The dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels

Veatch, William R.; Stryer, Lubert

doi:10.1016/0022-2836(77)90042-0

Cited by 241 publications

(167 citation statements)

References 14 publications

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“…The energy transfer was determined as described for the microplate BRET assay in the absence and presence of various ligands. Data were fitted by adapting the dimer, trimer, and tetramer model of energy transfer quenching proposed by Veatch and Stryer (15). Assuming that oligomer formation is random between the different receptor species, the following equations can be used:…”

Section: Methodsmentioning

confidence: 99%

“…Data were fitted by adapting the dimer (n ϭ 2), trimer (n ϭ 3), and tetramer (n ϭ 4) model of energy transfer quenching proposed by Veatch and Stryer (15) (see "Material and Methods"). These models can be applied with the assumption that oligomerization occurs randomly between tagged and non-tagged receptors (15). As shown in Fig.…”

Section: Detection Of Mt1r and Mt2r In Hek 293mentioning

confidence: 99%

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Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

Ayoub¹,

Couturier²,

Lucas-Meunier³

et al. 2002

Journal of Biological Chemistry

287

254

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Several G protein-coupled receptors have been shown to exist as homo-and hetero-oligomeric complexes in living cells. However, the link between ligand-induced receptor activation and its oligomerization state as well as the proportion of the total receptor population that can engage in oligomeric complexes remain open questions. Here, the closely related human MT1 and MT2 melatonin receptors (MT1R, MT2R) were used to address these issues. Bioluminescence resonance energy transfer (BRET) experiments in living HEK 293 cells revealed that these receptors form homo-and hetero-oligomers. Constitutive energy transfer was observed for all receptor combinations at physiological expression levels and could be detected in single cell BRET experiments. Inhibition of the energy transfer by dilution of the BRET partners identified MT1R and MT2R dimers as the predominant receptor species, and this oligomerization state did not change upon agonist and antagonist binding. Agonists, neutral antagonists, and inverse agonists all promoted increases in BRET values for MT2R but not for MT1R homodimers in living cells and isolated plasma membranes. This indicates that no correlation could be inferred between the receptor activation state and the dimerization state of the receptor. This also suggests that ligand-promoted BRET increases represent specific ligand-induced conformational changes of pre-existing dimers rather then increased dimerization. The observation that ligands favored the energy transfer within the hetero-oligomer from MT1R to MT2R but not in the reverse orientation, from MT2R to MT1R, supports this view.Membrane proteins such as tyrosine kinase, cytokine, or transforming growth factor receptors have been known for many years to form oligomers, and the link between their oligomerization and activity states has been well established (1). In contrast, G protein-coupled receptor (GPCR) 1 oligomerization has been documented only recently (2), and the relation between receptor activation and oligomerization is still poorly understood. Even the proportion between monomeric and oligomeric receptor species and the exact oligomerization state (dimer, trimer, tetramer, etc.) remains a matter of controversy. Currently, two models are proposed. In the first model GPCR are monomeric in their inactive state, and agonist activation induces the formation of receptor oligomers. This model is based on low basal and strong agonist-induced energy transfer signals observed in fluorescence and bioluminescence resonance energy transfer (FRET, BRET) experiments for the gonadotropin-releasing hormone (3), the somatostatin SSTR5 (4), and the SSTR5/dopamin D2R receptor oligomers (5). The second model proposes that GPCR are constitutively oligomerized and is supported by studies reporting high basal BRET or FRET signals for ␣-mating factor (6), ␤2-adrenergic (␤2AR) (7), tyrothropin-releasing hormone (8), ␦-opioid (9), type A cholecystokinin (10), and dopamine D2 receptors (11). Agonist-promoted increases in signals were observed in some of thes...

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Section: Methodsmentioning

confidence: 99%

Section: Detection Of Mt1r and Mt2r In Hek 293mentioning

confidence: 99%

Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

Ayoub¹,

Couturier²,

Lucas-Meunier³

et al. 2002

Journal of Biological Chemistry

287

254

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“…It was mainly employed to measure relative molecular distances [22] and less frequently to determine kinetic and thermodynamic properties of molecular processes [23,24]. Particularly, protein dimerization can be measured by using two populations of the protein, each one labeled with a £uorophore, constituting a FRET donor^ac-ceptor pair [25,26].…”

Section: Monitoring Pmca Dimerization By Using Fretmentioning

confidence: 99%

Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability

et al. 2000

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Ca2+ pump dimerization was studied by using a combined approach of thermal denaturation and fluorescence resonance energy transfer. The measurement of calcium pump ability to dimerize after the unfolding of individual functional domains of the enzyme demonstrated the existence of two different regions involved in the self-association process. One of these regions is highly susceptible to thermal unfolding and was identified as the calmodulin (CaM)-binding domain. The other region whose thermal stability is higher than those of the catalytic and CaM-binding domains could be related with the previously found C28W-binding regions. ß

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“…0108-7681/90/030440-07503.00 membranes; these channels are specific for the conductance of monovalent cations (Hladky & Haydon, 1972;Veatch & Stryer, 1977). Until recently (Wallace & Ravikumar, 1988;Langs, 1988), no crystal structure of gramicidin had been solved at the molecular level, because the molecule falls in a difficult size range for crystallographic studies: rather large for direct methods, and because of the difficulty in forming isomorphous derivatives of this flexible molecule, somewhat small for multiple isomorphous replacement phasing.…”

Section: Introductionmentioning

confidence: 99%

The use of single-wavelength anomalous scattering to solve the crystal structure of a gramicidin A/caesium chloride complex

Wallace¹,

Hendrickson²,

Ravikumar³

1990

Acta Crystallogr Sect B

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Single-wavelength Cu Ka anomalous scattering has been used to determine the structure of a crystalline complex of gramicidin A and caesium chloride. The asymmetric unit in these crystals, with space group P212~2~ and a= 32.118 (6), b= 52.103 (12), c= 31.174 (7)A, contains four independent monomers (two dimers) of the pentadecapeptide. This structure falls in an intermediate size range for which direct methods and multiple isomorphous replacement are generally not successful for obtaining phase information. However, using the Bijvoet differences and the partial structure of the caesium atoms which have been incorporated in the crystals, it has been possible to obtain information on this crystal form. Because the caesium atoms dominate the scattering of these crystals, inclusion of the Friedel mate information in the restrained least-squares refinement has been essential. These studies extend the utility of singlewavelength anomalous-scattering phase determination to a macromolecular structure in which the partial structure of the anomalous scatterer is large.

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The dimeric nature of the gramicidin A transmembrane channel: Conductance and fluorescence energy transfer studies of hybrid channels

Cited by 241 publications

References 14 publications

Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

Oligomerization of the plasma membrane calcium pump involves two regions with different thermal stability

The use of single-wavelength anomalous scattering to solve the crystal structure of a gramicidin A/caesium chloride complex

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