Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit Counting

Durisic, Nela; Godin, Antoine G.; Wever, Claudia M.; Heyes, Colin D.; Lakadamyali, Melike; Dent, Joseph A.

doi:10.1523/jneurosci.2050-12.2012

Cited by 119 publications

(119 citation statements)

References 37 publications

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“…S1 and S2). This probability value of mEGFP being fluorescent is similar to what was observed previously for mEGFP fused to a number of other membrane proteins (28)(29)(30)(31)(32)(33). Binomial predictions based on four subunits also accounted well for the other hBest family members ( Fig.…”

Section: Resultssupporting

confidence: 87%

“…Although earlier biochemical studies concluded that the Best1 channel is an oligomer, the estimate of the number of subunits ranged from 2 to 5 (22,27). To resolve this question, we used the single-molecule subunitcounting method (28)(29)(30)(31)(32)(33) to determine the number of subunits by counting the number of fluorescence-bleaching steps of monomeric enhanced green fluorescent protein (mEGFP)-tagged hBest1 in Xenopus oocytes [EGFP-tagged hBest1 was previously shown to be functional (34)]. The main advantages of this method are that: (i) the counting uses Total Internal Reflection Fluorescence Microscopy (TIRFM) to focus exclusively on functional channels in their native physiological environment, the plasma membrane of live cells, and (ii) because the level of expression can be easily controlled in oocytes, it is possible to obtain low enough densities so that each channel appears as an individual fluorescent spot that can be readily resolved from its neighbors.…”

Section: Resultsmentioning

confidence: 99%

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Stoichiometry and specific assembly of Best ion channels

Bharill

Palty

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Human Bestrophin 1 (hBest1) is a calcium-activated chloride channel that regulates neuronal excitability, synaptic activity, and retinal homeostasis. Mutations in hBest1 cause the autosomal-dominant Best macular dystrophy (BMD). Because hBest1 mutations cause BMD, but a knockout does not, we wondered if hBest1 mutants exert a dominant negative effect through interaction with other calciumactivated chloride channels, such as hBest2, 3, or 4, or transmembrane member 16A (TMEM16A), a member of another channel family. The subunit architecture of Best channels is debated, and their ability to form heteromeric channel assemblies is unclear. Using single-molecule subunit analysis, we find that each of hBest1, 2, 3, and 4 forms a homotetrameric channel. Despite considerable conservation among hBests, hBest1 has little or no interaction with other hBests or mTMEM16A. We identify the domain responsible for assembly specificity. This domain also plays a role in channel function. Our results indicate that Best channels preferentially self-assemble into homotetramers.

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Section: Resultssupporting

confidence: 87%

Section: Resultsmentioning

confidence: 99%

Stoichiometry and specific assembly of Best ion channels

Bharill

Palty

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…As shown in previous studies, blinking of PAFPs can be corrected by combining fluorescent bursts from a single molecule based on a cutoff time determined from the measured characteristic darktime distribution (25,27). In addition, it has been previously reported that the detectable fraction of PAFPs can be substantially smaller than 100% (29)(30)(31). Both the blink characteristics and the detectable fraction are sensitive to the experimental conditions.…”

Section: Calibration Of Single-molecule Superresolution Microscopy Elmentioning

confidence: 85%

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Puchner

Walter

Kasper

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

172

213

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Cells tightly regulate trafficking of intracellular organelles, but a deeper understanding of this process is technically limited by our inability to track the molecular composition of individual organelles below the diffraction limit in size. Here we develop a technique for intracellularly calibrated superresolution microscopy that can measure the size of individual organelles as well as accurately count absolute numbers of molecules, by correcting for undercounting owing to immature fluorescent proteins and overcounting owing to fluorophore blinking. Using this technique, we characterized the size of individual vesicles in the yeast endocytic pathway and the number of accessible phosphatidylinositol 3-phosphate binding sites they contain. This analysis reveals a characteristic vesicle maturation trajectory of composition and size with both stochastic and regulated components. The trajectory displays some cell-to-cell variability, with smaller variation between organelles within the same cell. This approach also reveals mechanistic information on the order of events in this trajectory: Colocalization analysis with known markers of different vesicle maturation stages shows that phosphatidylinositol 3-phosphate production precedes fusion into larger endosomes. This single-organelle analysis can potentially be applied to a range of small organelles to shed light on their precise composition/structure relationships, the dynamics of their regulation, and the noise in these processes.S ingle-cell analysis of protein abundance has revealed cell-tocell variation in the form of phenotypic (extrinsic) heterogeneity and intrinsic variation (1). Dynamic processes such as the cell cycle, endocytosis, and meiosis are regulated but also influenced by stochastic events involving small numbers of molecules (e.g., DNA transcription) (2, 3). Similarly, the size and molecular composition of small subcellular organelles are dynamically regulated but still subject to stochastic noise. Studying the biomolecular composition and size of organelles will illuminate the regulation and noise in these dynamically stable systems. A major challenge for such exploration, however, is the development of a combined approach for both resolving small structures below the optical diffraction limit and simultaneously counting biomolecules over several orders of magnitude (Fig. 1A).In the endocytic pathway, both vesicle morphology and biomolecular composition are dynamically regulated along a maturation path. Formation of vesicles, tethering, fusion, and maturation to endosomes are controlled by over 60 proteins in concert with conversion of phosphoinositides (PIs) (4, 5). Phosphatases and PI-kinases produce phosphatidylinositol 3-phosphate (PI3P) from plasma membrane phospholipids (6). PI3P is required for endocytosis (7) and membrane transport to early and late endosomes (8, 9). PI3P regulates the recruitment of proteins (10), such as Rab GTPases, which coordinate many aspects of vesicle identity (11, 12) and maturation to endosomes, including tethe...

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“…Ϫ26Ј -␣1 19Ј coupling energy implies the presence of at least one ␣1-␣1 interface. The stoichiometry of ␣1␤ GlyRs is in dispute, with some studies finding evidence for a 3␣:2␤ ratio (42,43) and others for a 2␣:3␤ ratio (26,27). If ␣ and ␤ subunits alternate in the pentamer, as proposed by both the Grudzinska and Yang studies (26,27), the presence of at least one ␣1-␣1 interface necessitates a 3␣:2␤ ratio.…”

Section: Discussionmentioning

confidence: 99%

Correlating Structural and Energetic Changes in Glycine Receptor Activation

Scott

Lynch

Keramidas

2015

Journal of Biological Chemistry

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Background: Interactions between GlnϪ26Ј (in M1 domain), Arg 19Ј (in M2 domain), and Lys 24Ј (in M2-M3 linker) may reveal molecular mechanisms of glycine receptor activation. Results: ␣1QϪ26ЈE -containing receptors have longer active periods and lower conductances. Conclusion:The energy for activation is distributed broadly at the transduction zone. Significance: These energetic interactions are likely present in multiple pentameric ligand-gated ion channels.

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Stoichiometry of the Human Glycine Receptor Revealed by Direct Subunit Counting

Cited by 119 publications

References 37 publications

Stoichiometry and specific assembly of Best ion channels

Stoichiometry and specific assembly of Best ion channels

Counting molecules in single organelles with superresolution microscopy allows tracking of the endosome maturation trajectory

Correlating Structural and Energetic Changes in Glycine Receptor Activation

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