes in phosphoinositide pools. We find that Ci-VSP is a functional monomer which undergoes complex voltage-dependent conformational changes to control a cytosolic phosphoinositide phosphatase domain. As Ci-VSP catalyzes several reactions, we also developed fluorescent-based methods to study Ci-VSP substrate specificity and monitor Ci-VSP-mediated changes in multiple phosphoinositide pools in a single cell. Finally, we find that basic residues in the interdomain linker connecting the voltage sensing domain and phosphatase domains in Ci-VSP are essential for coupling the two domains. Our results indicate that a single voltage sensing domain can function in the membrane on its own and suggests that voltage sensing domains are modular units that can impart voltage sensitivity to a variety of effector domains
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