Publications by authors named "Manuel D Leonetti"

18 Publications

  • Page 1 of 1

Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing.

Cell 2021 Apr 9;184(9):2503-2519.e17. Epub 2021 Apr 9.

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA; Howard Hughes Medical Institute, University of California, San Francisco, CA 94158, USA; Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge 02142, USA. Electronic address:

A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
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http://dx.doi.org/10.1016/j.cell.2021.03.025DOI Listing
April 2021

Split-wrmScarlet and split-sfGFP: tools for faster, easier fluorescent labeling of endogenous proteins in Caenorhabditis elegans.

Genetics 2021 Apr;217(4)

Calico Life Sciences LLC, South San Francisco, CA 94080, USA.

We create and share a new red fluorophore, along with a set of strains, reagents and protocols, to make it faster and easier to label endogenous Caenorhabditis elegans proteins with fluorescent tags. CRISPR-mediated fluorescent labeling of C. elegans proteins is an invaluable tool, but it is much more difficult to insert fluorophore-size DNA segments than it is to make small gene edits. In principle, high-affinity asymmetrically split fluorescent proteins solve this problem in C. elegans: the small fragment can quickly and easily be fused to almost any protein of interest, and can be detected wherever the large fragment is expressed and complemented. However, there is currently only one available strain stably expressing the large fragment of a split fluorescent protein, restricting this solution to a single tissue (the germline) in the highly autofluorescent green channel. No available C. elegans lines express unbound large fragments of split red fluorescent proteins, and even state-of-the-art split red fluorescent proteins are dim compared to the canonical split-sfGFP protein. In this study, we engineer a bright, high-affinity new split red fluorophore, split-wrmScarlet. We generate transgenic C. elegans lines to allow easy single-color labeling in muscle or germline cells and dual-color labeling in somatic cells. We also describe a novel expression strategy for the germline, where traditional expression strategies struggle. We validate these strains by targeting split-wrmScarlet to several genes whose products label distinct organelles, and we provide a protocol for easy, cloning-free CRISPR/Cas9 editing. As the collection of split-FP strains for labeling in different tissues or organelles expands, we will post updates at doi.org/10.5281/zenodo.3993663.
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http://dx.doi.org/10.1093/genetics/iyab014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049552PMC
April 2021

Spatiotemporal dissection of the cell cycle with single-cell proteogenomics.

Nature 2021 02 24;590(7847):649-654. Epub 2021 Feb 24.

Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH - Royal Institute of Technology, Stockholm, Sweden.

The cell cycle, over which cells grow and divide, is a fundamental process of life. Its dysregulation has devastating consequences, including cancer. The cell cycle is driven by precise regulation of proteins in time and space, which creates variability between individual proliferating cells. To our knowledge, no systematic investigations of such cell-to-cell proteomic variability exist. Here we present a comprehensive, spatiotemporal map of human proteomic heterogeneity by integrating proteomics at subcellular resolution with single-cell transcriptomics and precise temporal measurements of individual cells in the cell cycle. We show that around one-fifth of the human proteome displays cell-to-cell variability, identify hundreds of proteins with previously unknown associations with mitosis and the cell cycle, and provide evidence that several of these proteins have oncogenic functions. Our results show that cell cycle progression explains less than half of all cell-to-cell variability, and that most cycling proteins are regulated post-translationally, rather than by transcriptomic cycling. These proteins are disproportionately phosphorylated by kinases that regulate cell fate, whereas non-cycling proteins that vary between cells are more likely to be modified by kinases that regulate metabolism. This spatially resolved proteomic map of the cell cycle is integrated into the Human Protein Atlas and will serve as a resource for accelerating molecular studies of the human cell cycle and cell proliferation.
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http://dx.doi.org/10.1038/s41586-021-03232-9DOI Listing
February 2021

Conserved Functions of Ether Lipids and Sphingolipids in the Early Secretory Pathway.

Curr Biol 2020 10 27;30(19):3775-3787.e7. Epub 2020 Aug 27.

NCCR Chemical Biology, Department of Biochemistry, University of Geneva, 1211 Geneva, Switzerland. Electronic address:

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway.
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http://dx.doi.org/10.1016/j.cub.2020.07.059DOI Listing
October 2020

Mapping the nucleolar proteome reveals a spatiotemporal organization related to intrinsic protein disorder.

Mol Syst Biol 2020 08;16(8):e9469

Science for Life Laboratory, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden.

The nucleolus is essential for ribosome biogenesis and is involved in many other cellular functions. We performed a systematic spatiotemporal dissection of the human nucleolar proteome using confocal microscopy. In total, 1,318 nucleolar proteins were identified; 287 were localized to fibrillar components, and 157 were enriched along the nucleoplasmic border, indicating a potential fourth nucleolar subcompartment: the nucleoli rim. We found 65 nucleolar proteins (36 uncharacterized) to relocate to the chromosomal periphery during mitosis. Interestingly, we observed temporal partitioning into two recruitment phenotypes: early (prometaphase) and late (after metaphase), suggesting phase-specific functions. We further show that the expression of MKI67 is critical for this temporal partitioning. We provide the first proteome-wide analysis of intrinsic protein disorder for the human nucleolus and show that nucleolar proteins in general, and mitotic chromosome proteins in particular, have significantly higher intrinsic disorder level compared to cytosolic proteins. In summary, this study provides a comprehensive and essential resource of spatiotemporal expression data for the nucleolar proteome as part of the Human Protein Atlas.
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http://dx.doi.org/10.15252/msb.20209469DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7397901PMC
August 2020

Revealing architectural order with quantitative label-free imaging and deep learning.

Elife 2020 07 27;9. Epub 2020 Jul 27.

Chan Zuckerberg Biohub, San Francisco, United States.

We report quantitative label-free imaging with phase and polarization (QLIPP) for simultaneous measurement of density, anisotropy, and orientation of structures in unlabeled live cells and tissue slices. We combine QLIPP with deep neural networks to predict fluorescence images of diverse cell and tissue structures. QLIPP images reveal anatomical regions and axon tract orientation in prenatal human brain tissue sections that are not visible using brightfield imaging. We report a variant of U-Net architecture, multi-channel 2.5D U-Net, for computationally efficient prediction of fluorescence images in three dimensions and over large fields of view. Further, we develop data normalization methods for accurate prediction of myelin distribution over large brain regions. We show that experimental defects in labeling the human tissue can be rescued with quantitative label-free imaging and neural network model. We anticipate that the proposed method will enable new studies of architectural order at spatial scales ranging from organelles to tissue.
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http://dx.doi.org/10.7554/eLife.55502DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431134PMC
July 2020

Pervasive functional translation of noncanonical human open reading frames.

Science 2020 03;367(6482):1140-1146

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158, USA.

Ribosome profiling has revealed pervasive but largely uncharacterized translation outside of canonical coding sequences (CDSs). In this work, we exploit a systematic CRISPR-based screening strategy to identify hundreds of noncanonical CDSs that are essential for cellular growth and whose disruption elicits specific, robust transcriptomic and phenotypic changes in human cells. Functional characterization of the encoded microproteins reveals distinct cellular localizations, specific protein binding partners, and hundreds of microproteins that are presented by the human leukocyte antigen system. We find multiple microproteins encoded in upstream open reading frames, which form stable complexes with the main, canonical protein encoded on the same messenger RNA, thereby revealing the use of functional bicistronic operons in mammals. Together, our results point to a family of functional human microproteins that play critical and diverse cellular roles.
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http://dx.doi.org/10.1126/science.aay0262DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289059PMC
March 2020

Compromised function of the ESCRT pathway promotes endolysosomal escape of tau seeds and propagation of tau aggregation.

J Biol Chem 2019 12 2;294(50):18952-18966. Epub 2019 Oct 2.

Institute for Neurodegenerative Diseases, University of California, San Francisco, California 94158

Intercellular propagation of protein aggregation is emerging as a key mechanism in the progression of several neurodegenerative diseases, including Alzheimer's disease and frontotemporal dementia (FTD). However, we lack a systematic understanding of the cellular pathways controlling prion-like propagation of aggregation. To uncover such pathways, here we performed CRISPR interference (CRISPRi) screens in a human cell-based model of propagation of tau aggregation monitored by FRET. Our screens uncovered that knockdown of several components of the endosomal sorting complexes required for transport (ESCRT) machinery, including charged multivesicular body protein 6 (CHMP6), or CHMP2A in combination with CHMP2B (whose gene is linked to familial FTD), promote propagation of tau aggregation. We found that knocking down the genes encoding these proteins also causes damage to endolysosomal membranes, consistent with a role for the ESCRT pathway in endolysosomal membrane repair. Leakiness of the endolysosomal compartment significantly enhanced prion-like propagation of tau aggregation, likely by making tau seeds more available to pools of cytoplasmic tau. Together, these findings suggest that endolysosomal escape is a critical step in tau propagation in neurodegenerative diseases.
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http://dx.doi.org/10.1074/jbc.RA119.009432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6916486PMC
December 2019

Large dataset enables prediction of repair after CRISPR-Cas9 editing in primary T cells.

Nat Biotechnol 2019 09 29;37(9):1034-1037. Epub 2019 Jul 29.

Chan-Zuckerberg Biohub, San Francisco, CA, USA.

Understanding of repair outcomes after Cas9-induced DNA cleavage is still limited, especially in primary human cells. We sequence repair outcomes at 1,656 on-target genomic sites in primary human T cells and use these data to train a machine learning model, which we have called CRISPR Repair Outcome (SPROUT). SPROUT accurately predicts the length, probability and sequence of nucleotide insertions and deletions, and will facilitate design of SpCas9 guide RNAs in therapeutically important primary human cells.
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http://dx.doi.org/10.1038/s41587-019-0203-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388783PMC
September 2019

Epi-illumination SPIM for volumetric imaging with high spatial-temporal resolution.

Nat Methods 2019 06 6;16(6):501-504. Epub 2019 May 6.

Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA.

We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.
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http://dx.doi.org/10.1038/s41592-019-0401-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6557432PMC
June 2019

Reprogramming human T cell function and specificity with non-viral genome targeting.

Nature 2018 07 11;559(7714):405-409. Epub 2018 Jul 11.

HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute, Frederick, MD, USA.

Decades of work have aimed to genetically reprogram T cells for therapeutic purposes using recombinant viral vectors, which do not target transgenes to specific genomic sites. The need for viral vectors has slowed down research and clinical use as their manufacturing and testing is lengthy and expensive. Genome editing brought the promise of specific and efficient insertion of large transgenes into target cells using homology-directed repair. Here we developed a CRISPR-Cas9 genome-targeting system that does not require viral vectors, allowing rapid and efficient insertion of large DNA sequences (greater than one kilobase) at specific sites in the genomes of primary human T cells, while preserving cell viability and function. This permits individual or multiplexed modification of endogenous genes. First, we applied this strategy to correct a pathogenic IL2RA mutation in cells from patients with monogenic autoimmune disease, and demonstrate improved signalling function. Second, we replaced the endogenous T cell receptor (TCR) locus with a new TCR that redirected T cells to a cancer antigen. The resulting TCR-engineered T cells specifically recognized tumour antigens and mounted productive anti-tumour cell responses in vitro and in vivo. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells.
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http://dx.doi.org/10.1038/s41586-018-0326-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6239417PMC
July 2018

Improved split fluorescent proteins for endogenous protein labeling.

Nat Commun 2017 08 29;8(1):370. Epub 2017 Aug 29.

Department of Pharmaceutical Chemistry, University of California in San Francisco, San Francisco, CA, 94143, USA.

Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2 that improves the ratio of complemented signal to the background of FP-expressing cells compared to the commonly used split GFP; as well as a 10-fold brighter red-colored split-sfCherry2. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2 and GFP, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.
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http://dx.doi.org/10.1038/s41467-017-00494-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5575300PMC
August 2017

A scalable strategy for high-throughput GFP tagging of endogenous human proteins.

Proc Natl Acad Sci U S A 2016 06 6;113(25):E3501-8. Epub 2016 Jun 6.

Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94143

A central challenge of the postgenomic era is to comprehensively characterize the cellular role of the ∼20,000 proteins encoded in the human genome. To systematically study protein function in a native cellular background, libraries of human cell lines expressing proteins tagged with a functional sequence at their endogenous loci would be very valuable. Here, using electroporation of Cas9 nuclease/single-guide RNA ribonucleoproteins and taking advantage of a split-GFP system, we describe a scalable method for the robust, scarless, and specific tagging of endogenous human genes with GFP. Our approach requires no molecular cloning and allows a large number of cell lines to be processed in parallel. We demonstrate the scalability of our method by targeting 48 human genes and show that the resulting GFP fluorescence correlates with protein expression levels. We next present how our protocols can be easily adapted for the tagging of a given target with GFP repeats, critically enabling the study of low-abundance proteins. Finally, we show that our GFP tagging approach allows the biochemical isolation of native protein complexes for proteomic studies. Taken together, our results pave the way for the large-scale generation of endogenously tagged human cell lines for the proteome-wide analysis of protein localization and interaction networks in a native cellular context.
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http://dx.doi.org/10.1073/pnas.1606731113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4922190PMC
June 2016

Versatile protein tagging in cells with split fluorescent protein.

Nat Commun 2016 Mar 18;7:11046. Epub 2016 Mar 18.

Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, USA.

In addition to the popular method of fluorescent protein fusion, live cell protein imaging has now seen more and more application of epitope tags. The small size of these tags may reduce functional perturbation and enable signal amplification. To address their background issue, we adapt self-complementing split fluorescent proteins as epitope tags for live cell protein labelling. The two tags, GFP11 and sfCherry11 are derived from the eleventh β-strand of super-folder GFP and sfCherry, respectively. The small size of FP11-tags enables a cost-effective and scalable way to insert them into endogenous genomic loci via CRISPR-mediated homology-directed repair. Tandem arrangement FP11-tags allows proportional enhancement of fluorescence signal in tracking intraflagellar transport particles, or reduction of photobleaching for live microtubule imaging. Finally, we show the utility of tandem GFP11-tag in scaffolding protein oligomerization. These experiments illustrate the versatility of FP11-tag as a labelling tool as well as a multimerization-control tool for both imaging and non-imaging applications.
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http://dx.doi.org/10.1038/ncomms11046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4802074PMC
March 2016

Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel.

Proc Natl Acad Sci U S A 2012 Nov 5;109(47):19274-9. Epub 2012 Nov 5.

Laboratory of Molecular Neurobiology and Biophysics and Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10065, USA.

The activation of eukaryotic SLO K(+) channels by intracellular cues, mediated by a cytoplasmic structure called the gating ring, is central to their physiological roles. SLO3 channels are exclusively expressed in mammalian sperm, where variations of intracellular pH are critical to cellular function. Previous studies primarily focused on the mouse SLO3 orthologue and revealed that, in murine sperm, SLO3 mediates a voltage- and alkalization-activated K(+) current essential to male fertility. Here we investigate the activation of the human SLO3 channel by intracellular pH at the functional and structural level. By using electrophysiology in a heterologous system, we show that human SLO3 opens upon intracellular pH increase and that its expression and functional properties are modulated by LRRC52, a testis-specific accessory subunit. We next present the crystal structure of the human SLO3 gating ring. Comparison with the known structures of the corresponding domain from SLO1, a Ca(2+)-activated homologue, suggests that the SLO3 gating ring structure may represent an open state. Together, these results present insights into the function of a protein expected to be critical for human reproduction and provide a framework to study the mechanism of pH gating in SLO3 channels.
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http://dx.doi.org/10.1073/pnas.1215078109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3511096PMC
November 2012

Open structure of the Ca2+ gating ring in the high-conductance Ca2+-activated K+ channel.

Nature 2011 Dec 4;481(7379):94-7. Epub 2011 Dec 4.

Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, New York 10065, USA.

High-conductance voltage- and Ca(2+)-activated K(+) channels function in many physiological processes that link cell membrane voltage and intracellular Ca(2+) concentration, including neuronal electrical activity, skeletal and smooth muscle contraction, and hair cell tuning. Like other voltage-dependent K(+) channels, Ca(2+)-activated K(+) channels open when the cell membrane depolarizes, but in contrast to other voltage-dependent K(+) channels, they also open when intracellular Ca(2+) concentrations rise. Channel opening by Ca(2+) is made possible by a structure called the gating ring, which is located in the cytoplasm. Recent structural studies have defined the Ca(2+)-free, closed, conformation of the gating ring, but the Ca(2+)-bound, open, conformation is not yet known. Here we present the Ca(2+)-bound conformation of the gating ring. This structure shows how one layer of the gating ring, in response to the binding of Ca(2+), opens like the petals of a flower. The degree to which it opens explains how Ca(2+) binding can open the transmembrane pore. These findings present a molecular basis for Ca(2+) activation of K(+) channels and suggest new possibilities for targeting the gating ring to treat conditions such as asthma and hypertension.
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http://dx.doi.org/10.1038/nature10670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3319005PMC
December 2011

Structure of the human BK channel Ca2+-activation apparatus at 3.0 A resolution.

Science 2010 Jul 27;329(5988):182-6. Epub 2010 May 27.

Laboratory of Molecular Neurobiology and Biophysics, Rockefeller University, Howard Hughes Medical Institute, 1230 York Avenue, New York, NY 10065, USA.

High-conductance voltage- and Ca2+-activated K+ (BK) channels encode negative feedback regulation of membrane voltage and Ca2+ signaling, playing a central role in numerous physiological processes. We determined the x-ray structure of the human BK Ca2+ gating apparatus at a resolution of 3.0 angstroms and deduced its tetrameric assembly by solving a 6 angstrom resolution structure of a Na+-activated homolog. Two tandem C-terminal regulator of K+ conductance (RCK) domains from each of four channel subunits form a 350-kilodalton gating ring at the intracellular membrane surface. A sequence of aspartic amino acids that is known as the Ca2+ bowl, and is located within the second of the tandem RCK domains, creates four Ca2+ binding sites on the outer perimeter of the gating ring at the "assembly interface" between RCK domains. Functionally important mutations cluster near the Ca2+ bowl, near the "flexible interface" between RCK domains, and on the surface of the gating ring that faces the voltage sensors. The structure suggests that the Ca2+ gating ring, in addition to regulating the pore directly, may also modulate the voltage sensor.
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http://dx.doi.org/10.1126/science.1190414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3022345PMC
July 2010