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Detecting organelle-specific activity of potassium channels with a DNA nanodevice

Nature Biotechnology (2023)Cite this article

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Abstract

Cell surface potassium ion (K+) channels regulate nutrient transport, cell migration and intercellular communication by controlling K+ permeability and are thought to be active only at the plasma membrane. Although these channels transit the trans-Golgi network, early and recycling endosomes, whether they are active in these organelles is unknown. Here we describe a pH-correctable, ratiometric reporter for K+ called pHlicKer, use it to probe the compartment-specific activity of a prototypical voltage-gated K+ channel, Kv11.1, and show that this cell surface channel is active in organelles. Lumenal K+ in organelles increased in cells expressing wild-type Kv11.1 channels but not after treatment with current blockers. Mutant Kv11.1 channels, with impaired transport function, failed to increase K+ levels in recycling endosomes, an effect rescued by pharmacological correction. By providing a way to map the organelle-specific activity of K+ channels, pHlicKer technology could help identify new organellar K+ channels or channel modulators with nuanced functions.

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Fig. 1: pHlicKer is a selective, combination reporter for pH and K+.
Fig. 2: Intracellular calibration of pHlicKer.
Fig. 3: pH and K+ maps in endocytic organelles.
Fig. 4: pH and K+ maps in RE of WT and TWIK2−/− BMDM cells.
Fig. 5: pHlicKer reveals Kv11.1 channel activity in TGN.
Fig. 6: pHlicKer probes channel activity, trafficking defects and rescue of trafficking.

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Data availability

All data related to the study are included in the article and supporting information. The raw data supporting Figs. 1–6, extended data figures and supplementary figures, respectively, are available for public access at figshare54,55,56,57,58,59,60,61(https://figshare.com/articles/dataset/Figure_1/23713359, https://figshare.com/articles/dataset/Figure_3/23713776, https://figshare.com/articles/dataset/Figure_4/23713821, https://figshare.com/articles/dataset/Figure_5/23713833, https://figshare.com/articles/dataset/Figure_6/23713851, https://figshare.com/articles/dataset/Extended_data_figures/23713899 and https://figshare.com/articles/dataset/Supplementary_Figures/23713947). Source data are provided with this paper.

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Acknowledgements

We thank E. Perozo and A. Lin Chun for valuable comments on the paper. We thank the integrated light microscopy facilities at the University of Chicago. Y.K. acknowledges funding from NIH grants DP1GM149751, 1R01NS112139-01A1, R21HL161825-01A1 (Y.K. and B.P.D), 1R01GM147197-01 and FA9550-19-0003 from the AFOSR, HFSP grant no. RGP0032/2022, and the Ono Pharma Foundation.

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Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Palapuravan Anees, Anand Saminathan & Yamuna Krishnan

  2. Grossman Center for Quantitative Biology and Human Behavior, The University of Chicago, Chicago, IL, USA

    Palapuravan Anees, Anand Saminathan & Yamuna Krishnan

  3. Institute of Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Palapuravan Anees & Yamuna Krishnan

  4. Department of Physiology, University of Kentucky College of Medicine, Lexington, KY, USA

    Ezekiel R. Rozmus & Brian P. Delisle

  5. Department of Pharmacology and Regenerative Medicine, The University of Illinois College of Medicine, Chicago, IL, USA

    Anke Di & Asrar B. Malik

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Contributions

P.A., A.S. and Y.K. designed the sensor and experiments related to its validation. P.A. designed and synthesized the TAC-Rh dye. P.A., B.P.D. and Y.K. designed experiments related to Kv11.1 channels. E.R.R. performed electrophysiology. P.A. performed all other experiments. P.A. and E.R.R. analyzed data. A.D. and A.B.M. provided TWIK2 KO BMDM cells. P.A., B.P.D. and Y.K. wrote the paper. All authors provided input on the paper.

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Correspondence to Brian P. Delisle or Yamuna Krishnan.

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Y.K. is co-founder of Esya Inc. and MacroLogic Inc., which use DNA nanodevices for diagnostics and therapeutics, respectively. The other authors declare no competing interests.

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Nature Biotechnology thanks Haoxing Xu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 TAC-Rh fluorescence response as a function of both pH and K+.

a, Working principle of K+ sensing by TAC-Rh. b, Excitation (black) and emission (green) spectra of TAC-Rh. Emission intensity increased with increasing K+ concentration at pH = 7.0. c, Normalized O/R ratio of TAC-Rh/Alexa Fluor 647 with increasing [K+] at pH 7.0 and 6.0. Error bar represents mean + s.e.m. of three independent experiments. d, Fluorescence emission spectra of pHlicKerRE corresponding to TAC-Rh (green) and Alexa Fluor 647 (magenta) with increasing [K+] at pH = 7.0. e, Normalized O/R ratio of TAC-Rh/Alexa Fluor 647 with increasing [K+] at pH 7.0 Error bar represents mean + s.e.m. of three independent experiments.

Source data

Extended Data Fig. 2 Targeting modules (T) in organelle-specific pHlicKer variants.

a, pHlicKerRE localizes in recycling endosomes (REs) by transferrin receptor (TfR)-mediated endocytosis. T is an aptamer that binds TfR. b, pHlicKerTGN is retrogradely trafficked by an scFv-furin chimera to the trans Golgi network (TGN). T is a d(AT)4 sequence (cyan) in pHlicKer that sequence-specifically binds a single chain variable fragment (scFv) fused to the extracellular domain of furin. c, pHlicKerEE localizes in early endosomes (EEs) by scavenger receptor-mediated endocytosis. T is a duplex DNA domain, which is an excellent ligand for scavenger receptors. d–f, Three way (3W) junctions 3WRE, 3WTGN and 3WEE are made from the same sequences as pHlicKerRE, pHlicKerTGN and pHlicKerEE and lack the Alexa488 and TAC-Rh fluorophores for colocalization studies with fluorescent markers of the RE, TGN and EE. g, pHlicKerBiotin incorporates a biotin (grey pentagons) as indicated for immobilization on streptavidin coated beads.

Extended Data Fig. 3 Calibration of pHlicKer on beads.

a, Representative images of pHlicKerBiotin on beads clamped at indicated pH and K+ levels, imaged in the donor channel (D), acceptor channel (A), TMR (O), and Alexa Fluor 647 (R) channels. D/A and O/R are the corresponding pixel-wise pseudocolor images. (n = 100 beads). Scale bars, 5 μm. b–d, 2-IM profiles of beads clamped at indicated pH and [K+]. Experiments were performed in triplicate (n = 70–200 beads).

Source data

Extended Data Fig. 4 Targetability of 3WEE and 3WRE.

a, Uptake by HEK 293T cells expressing human scavenger receptor (hMSR1). Representative images of the uptake of Alexa 647 labelled 3WEE in untransfected and hMSR1 transfected HEK 293T cells. Scale bars, 5 μm. b, Normalized whole cell intensities for (a). Data represent mean ± s.e.m (n = 20–22 cells). hMSR1 expressing HEK 293T cells showed effective internalization of pHlicKerEE, revealing uptake is by scavenger receptors. c, Competition experiments with 3WRE and excess unlabelled transferrin (Tf) in HEK 293T cells. Representative fluorescence images of HEK 293T cells pulsed with 3WRE (500 nM) in the presence (+Tf, 20 μM) and absence (-Tf) of Tf. Cells are imaged in the Alexa 647 channel. AF, autofluorescence. Scale bars, 5 μm. d, Normalized intensities for (c). Data represent mean ± s.e.m (n = 18–22 cells). pHlicKerRE internalization by HEK 293T cell is competed out by excess Tf, revealing that uptake is mediated by transferrin receptor-mediated endocytosis.

Source data

Extended Data Fig. 5 Kv11.1 channel activity under transmembrane ion gradients equivalent to plasma membrane and recycling endosome.

a, b, Representative families of currents measured from cells stably expressing WT-Kv11.1 (black) or G601S- Kv11.1 (magenta) channel proteins using the voltage protocol shown in inset. a, Traces recorded using the standard extracellular saline or b, the modified extracellular saline to mimic recycling endosomes. Individual I-V relations were generated for each cell in each condition by plotting the peak current recorded during the test-pulse as a function of the pre-pulse (Fig. 6i,j). The individual I-V relations were described using a Boltzmann equation to calculate the IMAX (Fig. 6i–j), c, midpoint potential for IKv11.1 activation (V1/2), or d, the slope factor for IKv11.1 current activation (k). (Extended Data Fig. 5c: n = 9 for WT and n = 6 for G601S; Extended Data Fig. 5d: n = 4 for WT and n = 4 for G601S). Embedded box plots indicate the 25th–75th percentile. Boxes and bars represent the s.e.m. and standard deviation, respectively. *p=0.018; **p=0.001 (one-way ANOVA with Tukey post hoc test).

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Anees, P., Saminathan, A., Rozmus, E.R. et al. Detecting organelle-specific activity of potassium channels with a DNA nanodevice. Nat Biotechnol (2023). https://doi.org/10.1038/s41587-023-01928-z

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