Zinc activation of OTOP proton channels identifies structural elements of the gating apparatus

  1. Bochuan Teng
  2. Joshua P Kaplan
  3. Ziyu Liang
  4. Kevin Saejin Chyung
  5. Marcel P Goldschen-Ohm
  6. Emily R Liman  Is a corresponding author
  1. Section of Neurobiology, Department of Biological Sciences, University of Southern California, United States
  2. Program in Neuroscience, University of Southern California, United States
  3. University of Texas at Austin, Department of Neuroscience, United States
10 figures, 1 table and 2 additional files

Figures

Zn2+ blocks and potentiates mOTOP3 currents.

(A) Representative traces show Zn2+ both inhibits and potentiates mOTOP3 currents. Proton currents were elicited in HEK293 cells expressing each of the three mOTOP channels in response to lowering …

Pre-exposure to Zn2+ potentiates mOTOP3 currents in a dose- and time-dependent manner.

(A) Solution exchange protocol designed to measure effects of Zn2+ on gating of OTOP currents without confounds due to its inhibitory effects. Vm was held at –80 mV. In this example, currents were …

Time dependence of the recovery of mOTOP3 currents from Zn2+ pre-potentiation.

(A) Solution exchange protocol designed to measure the recovery of mOTOP3 currents following exposure to Zn2+. In this example, the cell expressing mOTOP3 was first exposed to 1 mM Zn2+ for 16 s …

mOTOP1 is potentiated by Zn2+ when activated by a mild acid stimulus.

(A) Proton currents recorded from HEK293 cells expressing each of the three mOTOP channels as indicated, in response to pH 5.5 with (red) or without (black) Zn2+ pre-exposure. Vm was held at –80 mV. …

Figure 5 with 1 supplement
Divalent transition metal ions also potentiate mOTOP3.

(A) Proton currents in response to a pH 5.5 stimulus following exposure (1 mM, 16 s) to various d-block transition metals recorded from HEK293 cells expressing wildtype mOTOP3. Vm was held at –80 …

Figure 5—figure supplement 1
Divalent transition metals also inhibit mOTOP3.

(A) Representative traces show Ni2+, Cd2+, and Cu2+ all inhibit mOTOP3 currents. Proton currents were elicited in HEK293 cells expressing wildtype mOTOP3 in response to lowering the extracellular pH …

Figure 6 with 1 supplement
The tm 11–12 linker is both necessary and sufficient for Zn2+ potentiation.

(A, B) Proton currents in response to a pH 5.5 stimulus with (red) or without (black) Zn2+ pre-exposure (1 mM, 16 s) recorded from HEK293 cells expressing either wildtype (WT) OTOP channels or …

Figure 6—figure supplement 1
O3/O2(L11-12) chimera is completely insensitive to potentiation by Zn2+.

(A) Representative traces show O3/O2(L11-12) currents in the absence of Zn2+ (black) and with pre-exposure times as indicated. (B,C) Average data for fold potentiation (B) and time to peak (C) of …

Inhibition of mOTOP3 by Zn2+ is retained in chimeric channels.

(A) Zn2+ sensitivity of chimeric mOTOP3-mOTOP2 channels as measured with a pre-exposure protocol (left panel in each) or by adding 1 mM Zn2+ to the pH 5.5 stimulus (blocking protocol; right panel in …

Figure 8 with 1 supplement
H531 in mOTOP3 L11–12 is essential for Zn2+ potentiation.

(A) Sequence alignment of three mOTOP channels. The residues that were exchanged between mOTOP2 and mOTOP3 in the L11–12 chimeras is indicated with a blue box. Residues that differed between the two …

Figure 8—figure supplement 1
Introduction of histidine into mOTOP1 partially confers sensitivity to potentiation by Zn2+.

(A,B) Representative traces of OTOP2 and OTOP2_R517H and OTOP1 and OTOP1_R554H currents in response to pH 5.5 with (red) and without (black) pre-exposure to Zn2+ (1 mM, 16 s). Vm was held at –80 mV. …

H234 and E238 in transmembrane domain 5 contribute to Zn2+ potentiation of mOTOP3.

(A) Sequence alignment of the three mOTOP channels. Alpha helices shown above the sequence are based on the AlphaFold prediction of the structure of mOTOP3. Red arrows indicate residues neutralized …

Figure 10 with 3 supplements
mOTOP3 kinetic model for Zn2+ potentiation and block.

(A) Kinetic model for activation of mOTOP3 by H+ and Zn2+. The channel moves from a closed state (C) upon binding H+ or Zn2+ to an open state (Zn-O, H–O) in which it permeates protons. The doubly …

Figure 10—figure supplement 1
Simulation time course for individual gating and binding domains in response to a pulse of Zn2+ during an acid stimulus.

Simulated responses to the stimulus protocol shown in Figure 1C for the model in Figure 9A. The top plot shows simulated current responses for various concentrations of Zn2+ (same as depicted in the …

Figure 10—figure supplement 2
Simulated decay of current potentiation largely reflects Zn2+ unbinding from the activating site.

Comparison of the simulated time courses for the model in Figure 9 to be conducting (i.e. pore is both open and unblocked; solid lines) and/or the Zn2+ activating site to be occupied (dashed lines) …

Figure 10—figure supplement 3
Simulation time course for individual gating and binding domains in response to an acid stimulus following pre-exposure to Zn2+.

Simulated responses for the model in Figure 9A to the stimulus protocol shown in Figure 2 for pre-incubation with 1 mM Zn2+. The top plot shows simulated current responses for various pre-incubation …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Gene (Mus musculus)Otop1, Otop2, and Otop3Tu et al., 2018. PMID:29371428
Cell line (Homo sapiens)HEK293ATCCCRL-1573
Cell line (Homo sapiens)PAC-KO HEK293 cellsYang et al., 2019. PMID:31023925
Recombinant DNA reagentOtop1, Otop2 and Otop3 in pcDNA3.1Tu et al., 2018. PMID:29371428
Recombinant DNA reagentOtop1, Otop2 and Otop3 – GFPSaotome et al., 2019. PMID:31160780
Recombinant DNA reagentmO2_O3 loop swap mutationsThis papercDNAs encode chimeric channels (see Materials and methods and Figure 6—figure supplement 1). Available upon request
Recombinant DNA reagentmO3_O2 loop swap mutationsThis papercDNAs encode chimeric channels (see Materials and methods and Figure 6—figure supplement 1). Available upon request
Recombinant DNA reagentpHluorin in pcDNA3Miesenbock, et al., 1998. PMID:9671304
Chemical compound, drugCHESSigmaC2885
Chemical compound, drugPIPESSigmaP6757
Chemical compound, drugHomopiperazine-1,4-bis(2-ethanesulfonic acid)Sigma53588
Software, algorithmGraphPad Prism 8 and 9GraphPadRRID:SCR_002798
Software, algorithmpClamp and clampfitMolecular DevicesRRID:SCR_011323
Software, algorithmOriginOriginLab corporationRRID:SCR_002815
Software, algorithmCorelDrawCorelRRID:SCR_014235
Software, algorithmSimplePCIHCImagehttps://hcimage.com/simple-pci-legacy/

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