(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 …
Source data for Figure 1.
(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 …
Source data for Figure 2.
(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 …
Source data for Figure 3.
(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. …
Source data for Figure 4.
(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 …
Source data for Figure 5.
(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 …
Source data for Figure 5—figure supplement 1.
(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 …
Source data for Figure 6.
(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 …
Source data for Figure 6—figure supplement 1.
(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 …
Source data for Figure 7.
(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 …
Source data for Figure 8.
(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. …
Source data for Figure 8—figure supplement 1.
(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 …
Source data for Figure 9.
(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 …
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) …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Mus musculus) | Otop1, Otop2, and Otop3 | Tu et al., 2018. PMID:29371428 | ||
Cell line (Homo sapiens) | HEK293 | ATCC | CRL-1573 | |
Cell line (Homo sapiens) | PAC-KO HEK293 cells | Yang et al., 2019. PMID:31023925 | ||
Recombinant DNA reagent | Otop1, Otop2 and Otop3 in pcDNA3.1 | Tu et al., 2018. PMID:29371428 | ||
Recombinant DNA reagent | Otop1, Otop2 and Otop3 – GFP | Saotome et al., 2019. PMID:31160780 | ||
Recombinant DNA reagent | mO2_O3 loop swap mutations | This paper | cDNAs encode chimeric channels (see Materials and methods and Figure 6—figure supplement 1). Available upon request | |
Recombinant DNA reagent | mO3_O2 loop swap mutations | This paper | cDNAs encode chimeric channels (see Materials and methods and Figure 6—figure supplement 1). Available upon request | |
Recombinant DNA reagent | pHluorin in pcDNA3 | Miesenbock, et al., 1998. PMID:9671304 | ||
Chemical compound, drug | CHES | Sigma | C2885 | |
Chemical compound, drug | PIPES | Sigma | P6757 | |
Chemical compound, drug | Homopiperazine-1,4-bis(2-ethanesulfonic acid) | Sigma | 53588 | |
Software, algorithm | GraphPad Prism 8 and 9 | GraphPad | RRID:SCR_002798 | |
Software, algorithm | pClamp and clampfit | Molecular Devices | RRID:SCR_011323 | |
Software, algorithm | Origin | OriginLab corporation | RRID:SCR_002815 | |
Software, algorithm | CorelDraw | Corel | RRID:SCR_014235 | |
Software, algorithm | SimplePCI | HCImage | https://hcimage.com/simple-pci-legacy/ |
MATLAB scripts for running the kinetic model simulations shown in Figure 10.