Surface Plasmon Resonance Study of G Protein/Receptor Coupling in a Lipid Bilayer-Free System

2006 ◽  
Vol 78 (4) ◽  
pp. 1228-1234 ◽  
Author(s):  
Konstantin E. Komolov ◽  
Ivan I. Senin ◽  
Pavel P. Philippov ◽  
Karl-Wilhelm Koch
Keyword(s):  
Surface Plasmon Resonance ◽  
G Protein ◽  
Lipid Bilayer ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Free System ◽  
Receptor Coupling ◽  
Protein Receptor

Independent and synergistic interaction of retinal G-protein subunits with bovine rhodopsin measured by surface plasmon resonance

2001 ◽  
Vol 358 (2) ◽  
pp. 389-397 ◽  
Author(s):  
William A. CLARK ◽  
Xiaoying JIAN ◽  
Loren CHEN ◽  
John K. NORTHUP
Keyword(s):  
Surface Plasmon Resonance ◽  
G Protein ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Specific Activity ◽  
Receptor Interaction ◽  
Nucleotide Exchange ◽  
Α Subunit ◽  
Protein Subunits ◽  
Bovine Rhodopsin

We have used surface plasmon resonance (SPR) measurements for the kinetic analysis of G-protein-receptor interaction monitored in real time. Functionally active rhodopsin was immobilized on an SPR surface, with full retention of biochemical specific activity for catalysis of nucleotide exchange on the retinal G-protein α subunit, via binding to immobilized concanavalin A. The binding interactions of bovine retinal αt and β1γ1 subunits with rhodopsin measured by SPR were profoundly synergistic. Synergistic binding of the retinal G-protein subunits to rhodopsin was not observed for guanosine 5′-[γ-thio]triphosphate-bound Gαt, nor was binding observed with squid retinal Gαq, which is not activated by bovine rhodopsin. The binding affinity (336±171nM; mean value±S.D.) of retinal βγ for rhodopsin in the presence of retinal α subunit measured by SPR confirmed the apparent affinity of 254nM determined previously by nucleotide exchange assays. Binding of β1γ1, β1γ2, and β1γ8-olf dimers to rhodopsin, independently of the α subunit, was readily observable by SPR. Further, these dimers, differing only in their γ subunit compositions, displayed markedly distinct binding affinities and kinetics. The β1γ2 dimer bound with a kinetically determined Kd of 13±3nM, a value nearly identical with the biochemically determined K1/2 of 10nM. The physiologically appropriate β1γ1 displayed rapid association and dissociation kinetics, whereas the other β1γ dimers dissociated at a rate less than 1/100 as fast. Thus rhodopsin interaction with its native signalling partners is both rapid and transient, whereas the interaction of rhodopsin with heterologous Gβγ dimers is markedly prolonged. These results suggest that the duration of a G-protein-coupled receptor signalling event is an intrinsic property of the G-protein coupling partners; in particular, the βγ dimer.

scholarly journals Association of Model Neurotransmitters with Lipid Bilayer Membranes

2019 ◽  
Author(s):  
B. Josey ◽  
F. Heinrich ◽  
V. Silin ◽  
M. Lösche
Keyword(s):  
Surface Plasmon Resonance ◽  
Ion Channels ◽  
Lipid Bilayer ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Dissociation Constants ◽  
Measurement Techniques ◽  
Neutron Reflectometry ◽  
Membrane Association ◽  
Lipid Bilayer Membranes

AbstractAimed to reproduce the results of electrophysiological studies of synaptic signal transduction, conventional models of neurotransmission are based on the specific binding of neurotransmitters to ligand-gated receptor ion channels. However, the complex kinetic behavior observed in synaptic transmission cannot be reproduced in a standard kinetic model without the ad hoc postulation of additional conformational channel states. On the other hand, if one invokes unspecific neuro-transmitter adsorption to the bilayer—a process not considered in the established models—the electrophysiological data can be rationalized with only the standard set of three conformational receptor states that also depend on this indirect coupling of neurotransmitters via their membrane interaction. Experimental verification has been difficult because binding affinities of neuro-transmitters to the lipid bilayer are low. We quantify this interaction with surface plasmon resonance to measure equilibrium dissociation constants in neurotransmitter membrane association. Neutron reflectometry on artificial membranes reveals the structural aspects of neurotransmitters association with zwitterionic and anionic bilayers. We establish that serotonin interacts non-specifically with the membrane at physiologically relevant concentrations whilst GABA (γ-aminobutyric acid) does not. Surface plasmon resonance shows that serotonin adsorbs with millimolar affinity and neutron reflectometry shows that it penetrates the membrane deeply whereas GABA is excluded from the bilayer.SignificanceReceptor ion channels in the postsynaptic membrane and their neurotransmitter agonists enable fast communication between neuronal cells. Electrophysiology studies reveal surprisingly complex kinetics that apparently require a variety of protein conformational states for their quantitative interpretation, but an alternate hypothesis invoking neurotransmitter membrane association reduces the complexity of the underlying reaction schemes significantly. While their affinity may be low, and is hard to quantify experimentally, neurotransmitter membrane association can be relevant because of their large temporary concentration in the synaptic cleft. With thermodynamic and structural measurements we quantify membrane-bound states of serotonin, establishing this neurotransmitter as membrane-affine, whereas the affinity of the more hydrophilic GABA is too low to register in our sensitivity-optimized measurement techniques.

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