Riccardo Olcese, PhD - US grants

DESCRIPTION (provided by applicant): Large conductance Voltage and Ca2+ activated K+ channels (BK) are membrane proteins that play a fundamental role in controlling smooth muscle tone and neuronal excitability. In most of the tissues, they form complexes composed by the pore-forming alpha subunit and by regulatory subunits. Similarly the other voltage dependent ion channels, BK posses a voltage sensor that is mainly represented by the S4 transmembrane segment. Changes in potential across the membrane displace the voltage sensor, producing a conformational change of the protein. Eventually, for adequate depolarizations, the consequent conformational change brings the channel into a state that allows ion conduction. Very little is known about structures that regulate the opening and closing of BK channels, and no information exists about the dynamical rearrangements produced in BK channels by changes in the membrane potential. One of the aims of this project is to investigate the structural changes underlying the operation of BK channel. Conformational changes of both a and b subunits will be assessed by residue specific fluorescent labeling of the channel protein. The short-term objective is to obtain a more realistic view of BK protein by identifying regions of motion that underlie voltage sensing, and that are couple to activation, inactivation and deactivation, and regions of relative staticity, involved in other channel functions. In addition the role of charged residues in pore region will be investigated. BK channel activators are now under close investigation for treatment of urinary incontinence and as stroke neuroprotectant. This study will contribute to set a framework for the design of new therapeutic agents or for the amelioration of the one already adopted by the medical practice.

The goal of this Program Project is to develop novel antiarrhythmic approaches based on improved understanding of the arrhythmia mechanisms causing sudden cardiac death. Project 4 will combine biological experiments and mathematical modeling to study how the interaction between the L-type Ca curret (l{Ca,L}), the Ca{i} transient and other Ca-sensitive currents lead to early afterdepolarizations (EADs) in normal and failing ventricular myocytes (Aim 1). This analysis will then be used to guide development of therapeutic strategies to suppress EADs and EAD-mediated arrhythmias by modifying I{Ca,L} properties (Aim 2). We will utilize the dynamic patch clamp approach which permits virtual currents to be added and interact bidirectionally with the endogenous currents of a live myocyte. EADs will be induced with various interventions, and then suppressed by the Ca channel blocker nifedepine. In stages, the dynamic clamp will add back a virtual I{Ca,L} virtual Ca; transient, and other Ca-sensitive currents to determine the necessary interactions required to reconstitute EADs. Given the critical importance of the I{Ca,L} window current in EAD formation, we will use the dynamic clamp approach to explore how the kinetic and/or voltage dependent properties of I{Ca,L} can be modified to suppress reconstituted EADs in isolated myocytes. The normal I{Ca,L} in the dynamic clamp will be replaced with an appropriately modified virtual I{Ca,L} to identify which modifications eliminate EADs while preserving a normal Ca{i} transient (Aim 2a). Using this information, we will explore genetic modifications of I{Ca,L} in rabbit ventricular myocytes to identify interventions which suppress EADs without adversely affecting E-C coupling, using two strategies: i) genetic overexpression of ancillary Ca channel subunits to replace the corresponding native Ca channel subunits. ii) downregulation of native Ca channel subunits in the adult rabbit ventricular myocytes using appropriate viral vectors. These hybrid modeling/experimental studies promise to both advance our understanding of the mechanisms of EAD formation and identify novel antiarrhythmic strategies. Project 4 will be complemented by the modeling studies in Project 1, cellular level studies in P2, and tissue level studies in Project 3.

DESCRIPTION (provided by applicant): Large-conductance Ca2+- and voltage-gated K+ (Slo1 BK) channels play numerous physiological and pathophysiological roles and their allosteric gating mechanism is subject to modulation by a variety of cellular signaling pathways. Increasing evidence suggests that certain lipids may serve as signaling molecules. We propose to reveal the biophysical and physicochemical mechanisms of modulation of Slo1 BK channels by two lipid messengers, phosphatidylinositol 4,5-bisphosphate (PIP2) and docosahexaenoic acid (DHA), an omega-3 long-chain polyunsaturated fatty acid enriched in oily fish. The effect of PIP2 on the Slo1 BK channel was reported recently but the mechanism is only poorly known. We will fill this critical knowledge gap by performing thorough mechanistic electrophysiological measurements. Furthermore, we will identify the structural determinants of the auxiliary beta subunit important for the action of PIP2. Our electrophysiological measurements will be complemented with measurements of tryptophan fluorescence of the isolate and purified Slo1 gating ring protein. The biophysical and physicochemical mechanisms of the action of DHA, an emerging lipid messenger, on the Slo1 channel will be also investigated similarly using the electrophysiological and fluorescence methods. The research outcome is expected to provide definitive mechanisms of the PIP and DHA actions on the allosteric gating mechanism of the Slo1 channel and establish the novel paradigm that the Slo1 channel is an omega-3 fatty acid receptor. The modulation of the Slo1 BK channel by DHA may underlie the health-promoting effects of omega-3 long-chain fatty acids.

DESCRIPTION (provided by applicant): The large-conductance, Ca2+-activated K+ channel from cardiac mitochondria (mitoBKCa) is thought to play a role in cardioprotection. MitoBKCa molecular size is uncertain with reported immunochemical signals at ~55 and ~125 kDa. In addition, mitoBKCa molecular identity and its mitochondrial targeting mechanisms remain unknown, while there is scarce information about its functional properties or direct evidence for their role in cardioprotection. Because cardiac mitoBKCa shares conductance, Ca2+ responsiveness, and sensitivity to pharmacological agents with its plasma membrane counterpart known as BKCa (or MaxiK), we expect that mitoBKCa is assembled like BKCa by four pore-forming a subunits with a monomeric mass of ~125 kDa. We will now test the hypotheses that: 1) mitoBKCa and plasma membrane BKCa are encoded by the same gene and splice variation provides BKCa with intrinsic signals for its preferential mitochondrial targeting; 2) the normal absence of BKCa from the cardiomyocyte plasmalemma and presence in mitochondria is ruled by both an intrinsic signal(s) within mitoBKCa backbone (i.e. splice insert) either directly or indirectly (i.e. via a chaperone), and by cell-specific mechanisms, and ) mitoBKCa contributes to cardioprotection by regulating mitochondrial calcium retention capacity (CRC) and permeability transition pore (mPTP) opening. Preliminary Data shows: 1) the detection of a ~125 kDa protein in mitochondria by specific anti-BKCa antibodies; 2) the detection of all 27 constitutive BKCa exons in isolated cardiomyocyte mRNAs; 3) that BKCa isoform containing splice insert DEC (C-terminal insert of 61 amino acids) but not the constitutive form of BKCa (insertless BKCa) is readily targeted to mitochondria in adult cardiomyocytes; 4) that mitoBKCa subproteome uncovered as a partner Hsp60, a heat shock protein relevant for folding of mitochondrial imported proteins; and 5) that BKCa gene ablation prevents the cardioprotective action of putative BKCa channel opener NS1619. Overall the data support the above hypotheses, which will be tested using multiple approaches and pursuing the following Specific Aims to: 1. Identify the molecular correlate of cardiac mitoBKCa; 2. Functionally validate the identity of cloned putative mitoBKCa; 3. Determine signal mechanisms involved in mitoBKCa mitochondrial targeting; and 4. Directly address the role of mitoBKCa in cardioprotection. The outcomes of this program will open the opportunity to study mitoBKCa at the molecular level and advance the cardiac field by: solving mitoBKCa identity, providing information on its targeting mechanisms, and defining its functional properties and role in cardioprotection. Further understanding of the underlying molecular mechanism(s) of mitoBKCa cardioprotective effects will provide new targets for translation into therapeutics.

DESCRIPTION (provided by applicant): A depolarization-initiated influx of Ca through voltage-gated Ca (CaV) channels gives rise to a plethora of physiological responses such as neurotransmitter release, muscle contraction and gene expression. Membrane depolarization is sensed by four transmembrane structures, the voltage sensor domains (VSDs), which surround and control the activation, deactivation and inactivation properties of a central Ca-selective pore governing the amount and timing of Ca influx. In contrast to homotetrameric KV channels, the CaV pore and four VSDs are encoded by a single long polypeptide chain (alpha1). Thus, each VSD has a unique primary amino acid sequence, suggesting distinct voltage-sensing properties. Critically, the voltage-sensing processes coupling membrane depolarization to Ca influx are still poorly understood and the molecular mechanisms by which auxiliary subunits, such as beta and alpha2delta, alter the voltage dependence of the channel, still need to be elucidated. This lack of knowledge persists in part because ionic and gating current measurements have not thus far captured the properties of individual VSDs in CaV channels. Using Voltage Clamp Fluorometry (VCF), we have resolved that the time- and voltage-dependent properties of each of the four VSDs of human CaV1.2 revealing their highly distinct functional properties. We now have the experimental tools and theoretical formulation to answer key unresolved questions on the operation of CaV1.2 channels, as delineated in four specific aims: (1) To establish the contribution of individual voltage sensing domains to CaV1.2 channel activation. (2) To establish the molecular mechanism by which accessory subunits regulate voltage-dependent activation of CaV1.2 channels. (2a) regulation by alpha2delta subunits (2b) regulation by beta subunits (3) To determine the role of each VSD in Voltage- and Ca-dependent Inactivation and (4) To develop a CaV1.2 model accounting for the operation and role of the four distinct VSDs. CaV1.2 channels specifically labeled at each VSD with small, environment-sensitive fluorophores will be voltage-clamped using the cut-open oocyte technique, so that voltage-evoked fluorescence changes will reflect local conformational rearrangements. A series of physically-relevant models consistent with the CaV1.2 structure and accounting for all experimentally- resolved aspects of CaV1.2 voltage-dependent operation, including the interactions governing excitation- evoked Ca influx. The innovative aspects of this proposal include (1) the experimental approach, unprecedented for the CaV superfamily; (2) the hypothesis that VSDs are the targets of regulation by modulatory subunits; (3) the premise, supported by striking preliminary results, that CaV1.2 VSDs are drivers and regulators for inactivation; (4) the theoretical approach proposes the first model consistent with the molecular architecture and asymmetry of CaV channels. Finally, this study will contribute to the understanding of the molecular mechanisms of pathological states caused by altered CaV1.2 voltage dependence, such as Timothy Syndrome.

PROJECT SUMMARY The holy grail of structural biology, i.e., the simultaneous quantitative determination of a protein?s structure and function, remains very difficult to attain. This application pertains to the development of a new, cutting-edge optical approach that allows the resolution of sub-nanometer-scale distances and distance changes in real time: distance-resolving Voltage Clamp Fluorometry (drVCF). drVCF combines the use of small, spectrally-identical, Cys-attached fluorophores of variable length with Trp-induced collisional quenching. Crucially, fluorophore range and flexibility are accounted for by radial probability density functions (pdfs) generated by fluorophore molecular dynamics (MD) simulations. The pdfs are used to simultaneously fit the optical signals of multiple labels and obtain highly constrained distance information immediately relevant to protein structure (from the Trp side-chain to the labeled Cys C? atom). drVCF encompasses the benefits of other optical structural approaches (FRET, LRET, etc.), such as wide applicability and physiologically-relevant experimental conditions; but also distinct advantages, such as (i) the ability to measure intramolecular distances and functionally-relevant distance changes with a very fine grain (<2 Å measurement error in a preliminary evaluation), practically excluding intersubunit and intermolecular signal contamination (<2.2 nm range); (ii) the acquisition of structural data in real time, allowing the simultaneous tracking of structure and the kinetics of structural change; (iii) the ability to acquire data from conducting channels without large protein adjuncts such as toxin-mounted fluorophores or large fluorescent proteins; (iv) no dependence on fluorophore dipole orientation. As all scientific approaches, drVCF carries assumptions and limitations. In this proposal, the capabilities and limitations of drVCF will be evaluated in established models of structural biology, over three Specific Aims. Aim 1: Validate a New Optical Approach to Measure Functionally-relevant Intramolecular Protein Distances (drVCF) Using Rigid Rod-like Peptides of Known Length. As Stryer and Haugland did to calibrate FRET, drVCF accuracy will be evaluated by measuring the length of rigid polyproline peptides. Aim 2: Validate drVCF in a Well Characterized Soluble Protein of Known Structure. drVCF will be used to measure intramolecular distances in T4 lysozyme, a gold standard in structural biology, to evaluate the applicability of this approach in proteins and its accuracy. Aim 3: Validate drVCF in a Voltage-sensitive Membrane Protein with Known Resting/Active Structures. The voltage-sensing domain of the voltage sensitive phosphatase (Ci-VSP) was recently crystallized in the Resting and Active states. drVCF will be combined with cut-open oocyte voltage clamp to test its ability to measure voltage-dependent distance changes. This approach was developed following highly-encouraging preliminary experiments. The proposed studies may reveal practical pitfalls and limitations, but with them also the opportunity to rectify and refine this highly innovative and potentially ground breaking approach.

PROJECT SUMMARY: The motivation for these studies is the pressing need for a new class of effective and safe antiarrhythmics to prevent VT/VF without compromising EC coupling. CaV channel blockers (Class IV antiarrhythmic agents, such as Diltiazem and Verapamil) have limited therapeutic value because of their negative inotropic effect. These investigators have recently discovered that arrhythmogenic EADs are potently suppressed by the minimal modification of the L-type Ca channel biophysical parameters. Crucially, what these maneuvers have in common is that they all reduce the late component of the L-type Ca current (late ICa,L). The selective reduction of late ICa,L has the benefit of leaving peak ICa,L largely intact, preserving contractility. Regrettably, while the relevance of the ICa,L window current to EAD formation was hypothesized 30 years ago, no therapy based on this idea has emerged yet. The antiarrhythmic strategies that emerged from the investigators? recent studies in animal models of VT/VF are now ready to be pharmacologically implemented: Aim 1 proposes to evaluate LTCC gating modifiers selectively reducing the late component of ICa,L as prototype members of a new class of antiarrhythmics that do not block peak ICa,L. Specifically, Aim 1A will validate the antiarrhythmic potential of pedestal ICa,L reduction using LTCC gating modifiers and determine the efficacy of pilot compounds that selectively decrease late ICa,L: roscovitine enantiomers, known to reduce ICa,L pedestal current. Aim 1B will ?validate the antiarrhythmic potential of ICa,L window current reduction using LTCC gating modifiers?. Gabapentinoids, found in preliminary studies to reduce ICa,L window current by shifting LTCC voltage-dependent activation to depolarized potentials, will be used as pilot compounds. Based on the success of preliminary studies in single cells and full hearts, the ability of these drugs to prevent or suppress VT/VF will be assessed in whole rabbit and rat hearts under EAD-favoring conditions (oxidative stress, hypokalemia). Establishing the VT/VF-preventing efficacy of late ICa,L reduction in two small animal models will justify translation to larger mammalian models and eventually humans. Aim 2, is designed ?to identify the molecular mechanisms underlying late ICa,L reduction by roscovitine and gabapentinoids, prototype members of a potential new class of antiarrhythmic action?. The investigators will take advantage of their recent breakthrough in optically tracking molecular transitions of the human CaV1.2 channel using Voltage Clamp Fluorometry to determine the mechanism by which pilot compounds modify the LTCC to reduce late ICa,L. Specifically, Aim 2A will ?identify the molecular mechanisms underlying pedestal ICa,L reduction by roscovitine? and Aim 2B will ?identify the molecular mechanisms underlying window ICa,L reduction by gabapentinoids?. This information will reveal how next-generation antiarrhythmics should act on the LTCC. Thus, this proposal has two main novel aspects: it sets the basis for a conceptually new class of antiarrhythmics (LTCC gating modifiers) and directs future rational drug design for the LTCC molecule.

PROJECT SUMMARY/ABSTRACT The motivation for these studies is the need to gain an understanding of the fundamental biophysical properties of the skeletal voltage-gated L-type Calcium channel CaV1.1. While ion conduction is a critical feature (and often the only duty) of the vast majority of ion channels, CaV1.1 is somewhat unique: the activation of its voltage sensors opens its pore, but Ca2+ entry is not required to trigger muscle contraction. Instead, the conformational changes of Cav1.1 voltage sensors directly gate Ryanodine receptors (RyR1) via a physical coupling between these two channels, to trigger the release of sarcoplasmic reticulum Ca2+. In this context, the four voltage-sensing elements of Cav1.1 are indeed the voltage sensors of RyR1 channels. As the possibility to express Cav1.1 channel in oocytes has recently become feasible thanks to the discovery of an essential adaptor protein (Stac3), the Olcese laboratory is in a privileged position to directly address the mechanism of voltage regulation in this protein, with a unique capability (to date) to implement the cutting-edge voltage clamp fluorometry approach to CaV channels. During the next five years, using electrophysiological, optical and computational techniques, the investigators will delineate the basis of voltage dependence in CaV1.1 channels, in both adult and embryonic splice variants. They will interrogate how this voltage dependence is modulated by the participation of auxiliary subunits (?, ?2?, and ?) in the CaV1.1 macromolecular complex. They will determine which of the four homologous, but non-identical CaV1.1 Voltage Sensing Domains confer voltage sensitivity to RyR1-mediated Ca release. Finally, the investigators will address the molecular mechanism of a malignant-hyperthermia-causing mutation that specifically affects the voltage-sensing apparatus of CaV1.1. The knowledge gathered by is critical to understand fundamental aspects of muscle physiology and the voltage-dependent control of contraction.