Structural rearrangements in single ion channels detected optically in living cells

Sonnleitner, Alois; Mannuzzu, Lidia M.; Terakawa, Susumu; Isacoff, Ehud Y.

doi:10.1073/pnas.192261499

Cited by 106 publications

(74 citation statements)

References 25 publications

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“…TIR microscopy was done as described (4,19). Devitellinized Xenopus oocytes were attached to a coverslip, and movies of 500 frames (10 -30 fps) were acquired with 532-nm illumination for ttTomato emission and 488 nm for GFP through an Olympus ϫ100/NA1.65 objective (see SI Text).…”

Section: Methodsmentioning

confidence: 99%

Rules of engagement for NMDA receptor subunits

Ulbrich

Isacoff²

2008

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

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150

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Canonical NMDA receptors assemble from two glycine-binding NR1 subunits with two glutamate-binding NR2 subunits to form glutamate-gated excitatory receptors that mediate synaptic transmission and plasticity. The role of glycine-binding NR3 subunits is less clear. Whereas in Xenopus laevis oocytes, two NR3 subunits coassemble with two NR1 subunits to form a glycine-gated receptor, such a receptor has yet to be found in mammalian cells. Meanwhile, NR1, NR2, and NR3 appear to coassemble into triheteromeric receptors in neurons, but it is not clear whether this occurs in oocytes. To test the rules that govern subunit assembly in NMDA receptors, we developed a single-molecule fluorescence colocalization method. The method focuses selectively on the plasma membrane and simultaneously determines the subunit composition of hundreds of individual protein complexes within an optical patch on a live cell. We find that NR1, NR2, and NR3 follow an exclusion rule that yields separate populations of NR1/NR2 and NR1/NR3 receptors on the surface of oocytes. In contrast, coexpression of NR1, NR3A, and NR3B yields triheteromeric receptors with a fixed stoichiometry of two NR1 subunits with one NR3A and one NR3B. At least part of this regulation of subunit stoichiometry appears to be caused by internal retention. Thus, depending on the mixture of subunits, functional receptors on the cell surface may follow either an exclusion rule or a stoichiometric combination rule, providing an important constraint on functional diversity. Cell-to-cell differences in the rules may help sculpt distinct physiological properties.NR3 ͉ total internal reflection ͉ single-molecule fluorescence ͉ subunit stoichiometry ͉ triheteromeric I on channels, receptors, and other proteins involved in transmembrane signaling are often composed of several different kinds of subunits. The composition can vary depending on the state of the cell and the availability of subunit types, which depends, in turn, on protein production and subcellular localization. Based on subunit availability and interaction affinity, complexes may form in one or more fixed stoichiometries or form with heterogeneous stoichiometries. Bulk assays may not be able to distinguish between these possibilities and could have difficulty in determining the pattern of assembly in specific subcellular compartments, in particular, separating plasma membrane fractions and intracellular membrane compartments. We used an optical approach to determine the subunit composition of integral membrane proteins complexes at the singlemolecule level. Measurements were made simultaneously for many complexes, yielding distributions from which it was possible to deduce the rule that governs complex assembly. The single-molecule approach is compatible with normal (low) levels of protein expression, where the formation of nonnative highorder complexes and aggregation are avoided.Our technique is based on the colocalization of single subunits of a macromolecular complex that are fused to fluorescent protein (FP) tags ...

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Section: Methodsmentioning

confidence: 99%

Rules of engagement for NMDA receptor subunits

Ulbrich

Isacoff²

2008

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

Self Cite

150

111

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“…To avoid the problem of autofluorescence from the cell cytoplasm, we used total internal reflection fluorescence microscopy, which restricts illumination to the interface between the coverslip and the cell resting on it 13 (Supplementary Methods). We imaged areas of the membrane where the density of fluorescent spots was between 20 and 200 in a 15 × 15 μm area.…”

mentioning

confidence: 99%

Subunit counting in membrane-bound proteins

2007

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The subunit number and stoichiometry of membrane-bound proteins are difficult to determine without disrupting their membrane environment. Here we describe a single-molecule technique for counting subunits of proteins in live cell membranes by observing bleaching steps of GFP fused to a protein of interest. After testing the method with proteins of known stoichiometry expressed in Xenopus laevis oocytes, we resolved the composition of NMDA receptors composed of NR1 and NR3 subunits.Many membrane proteins form multimers before they achieve a functional state and are transported to the plasma membrane of the cell. The stoichiometry of a complex is precisely regulated and is fundamental to its functional properties. Subunit stoichiometry is usually assessed via bulk biochemical and macroscopic functional analyses. For example, the multimeric state of K + channels in the squid giant axon was originally predicted from the shape of the current trace during opening of the channels in voltage clamp 1 . A kinetic analysis of inactivation 2 and an analysis of currents in mixed subunit channels 3 indicated that the channels are probably composed of four similar or identical subunits, which was later confirmed by crystallography 4 . Sometimes macroscopic functional recordings do not distinguish stoichiometry precisely. For example, CNG channels for years had been assumed to be composed of a 2:2 stoichiometry of CNGA1 and CNGB1 subunits 5 , but has been shown to actually be composed of a 3:1 CNGA1 to CNGB1 stoichiometry by biochemical analysis 6 and using fluorescence energy resonance transfer between fluorescent proteins fused to CNGA1 and CNGB1 ( ref. 7 ).The well-studied glutamate-gated NMDA receptors are tetramers containing two NR1 and two NR2 subunits. This stoichiometry had been deduced from macroscopic functional analysis, in which coexpression of wild-type and mutant subunits resulted in a triphasic response to agonists that could be explained by mixture of receptors containing zero, one or two mutant NR1 or NR2 subunits 8 , and from single-channel recordings that showed distinct behaviors consistent with the possible mixed stoichiometries 9 , and confirmed by crystallography 10 . Less is known about NMDA receptors containing the more recently discovered NR3 subunit, which is thought to be involved in synaptic development 11 . Initially it was thought that NR3 coassembles with NR1 and NR2 to form glutamate-gated receptors with unique properties 11 . More recently, and quite surprisingly, the NR3 subunit ligand binding domain had been found to bind glycine and not glutamate, and

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“…Similarly, advances in eukaryotic membrane protein purification and labeling have made fluorescence attractive for in vitro study of the agonist-induced signaling of G-protein coupled receptors (2). Progress in these techniques has extended from initial simple ensemble measurements to polarization studies (3), lifetimes (4), resonance energy transfer (5, 6), and single-molecule studies of functional membrane proteins (7,8).…”

mentioning

confidence: 99%

A fluorescent probe designed for studying protein conformational change

Cohen

Pralle

Yao

et al. 2005

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

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112

114

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The usefulness of fluorescence in studying protein motions derives from its sensitivity, kinetic resolution, and compatibility with both live cells and physiological assays. Recent advances in microscopy and membrane protein purification have permitted the observation of fluorescence changes that accompany the functional transitions of complex eukaryotic membrane proteins. These techniques rely on probes that can clearly report the environmental changes of specific residues, but most commonly available sidechain-reactive probes are not well suited for this purpose. Here, we introduce a red Cys-reactive probe, aminophenoxazone maleimide (APM), designed with improved chemical and spectral properties for reporting protein conformational change. APM is compact, uncharged, and has a short linker between probe and protein, all of which ensure that it can closely track the motions of the side chain to which it is attached. It undergoes large polarity-dependent changes in Stokes shift, as well as large bathochromic shifts in both excitation maximum (from 521 nm in toluene to 598 nm in water) and emission maximum (580 nm to 633 nm). These polaritydependent spectral changes offer a potentially simple means of relating fluorescence to local structure and motion, although they are partially offset by some complicating factors in APM fluorescence. We find that, like a rhodamine maleimide, APM senses the conformational changes underlying voltage sensing in the Shaker potassium channel, and it is superior at a site that shows limited reactivity to the rhodamine. The spectral characteristics of APM can also report subtle differences between aqueous positions in purified preparations of the ␤2 adrenergic receptor.fluorescene ͉ membrane proteins ͉ protein dynamics

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Structural rearrangements in single ion channels detected optically in living cells

Cited by 106 publications

References 25 publications

Rules of engagement for NMDA receptor subunits

Rules of engagement for NMDA receptor subunits

Subunit counting in membrane-bound proteins

A fluorescent probe designed for studying protein conformational change

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