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Open Access Publications from the University of California

Scholarly Works (22 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2022)

Ion channels are a large and diverse family of proteins broadly implicated in human physiology and that of other organisms. The study of the molecular mechanism of ion channels benefits from both “top-down” physiological and “bottom-up” biophysical studies. Here, we use a reductionist biophysical approach, with a focus on the use of cryo-electron microscopy (cryo-EM) to study the structure and function of three putative ion channels. The first, ORF3a is a putative ion channel encoded in the SARS-CoV-2 genome. Here, we determined the first ORF3a structure, and characterized ORF3a activity through electrophysiological recordings and fluorescent imaging of reconstituted ORF3a proteoliposomes. The second, Tweety-homolog family (TTYH) proteins, previously characterized as putative volume-regulated anion channels. Through parallel functional and structural studies, our work determined that TTYHs are not pore-forming subunits of anion channels. Additionally, we discovered a previously unknown “trans-dimeric” conformation of TTYH2. The third putative ion channel is TMEM87A, previously implicated in mechanosensitive conduction of cations. Here we determine the first structure of an TMEM87 protein, and through structural and bioinformatic analysis, identify related proteins forming the basis for a new protein family. Collectively, this work has demonstrated the value of cryo-EM for structural studies of small (<100 kDa) membrane proteins, while laying the foundation for deeper mechanistic understanding of each of these different proteins.

    Cover page: Structural and functional studies of putative ion channels
    • Peer Reviewed
    UC Berkeley Previously Published Works (2021)
    The SARS-CoV-2 3a protein is a putative ion channel implicated in virus life cycle and pathogenesis. We recently expressed, purified, and reconstituted 3a into lipid nanodiscs to solve its structure by cryo-EM to 2.1Å resolution. In this chapter, we describe methods we developed in order to facilitate the study of this protein in other laboratories. We emphasize factors that enabled rapid progression from gene sequence to reconstituted protein (3 weeks in the case of 3a) and provide general observations and tips for adapting these protocols to other membrane proteins of interest.
      Cover page: SARS-CoV-2 3a expression, purification, and reconstitution into lipid nanodiscs
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2020)
      TASK2 (also known as KCNK5) channels generate pH-gated leak-type K+ currents to control cellular electrical excitability1-3. TASK2 is involved in the regulation of breathing by chemosensory neurons of the retrotrapezoid nucleus in the brainstem4-6 and pH homeostasis by kidney proximal tubule cells7,8. These roles depend on channel activation by intracellular and extracellular alkalization3,8,9, but the mechanistic basis for TASK2 gating by pH is unknown. Here we present cryo-electron microscopy structures of Mus musculus TASK2 in lipid nanodiscs in open and closed conformations. We identify two gates, distinct from previously observed K+ channel gates, controlled by stimuli on either side of the membrane. Intracellular gating involves lysine protonation on inner helices and the formation of a protein seal between the cytoplasm and the channel. Extracellular gating involves arginine protonation on the channel surface and correlated conformational changes that displace the K+-selectivity filter to render it nonconductive. These results explain how internal and external protons control intracellular and selectivity filter gates to modulate TASK2 activity.
        Cover page: Structural basis for pH gating of the two-pore domain K+ channel TASK2
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2009)
        Nuclear pore complexes (NPCs) facilitate all nucleocytoplasmic transport. These massive protein assemblies are modular, with a stable structural scaffold supporting more dynamically attached components. The scaffold is made from multiple copies of the heptameric Y complex and the heteromeric Nic96 complex. We previously showed that members of these core subcomplexes specifically share an ACE1 fold with Sec31 of the COPII vesicle coat, and we proposed a lattice model for the NPC based on this commonality. Here we present the crystal structure of the heterotrimeric 134-kDa complex of Nup84-Nup145C-Sec13 of the Y complex. The heterotypic ACE1 interaction of Nup84 and Nup145C is analogous to the homotypic ACE1 interaction of Sec31 that forms COPII lattice edge elements and is inconsistent with the alternative 'fence-like' NPC model. We construct a molecular model of the Y complex and compare the architectural principles of COPII and NPC lattices.
          • Thesis
          • Peer Reviewed
          UC Berkeley Electronic Theses and Dissertations (2023)

          Cation-chloride-cotransporters (CCCs) play critical roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. Disruption of CCCs has been implicated in pathophysiology including epilepsy, hearing loss, and genetic disorders. In this study, we present the structure of a CCC, the Mus musculus K+-Cl- cotransporter (KCC) KCC4, determined by cryo-EM. The structure, captured in an inward-open conformation, reveals the architecture of KCCs and offers mechanistic insights into the function and regulation of this important transporter family. Our findings provide a structural explanation for the varied substrate specificity and ion transport ratio among CCCs, identify binding sites for substrate K+ and Cl- ions, and demonstrate the importance of key coordinating residues for transporter activity. These insights have significant implications for developing novel drugs and treatments targeting CCCs.

            Cover page: Structural and Functional Studies of the K+/Cl- Cotransporter 4
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2014)
            Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K(+) channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K(+) (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the "force-from-lipid" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K(+) channels.
              Cover page: Mechanosensitivity is mediated directly by the lipid membrane in TRAAK and TREK1 K+ channels
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2021)
              Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, in the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.
                Cover page: Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2014)
                Activation of mechanosensitive ion channels by physical force underlies many physiological processes including the sensation of touch, hearing and pain. TRAAK (also known as KCNK4) ion channels are neuronally expressed members of the two-pore domain K(+) (K2P) channel family and are mechanosensitive. They are involved in controlling mechanical and temperature nociception in mice. Mechanosensitivity of TRAAK is mediated directly through the lipid bilayer--it is a membrane-tension-gated channel. However, the molecular mechanism of TRAAK channel gating and mechanosensitivity is unknown. Here we present crystal structures of TRAAK in conductive and non-conductive conformations defined by the presence of permeant ions along the conduction pathway. In the non-conductive state, a lipid acyl chain accesses the channel cavity through a 5 Å-wide lateral opening in the membrane inner leaflet and physically blocks ion passage. In the conductive state, rotation of a transmembrane helix (TM4) about a central hinge seals the intramembrane opening, preventing lipid block of the cavity and permitting ion entry. Additional rotation of a membrane interacting TM2-TM3 segment, unique to mechanosensitive K2Ps, against TM4 may further stabilize the conductive conformation. Comparison of the structures reveals a biophysical explanation for TRAAK mechanosensitivity--an expansion in cross-sectional area up to 2.7 nm(2) in the conductive state is expected to create a membrane-tension-dependent energy difference between conformations that promotes force activation. Our results show how tension of the lipid bilayer can be harnessed to control gating and mechanosensitivity of a eukaryotic ion channel.
                  Cover page: Physical mechanism for gating and mechanosensitivity of the human TRAAK K+ channel
                  • Article
                  • Peer Reviewed
                  UC Berkeley Previously Published Works (2021)
                  TRAAK is a mechanosensitive two-pore domain K+ (K2P) channel localized to nodes of Ranvier in myelinated neurons. TRAAK deletion in mice results in mechanical and thermal allodynia, and gain-of-function mutations cause the human neurodevelopmental disorder FHEIG. TRAAK displays basal and stimulus-gated activities typical of K2Ps, but the mechanistic and structural differences between these modes are unknown. Here, we demonstrate that basal and mechanically gated openings are distinguished by their conductance, kinetics, and structure. Basal openings are low conductance, short duration, and due to a conductive channel conformation with the interior cavity exposed to the surrounding membrane. Mechanically gated openings are high conductance, long duration, and due to a channel conformation in which the interior cavity is sealed to the surrounding membrane. Our results explain how dual modes of activity are produced by a single ion channel and provide a basis for the development of state-selective pharmacology with the potential to treat disease.
                    Cover page: Physical basis for distinct basal and mechanically gated activity of the human K+ channel TRAAK
                    • Article
                    • Peer Reviewed
                    UC Berkeley Previously Published Works (2022)
                    TWIK1 (K2P1.1, KCNK1) is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a previously undescribed gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and illustrate how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.
                      Cover page: Structural Basis for pH-gating of the K+ channel TWIK1 at the selectivity filter
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