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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2021)

Potassium two-pore domain (K2P) family ion channels are increasingly appreciated as important electrogenic sensors that are used by diverse cell types including neurons, cardiomyocytes, and epithelial cells. The activity of these channels can be activated up to one-hundred fold by diverse physical and chemical stimuli including pH, intracellular calcium, temperature, phospholipids and fatty acids, volatile anesthetics, and membrane tension. In addition to this “stimulus gated” activity, channels in this family also have a basal “leak gated” activity that drives the cellular resting membrane close to the potassium ion reversal potential in the absence of their respective stimuli. While the structural basis of closed states and certain “stimulus gated” open states have been previously determined, the structural basis of a “leak gated” open state in K2P channels is unknown. Here, we show the physical basis for basal “leak gated” activity of the mechanosensitive K2P family ion channel TRAAK, and propose a gating model that includes both “leak gated” and “stimulus gated” openings. Next, we identify the structural basis of intracellular and extracellular alkaline pH “stimulus gated” activation in the acid-sensitive K2P family ion channel TASK2. We observe structural similarities between the “leak gated” structure of TRAAK and other K2P channel structures, including TASK2. This observation supports a general mechanism for “leak gated” activity in K2P family ion channels.

    Cover page: Mechanistic basis of gating in potassium two-pore domain ion channels
    • 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 (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 (2024)
        TRAAK, TREK-1, and TREK-2 are mechanosensitive two-pore domain K+ (K2P) channels that contribute to action potential propagation, sensory transduction, and muscle contraction. While structural and functional studies have led to models that explain their mechanosensitivity, we lack a quantitative understanding of channel activation by membrane tension. Here, we define the tension response of mechanosensitive K2Ps using patch-clamp recording and imaging. All are low-threshold mechanosensitive channels (T10%/50% 0.6-2.7 / 4.4-6.4 mN/m) with distinct response profiles. TRAAK is most sensitive, TREK-1 intermediate, and TREK-2 least sensitive. TRAAK and TREK-1 are activated broadly over a range encompassing nearly all physiologically relevant tensions. TREK-2, in contrast, activates over a narrower range like mechanosensitive channels Piezo1, MscS, and MscL. We further show that low-frequency, low-intensity focused ultrasound increases membrane tension to activate TRAAK and MscS. This work provides insight into tension gating of mechanosensitive K2Ps relevant to understanding their physiological roles and potential applications for ultrasonic neuromodulation.
          Cover page: Tension activation of mechanosensitive two-pore domain K+ channels TRAAK, TREK-1, and TREK-2.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2018)
          Mechanical force sensation is fundamental to a wide breadth of biology from the classic senses of touch, pain, hearing, and balance to less conspicuous sensations of proprioception, blood pressure, and osmolarity and basic aspects of cell growth, differentiation, and development. These diverse and essential systems use force-gated (or mechanosensitive) ion channels that convert mechanical stimuli into cellular electrical signals. TRAAK, TREK1, and TREK2 are K+-selective ion channels of the two-pore domain K+ (K2P) family that are mechanosensitive: they are gated open by increasing membrane tension. TRAAK and TREK channels are thought to play roles in somatosensory and other mechanosensory processes in neuronal and non-neuronal tissues. Here, we present protocols for three assays to study mechanical activation of these channels in cell membranes: (1) cell swelling, (2) cell poking, and (3) patched membrane stretching. Patched membrane stretching is also applicable to the study of mechanosensitive K2P channel activity in a cell-free system and a procedure for proteoliposome reconstitution and patching is also presented. These approaches are also readily applicable to the study of other mechanosensitive ion channels.
            Cover page: Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2021)
            Ultrasound modulates the electrical activity of excitable cells and offers advantages over other neuromodulatory techniques; for example, it can be noninvasively transmitted through the skull and focused to deep brain regions. However, the fundamental cellular, molecular, and mechanistic bases of ultrasonic neuromodulation are largely unknown. Here, we demonstrate ultrasound activation of the mechanosensitive K+ channel TRAAK with submillisecond kinetics to an extent comparable to canonical mechanical activation. Single-channel recordings reveal a common basis for ultrasonic and mechanical activation with stimulus-graded destabilization of long-duration closures and promotion of full conductance openings. Ultrasonic energy is transduced to TRAAK through the membrane in the absence of other cellular components, likely increasing membrane tension to promote channel opening. We further demonstrate ultrasonic modulation of neuronally expressed TRAAK. These results suggest mechanosensitive channels underlie physiological responses to ultrasound and could serve as sonogenetic actuators for acoustic neuromodulation of genetically targeted cells.
              Cover page: Ultrasound activates mechanosensitive TRAAK K+ channels through the lipid membrane
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