David T. Yue - US grants

Regulation of the processes that control the opening and closing, or gating, of Ca channels is crucially important to the control of the heartbeat. Classical ideas about how channels are regulated have focused upon changes in the gating behavior of channels that are almost always available to open upon short notice. In contrast, recent studies have hinted at the novel possibility that a predominant mechanism for modulating Ca channels is to shift channels slowly between two radically different modes of gating; (1) an active' mode in which channel openings are probable, and (2) a 'hibernating'mode in which channels are unlikely to open. The overall goal of this project is to use patch clamp techniques to establish a rigorous understanding, at the single channel and molecular level, of the slow transitions of the Ca channel between active and hibernating gating modes in the heart. Unitary "L-type" Ca channel currents will be measured by the patch voltage clamp technique in single mammalian ventricular cells. A new analytic approach, termed "sweep histogram analysis," will be applied to provide quantitative evidence for the genuine existence of distinct active and hibernating gating modes, between which channels cycle slowly. The approach will also enable the identification of explicit kinetic models to explain the transitions between gating modes. The validation of such a model opens the possibility to newly distinguish the specific kinetic steps that are affected by factors that modulate the Ca channel. These factors include regulators of channel phosphorylation (B-adrenergic and cholinergic agonists) or dephosphorylation (okadaic acid), as well as agents that interact with the channel directly (membrane voltage, synthetic Ca channel ligands, and G proteins). Sweep histogram comparison of the effects of these factors to the action of intracellularly applied proteases will provide important clues as to the existence of domains on the channel molecule that may be crucial to slow gating transitions between modes. Clarification of the mechanisms that bias Ca channels toward active or hibernating gating modes promises to provide fundamental insight into the molecular mechanisms by which the Ca channel is regulated.

This Presidential Faculty Fellow award will support studies at the interface of engineering and molecular physiology. The research will combine stochastic process analysis with molecular experimentation to understand the mechanisms of calcium channel opening. The award also will support the teaching activities of the investigator. The activities include writing a textbook that systematizes the application of engineering techniques to the understanding of molecular electrophysiology problems and the development of computer simulations of channel behavior.

Ca2+ serves not only as a charge carrier to depolarize cells, but also as a broadly-targeted second messenger molecule. Hence, Ca channels are particularly important as vital links in the control of essential specialized processes like contraction and exocytosis, as well as in the modulation of universal biological activities such as gene expression and metabolism. in heart and brain, Ca channels have been implicated in the pathogenesis of ischemic disorders and long-term functional derangement. For these reasons, gaining a clear understanding of the opening and closing, or 'gating' processes of Ca channels is a quest of enormous biological consequence. Yet, major questions remain about one pervasive class of these channels, known as L-type Ca channels. The uncertainty arises in large part from Ca-sensitive inactivation, a phenomenon whereby elevated intracellular [Ca2+] speeds channel inactivation. While the existence of this physiological feedback is unmistakable, many of its rudimentary features remain unclear and present unusual technical and theoretical challenges. The overall goal of this proposal is to elucidate the kinetic, biochemical, and molecular basis of Ca-sensitive inactivation in L-type Ca channels. Single-channel and whole-cell electrophysiology will be used to investigate both native L-type Ca channels, and those expressed in mammalian cells from cDNA. Using native Ca channels, we will test and refine a new modal hypothesis of Ca-sensitive inactivation, one in which Ca2+-entry shifts channel gating from high to low open probability modes. Experiments will be performed that distinguish among channel (de)phosphorylation, calmodulin activation, and direct Ca2+ binding to the channel as the actual molecular switch for intermodal shifts. We will next undertake extensive biophysical investigation of Ca2+ currents expressed in mammalian cells from Ca channel cDNAs. Any special subunit requirements for faithful reconstitution of native Ca inactivation will be determined. Finally, the molecular basis of Ca inactivation will be examined by an iterative process that combines construction of Ca channel mutants and chimeras together with electrophysiology. Ultimately, the precise spatial dimensions of Ca channel domains that mediate Ca inactivation are to be identified. Clarification of the kinetic, biochemical, and molecular mechanisms of Ca-sensitive inactivation promises to bridge fundamental gaps in Ca channel gating. As such, this research should provide new insight into Ca channel structure-function relations, along with important constraints on the role of Ca channels in diverse biological signalling pathways.

Voltage-gated channels are not only essential to countless physiological functions in heart, but also serve as the molecular target of diverse anti-arrhythmic agents. With the recent cloning, sequencing, and expression of voltage-gated Na and K channels, the stage is now set to identify the key amino acid residues primarily involved in anti- arrhythmic action on voltage-gated Na and K channels. In particular, wild-type and mutant versions of both a voltage-gated Na channel derived from adult skeletal muscle (mu1, Trimmer et al., 1990), as well as a voltage-gated K channel derived from human heart (HK1, or Kv1.4, Tamkun et al., 1991) will be expressed in a mammalian cell line and probed by patch-clamp methods to detect functional changes in the effects of internally applied QX-314 (for mu1) and clofilium (for HK1), permanently charged versions of anti-arrhythmic agents. Mutations will be made according to recent work localizing the probable site of action of internally active anti-arrhythmic agents to the cytoplasmic vestibule of Na and K channels (Gingrich et al., 1993; Backx et al., 1992, Choi et al., 1993). Unmasking the arrangement of amino acids that are important for drug action would be a crucial prerequisite to unravelling the mechanism of anti-arrhythmic action at the molecular level. Such understanding would be invaluable in rational drug therapy and design.

DESCRIPTION: The efficiency of information transfer from pre to postsynaptic neurons can be acutely modulated by recent presynaptic activity. Such short term synaptic plasticity figures prominently in the neuro-computational capabilities of cortical neurons; moreover, long term potentiation can act to modify short term plasticity, rather than to alter a uniform synaptic weight. Hence, transient changes in synaptic efficacy may be fundamental to how we process and store information. Because P/Q and N type calcium channels trigger neurotransmitter release, dynamic modulation of channels by G proteins and voltage inactivation could provide biophysical and molecular mechanisms for activity dependent changes in synaptic efficacy. This project will investigate this intriguing possibility by deepening the biophysical and structural understanding of G protein inhibition and voltage inactivation of neuronal Ca channels, and by bridging this new understanding to the operation of channels during activation by action potential waveforms. Electrophysiology, recombinant expression of P/Q and N type Ca channels, and molecular biological approaches will be combined in 5 specific aims, each of which targets a salient gap in understanding channel performance during action potentials. Because many Ca channel subunit combinations comprise the broad categories of P/Q and N type channels, recombinant Ca channels will be studied to permit deepened understanding of individual subunit combinations. 1) Test whether channels mainly open according to one gating pathway, despite a possible multiplicity of opening pathways in currently proposed mechanisms. The one pathway scenario would simplify prediction of Ca2+ current during action potentials. 2) Define and characterize fast and slow forms of voltage inactivation. Two forms are unmistakable in our preliminary studies, but have been poorly appreciated. 3) Establish whether G protein inhibition and voltage inactivation interact, or proceed independently. Because both processes evolve smultaneously, this is a key issue. 4) Determine whether specified domains of different Ca channel beta subunits have selective control over fast inactivation, slow inactivation , or G protein inhibition. The existence of such domains would argue that the three processes are distinct in terms of not only function, but molecular mechanism. 5) Understand the impact of G protein inhibition and voltage inactivation on channel performance during trains of action potential waveforms. This aim explicitly links the deepened basic understanding to channel performance during physiological paradigms of activation. Overall, this proposal will usher in new awareness and understanding of the potential role of Ca channels in short term changes of synaptic efficacy.

Functional hallmarks of heart failure include prolonged action-potential duration, depressed contraction and intracellular Ca2+ transients, as well as blunted adrenergic responsiveness. Among the molecules implicated as underlying players, the L-type channel is particularly important. For example, in cells isolated from dogs with pacing-induced heart failure (HF cells), a key factor underlying action potential prolongation appears to be decreased L-type channel inactivation. However, rigorous assessment of the role of L-type channels in heart failure is hampered by crucial gaps in understanding inactivation and functional modulation by auxiliary channel subunits. The overall goal is to deepen fundamental understanding of L-type channels in these gap areas, and to bridge the enhance basic knowledge to a new appreciation of how these channels contribute to heart failure. Using HF cells from dogs and humans, we will pursue the following 5 Aims, each targeting a salient gap in understanding channel performance during failure. 1. To clarify fundamental uncertainties about voltage-dependent inactivation of cardiac L-type channels. 2. To discover structure-based mechanisms of Ca2+- dependent inactivation of cardiac L-type channels. 3. To determine the potential for tuning L-type channel function and expression by Ca channel beta subunits. 4. To identify the profile of Ca channel beta subunits in normal and failing dog and human heart tissue. 5. To establish a functional profile of L-type channel properties in HF cells., and to test whether expression of recombinant beta subunits can alter or "normalize" identified irregularities in function. The latter experiments will help lay the groundwork for gene therapy of excitability disorders and heart failure.

DESCRIPTION (provided by applicant): Feedback, both positive and negative, is inherently critical to information networks, and Ca+ signaling is no exception. Over the past few years, the description of feedback involving Ca2+ regulation of voltage-gated Ca channels has shifted fundamentally in terms of scope, nature, and underlying mechanism. Within the family of high-voltage-activated (HVA) Ca channels (L-, P/Q-, N-, and R-type), only the L-type was widely renowned for strong Ca22+ regulation, and this regulation was believed to result from Ca22+ binding directly to the channel. But this simple view has begun to dissolve. Rather than Ca2+ acting directly on channels, it is calmodulin (CaM) interaction with L-type channels that triggers Ca2+-dependent inactivation (CDI), and perhaps facilitation (CDF). Moreover, the way in which CaM modulates Ca channels is unusual, showing unexpected capabilities only sparingly glimpsed elsewhere. Of further interest, CaM interaction with L-type channels relies on distinctive structural hallmarks that are largely conserved in other HVA channels, hinting that their Ca2+ regulation may not be dormant. Already, P/Q-type channels have recently shown Ca2+/CaM regulation. The overall goals are therefore to drive discovery of Ca2+ regulation across the HVA channel family, and to test for a common overall regulatory mechanism. Electrophysiology, expression of recombinant Ca channels, molecular biology/biochemistry, and FRET-based microscopy are combined in four aims, each targeting a salient area of functional and/or mechanistic discovery. (1) To clarify structural mechanisms for Ca2+ regulation of L-type (Cay 1.2) Ca channels via CaM. (2) To refine the functional dimensions of P/Q-type (Ca about2.1) Ca channel regulation by Ca2+, and to elucidate structure-based mechanisms underlying such regulation. (3) To discover Ca2+ regulation of the other HVA channels (N- and R-type; Ca about2.2 and Ca about2.3, respectively), and to delineate essential molecular elements for any such regulation. (4)To determine whether the carboxy tail of each type of o about subunit is a Ca2+ regulatory module-one that can confer a channel-specific phenotype of Ca2+ regulation to a foreign channel o about backbone. These aims promise new dimensions of neurobiology, and may provide the basis for drug discovery targeting psychiatric illness.

This application requests a Zeiss LSM510 laser scanning confocal microscope, outfitted with dedicated equipment permitting patch-clamp electrophysiological measurements to be made concurrently with confocal image acquisition. This particular instrument will feature two crucial advantages absent in all core-facility confocal microscopes at Johns Hopkins University. First, the microscope will possess the requisite flexibility in selective excitation and emission wavelengths to permit implementation of a novel 3(3)-FRET algorithm that rapidly and nondestructively specifies the FRET efficiency (E) between donor and acceptor fluorophores. (Conveniently done with GFP-color mutants like CFP and YFP), determination of E can specify interfluorophore distances between 10-100 Angstroms, thereby providing a powerful and quantitative measure of protein-protein interaction in living cells. The 3(3)-FRET algorithm overcomes a major obstacle to this approach, allowing E to be determined despite commonly observed variability in the expression levels of labeled proteins. The proposed confocal instrument will thus furnish spatially resolved maps of protein-protein interaction. Second, no core-facility confocal microscope currently accommodates simultaneous imaging and patch-clamp recording; the attachment of electrophysiological apparatus would prove disruptive to conventional users, and such apparatus would be easily damaged by a high-volume of non-electrophysiological participants. Yet, numerous scientific questions hinge critically upon simultaneous image acquisition and measurement/control of membrane voltage. By drawing from a user base that values this dual capability, the proposed confocal instrument with dedicated patch clamp would address this void. The Zeiss LSM510 platform accommodates optimal implementation of 3(3)-FRET and electrophysiology. The microscope will anchor a departmental facility supported by 3 major and 3 minor users, all of whose research would be fundamentally advanced by the platform's unique capabilities.

The overall thrust is to deploy new molecular constructs---many inspired from channel mechanistic studies-for the discovery of fundamental, newly accessible arenas of CaM/Ca channel physiology in heart. This thrust drives three aims, addressing successively more general realms of cardiac physiology, each with fundamental and therapeutic implications. (1) To clarify facilitation of cardiac L-type Ca channels by Ca2+/CaM. By contrast to CDI, a distinct process of facilitated channel opening by Ca 2+ (CDF) remains mysterious, despite its probable role in strengthening the heartbeat at faster heart rates. Still unclear is the actual strength of CDF in heart, and whether CDF shares rich CaM signaling features found in model experimental systems. Those systems permit study of engineered recombinant L-type channels that lack CDI and thereby permit maximal resolution of CDF. By contrast, incomplete separation of CDF from CDI seriously complicates study in heart. We will thus express engineered L-type channels (lacking CDI and dihydropyridine block) in myocytes. During dihydropyridine block of native channels, selective resolution of recombinant channels will permit unambiguous assessment and mechanistic dissection of CDF in the native setting. (2) To define the capabilities of cardiac L-type Ca channels to activate nuclear CREB. Such Ca 2+ signaling appears crucial to the dynamic regulation of cardiac genes. In neurons, CaM not only regulates the channel to which it is bound, such CaM may also bridge preferential signaling of L-type channels to CREB. Here, we will define basic aspects of CREB signaling in myocytes, using distinctive methodologies such as CaM/L-type channel fusions to test whether the very CaM that modulates a channel is essential for triggering CREB. Optical FRET-based sensors of CREB activation also promise rapid temporal correlation of Ca 2+ entry patterns and CREB activation. (3) To estimate the concentration of local endogenous CaM near L-type channels in heart cells. As CaMs responsive to local Ca 2+ influx through L-type channels may be the initiatory Ca 2+ sensors that ultimately trigger CREB and other nuclear factors, the number of CaMs privy to the local Ca 2+ signal from channels is key to downstream signaling strength. Here, we will utilize CaM/L-type channel fusions, with polymer chain theory, to estimate the local concentration of endogenous CaM near channels. Preliminary results hint at mM concentrations, suggesting that a 'school'of local CaMs resides near channels. Overall, this proposal will answer fundamental unknowns of CaM/Ca channel physiology in the heart.

DESCRIPTION (provided by applicant): Calmodulin (CaM) regulation of CaV1-2 channels-termed calmodulation has proven rich, both biologically and as a general modulatory prototype with discriminating Ca2+ decoding capabilities. In earlier cycles, Ca2+-free CaM (apoCaM) was found to be pre-associated to an IQ domain on the intracellular carboxy terminus of channels. Ca2+-binding to this resident CaM induces as-yet-unclear conformational changes that somehow facilitate (CDF) or inactivate (CDI) channel opening, casting calmodulation as a positive or negative feedback control system for Ca2+. Intriguingly, Ca2+-binding to the C- and N-terminal lobes of CaM can each induce distinct forms of channel regulation, echoing earlier findings of CaM 'functional bipartition' in Paramecium. More remarkably, the C-lobe responds to the ~100-mM Ca2+ pulses driven by the associated channel (local Ca2+ selectivity), whereas the N-lobe is somehow capable of sensing far weaker signals from distant Ca2+ sources (global selectivity). In the current cycle, we deduced in coarse outline the functional mechanisms of calmodulation by the C-terminal lobe of CaM (slow CaM scheme), and by the N-terminal lobe (SQS scheme). These mechanisms explain the notable spatial Ca2+ decoding properties of these two calmodulatory subsystems. Prominently absent, however, is a quantitative sense of the Ca2+ dependence of these low-resolution mechanistic sketches, owing to the complexity of Ca2+ channel influx that normally drives calmodulation. A greater void concerns the channel/CaM interfaces postulated by slow CaM and SQS schemes. If we follow the lead of preliminary data, and abandon a prevailing 'IQ-centric' view, where apoCaM and Ca2+/CaM both interact with the IQ domain to trigger channel regulation, little would be known. This indeterminacy obscures the linkage of our promising functional mechanisms to molecular reality, and confounds understanding of just discovered physiological tuning of CaM regulation via RNA editing of CaV channels. These prominent challenges will be addressed by three specific aims. 1) To quantify the Ca2+ dependence of calmodulation via dynamic control of Ca2+ inputs to channels, as afforded by UV Ca2+ uncaging. 2) To identify the molecular states underlying CaM regulation of CaV channels, using an approach termed individually Transformed Langmuir (iTL) analysis, combined with in silico prediction of CaM/channel configurations. 3) To deduce, using Aim 2 insight, the mechanism whereby RNA editing of CaV1.3 channels alters their CaM regulation.

DESCRIPTION (provided by applicant): Calmodulin (CaM) regulation of four-domain channels first emerged in CaV channels, and has proved rich both mechanistically and biologically. Our laboratory has been one of the leaders in unveiling this exciting chapter of CaV channel discovery. Given the sequence similarity of CaV and NaV channels, intriguing suspicions arose that NaV channels might also exhibit such CaM regulation. This possibility was most attractive, given the wide-ranging biological and clinical impact of NaV channels, which include mentation (epilepsy), muscle contraction (myotonia and arrhythmias), and sensation (neuropathic pain). Biochemistry and structural biology both underscored this similarity, but the reported functional effects of Ca2+ and/or CaM on NaV channels have been rather subtle, mostly limited to several-mV shifts of steady-state inactivation (h%) curves. Yet, channelopathic NaV mutations at putative structural determinants of Ca2+ and/or CaM regulation do confer severe disease phenotypes. One potential culprit for the apparent disconnect is that, unlike CaV channels, NaV channels do not conduct Ca2+, so they cannot directly trigger Ca2+ responses. Instead, the NaV field has used whole-cell dialysis to tonically manipulate Ca2+ levels, over several minutes or longer. Could the field be characterizing desensitized responses to Ca2+ and/or CaM, with poor similarity to short-term and larger physiological effects? Here, we use a different approach to dynamically perturb Ca2+, and our preliminary data unveil something long sought in the field>rapid and robust Ca2+/CaM-dependent inactivation of NaV channels (CDI). The advances now permitted may revolutionize understanding of the CaM regulation of NaV channels. This project will usher in an exciting era of discovery via three specific aims. (1) To unveil the existence of rapid Ca2+/CaM-mediated regulation across the family of NaV channels. (2) To elucidate the mechanism of dynamic CaM-mediated regulation of NaV channels. With long-sought robust readouts of NaV regulation in hand, Aim 2 will be uniquely poised to dissect the mechanistic underpinnings of regulation. (3) To assess the broader biological impact of CaM-mediated regulation of Nav channels. The mechanistic advances above hold numerous biological implications that Aim 3 will explore. In all, this project will facilitate a period of unprecedented progress in the Ca2+ regulation of NaV channels. As well, this proposal will explicitly link Ca2+ regulation of NaV channels to certain channelopathies, and perhaps to related but more generalized forms of disease.

DESCRIPTION (provided by applicant): CaV1.3 channels are low-threshold, dihydropyridine-sensitive L-type Ca2+ channels which mediate low-voltage signaling and rhythmicity throughout the body. They are essential for neurotransmitter release at ribbon synapses such as found in cochlear hair cells; they mediate pacemaking in the heart; and they modulate oscillatory behavior throughout the brain, such as the repetitive bursting in supra-chiasmatic (circadian pacemaking circuitry) and substantia nigra (locus of primary damage in Parkinson's) nuclei. As such, overactivity of these channels may predispose for Ca2+ overload precipitating Parkinson's, and downward modulation of these channels may enhance positive mood and affect. Clearly, small-molecule compounds that selectively inhibit or enhance CaV1.3 channels, rather than the other CaV1 L-type channels would be of enormous utility for basic studies of CaV1.3 roles, and for potentially amerliorating a number of CaV1.3-related pathologies. However, though excellent L-type channels antagonists and agonists have been discovered, none can truly select among the L-type channel subtypes. Here, in the search for selective modulators, we will exploit a unique molecular interaction between ICDI and IQ domains of CaV1.3 channels, where this interaction modulates the strength Ca2+ feedback inhibition (CDI) of these channels. This promising screen will be prosecuted according to three specific aims. 1) To perform a primary screen for small molecules that disrupt or enhance a functionally critical interaction between IQ and ICDI domains of CaV1.3 channels, using the MLSMR library of 350,000-500,00 compounds. 2) To confirm and identify candidate hits from Aim 1 using a microscope-based FRET analysis of single living cells. 3) To test candidate compounds for modulation of CaV1.3 Ca2+ regulation, using patch-clamp electrophysiology. Overall, this project promises lead candidates for selective modulators of CaV1.3 versus other CaV1 L-type calcium channels.

PROJECT SUMMARY PI: David Yue, MD, PhD One type of voltage-activated Ca2+-permeable ion channel, known as CaV1.3, is emerging as a preeminent Ca2+ entry pathway into neurons residing at the epicenter of brain rhythmicity and neurodegenerative disease. The lower transmembrane voltages required to open CaV1.3 allow these channels to contribute importantly to pacemaking and subthreshold voltage fluctuations. CaV1.3 channels thus constitute a dominant Ca2+ entry module into many neurons undergoing oscillatory and subthreshold activity. Nowhere is this Ca2+ entry function more salient than in substantia nigral neurons, where CaV1.3 channels furnish the lion's share of Ca2+ entry, while driving rapid pacemaking essential for movement control. Notably, degeneration of substantia nigral neurons is central to Parkinson's disease (PD), and intracellular Ca2+ dysregulation and overload are crucial to PD pathogenesis. Accordingly, a highly promising avenue for novel PD therapeutics involves the burgeoning search for small molecules that selectively inhibit the opening of CaV1.3 channels. Yet, comparatively little is known about the mechanisms controlling the open probability PO of CaV1.3 channels. Ongoing small-molecule screens thereby rely on rank empiricism, largely bereft of known channel interfaces to which drug binding would likely alter opening. Multiplying the challenge is the recent discovery that CaV1.3 channels are not monolithic, but comprised of numerous RNA-edited and splice variants, each with potentially distinct effects on the open probability PO of channels. The mechanism underlying variant-related PO modulation is currently obscure. Additionally, GPCR-mediated changes in the plasmalemmal lipid PIP2 powerfully regulates PO, but it is unknown how this occurs, and how it relates to edited/splice variation. Together, the mechanistic void relating to these two systems precludes quantitative understanding of how Ca2+ entry through these channels contributes to pathogenesis, and obscures the path to rational small-molecule screens for CaV1.3 modulators. Yet, forward progress has proven difficult by traditional means alone. This project thus proposes to clarify CaV1.3 PO modulation by melding electrophysiology with novel chemical-biological and live-cell FRET tools. Overall, this proposal promises elegant clarification, simplification, and unification of seemingly diverse mechanisms of CaV1.3 PO modulation; identification of channel interfaces that could be targeted for discovery of small-molecule PO modulators; and new chemical-biological and FRET-based tools of wide applicability.