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Open Access Publications from the University of California

Scholarly Works (58 results)

  • Article
  • Peer Reviewed
UC San Francisco Previously Published Works (2016)
Ligand binding usually moves the target protein from an ensemble of inactive states to a well-defined active conformation. Matthies et al. flip this scheme around, finding that, for the magnesium channel CorA, loss of ligand binding induces an ensemble of conformations that turn the channel on.
    Cover page: Let It Go and Open Up, an Ensemble of Ion Channel Active States
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2017)
    Beyond their roles in generating nerve impulses, ion channels are important for many facets of cell biology, including swelling and osmotic stress responses. Sabirov and colleagues now report that the prostaglandin transporter SLCO2A1 is a central component of the ubiquitous Maxi‐Cl anion channel. These findings add to a growing number of transporters displaying ion channel activity and not only provide a molecular handle for future Maxi‐Cl physiological and biophysical studies, but also underscore general questions about the principles that underlie such transporter/channel dual‐use behavior.
      Cover page: Channel surfing uncovers a dual‐use transporter
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2018)
      Ion channels turn diverse types of inputs, ranging from neurotransmitters to physical forces, into electrical signals. Channel responses to ligands generally rely on binding to discrete sensor domains that are coupled to the portion of the channel responsible for ion permeation. By contrast, sensing physical cues such as voltage, pressure, and temperature arises from more varied mechanisms. Voltage is commonly sensed by a local, domain-based strategy, whereas the predominant paradigm for pressure sensing employs a global response in channel structure to membrane tension changes. Temperature sensing has been the most challenging response to understand and whether discrete sensor domains exist for pressure and temperature has been the subject of much investigation and debate. Recent exciting advances have uncovered discrete sensor modules for pressure and temperature in force-sensitive and thermal-sensitive ion channels, respectively. In particular, characterization of bacterial voltage-gated sodium channel (BacNaV) thermal responses has identified a coiled-coil thermosensor that controls channel function through a temperature-dependent unfolding event. This coiled-coil thermosensor blueprint recurs in other temperature sensitive ion channels and thermosensitive proteins. Together with the identification of ion channel pressure sensing domains, these examples demonstrate that "local" domain-based solutions for sensing force and temperature exist and highlight the diversity of both global and local strategies that channels use to sense physical inputs. The modular nature of these newly discovered physical signal sensors provides opportunities to engineer novel pressure-sensitive and thermosensitive proteins and raises new questions about how such modular sensors may have evolved and empowered ion channel pores with new sensibilities.
        Cover page: Global versus local mechanisms of temperature sensing in ion channels
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2015)
        Voltage-gated sodium channels (Na(V)s) provide the initial electrical signal that drives action potential generation in many excitable cells of the brain, heart, and nervous system. For more than 60years, functional studies of Na(V)s have occupied a central place in physiological and biophysical investigation of the molecular basis of excitability. Recently, structural studies of members of a large family of bacterial voltage-gated sodium channels (BacNa(V)s) prevalent in soil, marine, and salt lake environments that bear many of the core features of eukaryotic Na(V)s have reframed ideas for voltage-gated channel function, ion selectivity, and pharmacology. Here, we analyze the recent advances, unanswered questions, and potential of BacNa(V)s as templates for drug development efforts.
          Cover page: Bacterial Voltage-Gated Sodium Channels (BacNaVs) from the Soil, Sea, and Salt Lakes Enlighten Molecular Mechanisms of Electrical Signaling and Pharmacology in the Brain and Heart
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2009)
          Two processes dominate voltage-gated calcium channel (Ca(V)) inactivation: voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). The Ca(V)beta/Ca(V)alpha(1)-I-II loop and Ca(2+)/calmodulin (CaM)/Ca(V)alpha(1)-C-terminal tail complexes have been shown to modulate each, respectively. Nevertheless, how each complex couples to the pore and whether each affects inactivation independently have remained unresolved. Here, we demonstrate that the IS6-alpha-interaction domain (AID) linker provides a rigid connection between the pore and Ca(V)beta/I-II loop complex by showing that IS6-AID linker polyglycine mutations accelerate Ca(V)1.2 (L-type) and Ca(V)2.1 (P/Q-type) VDI. Remarkably, mutations that either break the rigid IS6-AID linker connection or disrupt Ca(V)beta/I-II association sharply decelerate CDI and reduce a second Ca(2+)/CaM/Ca(V)alpha(1)-C-terminal-mediated process known as calcium-dependent facilitation. Collectively, the data strongly suggest that components traditionally associated solely with VDI, Ca(V)beta and the IS6-AID linker, are essential for calcium-dependent modulation, and that both Ca(V)beta-dependent and CaM-dependent components couple to the pore by a common mechanism requiring Ca(V)beta and an intact IS6-AID linker.
            Cover page: Disruption of the IS6-AID Linker Affects Voltage-gated Calcium Channel Inactivation and Facilitation
            • Thesis
            • Peer Reviewed
            UC San Francisco Electronic Theses and Dissertations (2019)

            Two-pore potassium (K2P) channels are responsible for regulating the resting membrane potential of excitable cells. Though K2P channels are heavily implicated in disease, no approved therapies exist to specifically target K2P channels. Defined pharmacology for these channels has lagged, as K2Ps differ from traditional potassium channels in a number of ways. By harnessing these differences, we aim to discover and characterize new K2P modulators to aid in the study of this unique channel class. Through the structural and functional characterization of small molecule K2P modulators, we will facilitate the understanding of how these channels contribute to physiology and disease.

            The introduction of this thesis aims to define and describe the scope of K2P pharmacology, with a specific focus on TREK family modulation. High throughput methods for identifying new classes of TREK family modulators are reviewed, and a synthesis of the selectivity of small-molecule TREK family modulators has been compiled, with special attention to those compounds for which structural information has been elucidated. In chapter two, the action and selectivity of K2P activator BL-1249 is presented and, using a chimeric functional approach, subtle differences between K2P2.1 (TREK-1) and K2P4.1 (TRAAK) channels and their differential response to BL-1249 were pinpointed to specific areas involving M2 and M4 helices. This work highlights how channels within K2P families can differ from one another in their pharmacological profiles and how selective molecules can help to tease apart these important differences. Chapter three presents structural and functional studies of polyruthenium amine inhibitors of K2Ps and creation of a ruthenium red super-binder with low nanomolar efficacy. Through our studies, we define the mechanism of polyruthenium amine inhibitors as an electrostatic block positioned above by coordinating acidic residues. Furthermore, we provide a template for future development of K2P modulators which may target this novel binding site. From these and other recent studies, a multi-site model for K2P pharmacological regulation has emerged and future work will require precise dissection to establish how each of these sites may preferentially affect gating in TREK, and more broadly, all K2Ps.

            Finally, chapter four presents the synthetic modulation of a protein-protein interaction (PPI) using stapled peptides in a different context, departing from a focus on K2P channels onto another voltage-gated ion channel (VGIC) family member, voltage-gated calcium channels (CaV). Stapled peptide mimics of the alpha-interacting domain (AID) were found to inhibit the high affinity interaction of the channel domain (CaV-alpha) with its cytosolic subunit (CaV-beta) to affect calcium channel properties. Establishing and improving the modulation of this type of PPI informs the future development of inhibitors of K2P interactions with important cellular partners.

              Cover page: Interrogating K2P Pharmacology: development and characterization of small-molecule modulators of two-pore potassium channels
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2021)
              K2P (KCNK) potassium channels form "background" or "leak" currents that have critical roles in cell excitability control in the brain, cardiovascular system, and somatosensory neurons. Similar to many ion channel families, studies of K2Ps have been limited by poor pharmacology. Of six K2P subfamilies, the thermo- and mechanosensitive TREK subfamily comprising K2P2.1 (TREK-1), K2P4.1 (TRAAK), and K2P10.1 (TREK-2) are the first to have structures determined for each subfamily member. These structural studies have revealed key architectural features that underlie K2P function and have uncovered sites residing at every level of the channel structure with respect to the membrane where small molecules or lipids can control channel function. This polysite pharmacology within a relatively small (~70 kDa) ion channel comprises four structurally defined modulator binding sites that occur above (Keystone inhibitor site), at the level of (K2P modulator pocket), and below (Fenestration and Modulatory lipid sites) the C-type selectivity filter gate that is at the heart of K2P function. Uncovering this rich structural landscape provides the framework for understanding and developing subtype-selective modulators to probe K2P function that may provide leads for drugs for anesthesia, pain, arrhythmia, ischemia, and migraine.
                Cover page: The Polysite Pharmacology of TREK K2P Channels
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2023)
                Gabapentinoid drugs for pain and anxiety act on the CaVα2δ-1 and CaVα2δ-2 subunits of high-voltage-activated calcium channels (CaV1s and CaV2s). Here we present the cryo-EM structure of the gabapentin-bound brain and cardiac CaV1.2/CaVβ3/CaVα2δ-1 channel. The data reveal a binding pocket in the CaVα2δ-1 dCache1 domain that completely encapsulates gabapentin and define CaVα2δ isoform sequence variations that explain the gabapentin binding selectivity of CaVα2δ-1 and CaVα2δ-2.
                  Cover page: Structural basis for CaVα2δ:gabapentin binding
                  • Article
                  • Peer Reviewed
                  UC San Francisco Previously Published Works (2020)
                  The trinuclear ruthenium amine ruthenium red (RuR) inhibits diverse ion channels, including K2P potassium channels, TRPs, the calcium uniporter, CALHMs, ryanodine receptors, and Piezos. Despite this extraordinary array, there is limited information for how RuR engages targets. Here, using X-ray crystallographic and electrophysiological studies of an RuR-sensitive K2P, K2P2.1 (TREK-1) I110D, we show that RuR acts by binding an acidic residue pair comprising the "Keystone inhibitor site" under the K2P CAP domain archway above the channel pore. We further establish that Ru360, a dinuclear ruthenium amine not known to affect K2Ps, inhibits RuR-sensitive K2Ps using the same mechanism. Structural knowledge enabled a generalizable design strategy for creating K2P RuR "super-responders" having nanomolar sensitivity. Together, the data define a "finger in the dam" inhibition mechanism acting at a novel K2P inhibitor binding site. These findings highlight the polysite nature of K2P pharmacology and provide a new framework for K2P inhibitor development.
                    Cover page: Polynuclear Ruthenium Amines Inhibit K2P Channels via a “Finger in the Dam” Mechanism
                    • Thesis
                    • Peer Reviewed
                    UC San Francisco Electronic Theses and Dissertations (2008)

                    Potassium (K+) channels are membrane-embedded proteins that selectively pass K+ ions in or out of cells in response to a variety of signals, such as membrane potential changes or binding of ligands. In an excitable cell, such as a neuron or cardiac muscle cell, delayed rectifier voltage-gated K+ channels respond to changes in membrane potential to restore the cell membrane to its resting state after an action potential.

                    In vertebrates, voltage-gated K+ (Kv) channels are tetramers of similar or identical subunits arranged around a central conducting pore. While these channels are primarily gated by membrane potential, their biophysical properties are set by the type of subunits in each tetramer and by interactions with other effector molecules, such as membrane phospholipids, calcium-binding proteins, kinases, and scaffolding proteins. In some cases, discrete intracellular domains control the specific assembly of pore-forming and accessory proteins. However, the molecular mechanisms that direct specific assembly of this wide range of components into a functional K channel complex are incompletely understood.

                    Chapter 2 of this thesis establishes the atomic-resolution structure of one such assembly domain from a Kv7 family channel (Kv7.4). This study suggests the structural basis for specific assembly properties and binding of scaffolding proteins by other members of the Kv7 channel family. Additional studies in Chapter 3 explore implications of the Kv7.4 assembly domain structure for oligomerization in other subtypes. The biochemical and functional effects of Kv7 mutations designed to disrupt or enhance assembly domain oligomerization further support the critical role for this domain in specific assembly of these channels.

                      Cover page: Structure and Assembly Properties of a Conserved C-terminal Domain in Kv7 Channels
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