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Biochim Biophys Acta
2016 Jan 01;18581:47-56. doi: 10.1016/j.bbamem.2015.10.002.
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Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.
Padilla-Morales LF
,
Colón-Sáez JO
,
González-Nieves JE
,
Quesada-González O
,
Lasalde-Dominicci JA
.
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In our previous study we examined the functionality and stability of nicotinic acetylcholine receptor (nAChR)-detergent complexes (nAChR-DCs) from affinity-purified Torpedo californica (Tc) using fluorescence recovery after photobleaching (FRAP) in Lipidic Cubic Phase (LCP) and planar lipid bilayer (PLB) recordings for phospholipid and cholesterol like detergents. In the present study we enhanced the functional characterization of nAChR-DCs by recording macroscopic ion channel currents in Xenopus oocytes using the two electrode voltage clamp (TEVC). The use of TEVC allows for the recording of macroscopic currents elicited by agonist activation of nAChR-DCs that assemble in the oocyteplasma membrane. Furthermore, we examined the stability of nAChR-DCs, which is obligatory for the nAChR crystallization, using a 30 day FRAP assay in LCP for each detergent. The present results indicate a marked difference in the fractional fluorescence recovery (ΔFFR) within the same detergent family during the 30 day period assayed. Within the cholesterol analog family, sodium cholate and CHAPSO displayed a minimum ΔFFR and a mobile fraction (MF) over 80%. In contrast, CHAPS and BigCHAP showed a marked decay in both the mobile fraction and diffusion coefficient. nAChR-DCs containing phospholipid analog detergents with an alkylphosphocholine (FC) and lysofoscholine (LFC) of 16 carbon chains (FC-16, LFC-16) were more effective in maintaining a mobile fraction of over 80% compared to their counterparts with shorter acyl chain (C12, C14). The significant differences in macroscopic current amplitudes, activation and desensitization rates among the different nAChR-DCs evaluated in the present study allow to dissect which detergent preserves both, agonist activation and ion channel function. Functionality assays using TEVC demonstrated that LFC16, LFC14, and cholate were the most effective detergents in preserving macroscopic ion channel function, however, the nAChR-cholate complex display a significant delay in the ACh-induce channel activation. In summary, these results suggest that the physical properties of the lipid analog detergents (headgroup and acyl chain length) are the most effective in maintaining both the stability and functionality of the nAChR in the detergent solubilized complex.
Asmar-Rovira,
Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments.
2008, Pubmed
Asmar-Rovira,
Biophysical and ion channel functional characterization of the Torpedo californica nicotinic acetylcholine receptor in varying detergent-lipid environments.
2008,
Pubmed
Beeson,
Synaptic dysfunction in congenital myasthenic syndromes.
2012,
Pubmed
Caballero-Rivera,
Tryptophan scanning mutagenesis reveals distortions in the helical structure of the δM4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.
2012,
Pubmed
,
Xenbase
Caballero-Rivera,
Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.
2011,
Pubmed
,
Xenbase
Caffrey,
Crystallizing membrane proteins using lipidic mesophases.
2009,
Pubmed
Chen,
In pursuit of the high-resolution structure of nicotinic acetylcholine receptors.
2010,
Pubmed
Cherezov,
LCP-FRAP Assay for Pre-Screening Membrane Proteins for in Meso Crystallization.
2008,
Pubmed
Cornelius,
Modulation of Na,K-ATPase and Na-ATPase activity by phospholipids and cholesterol. I. Steady-state kinetics.
2001,
Pubmed
Corringer,
Atomic structure and dynamics of pentameric ligand-gated ion channels: new insight from bacterial homologues.
2010,
Pubmed
Cruz-Martín,
Tryptophan substitutions at lipid-exposed positions of the gamma M3 transmembrane domain increase the macroscopic ionic current response of the Torpedo californica nicotinic acetylcholine receptor.
2001,
Pubmed
,
Xenbase
Dellisanti,
Crystal structure of the extracellular domain of nAChR alpha1 bound to alpha-bungarotoxin at 1.94 A resolution.
2007,
Pubmed
DeMichele-Sweet,
Genetics of psychosis in Alzheimer's disease: a review.
2010,
Pubmed
Dumas,
Consequences of hydrophobic mismatch between lipids and melibiose permease on melibiose transport.
2000,
Pubmed
Engel,
Current understanding of congenital myasthenic syndromes.
2005,
Pubmed
Fong,
Correlation between acetylcholine receptor function and structural properties of membranes.
1986,
Pubmed
Gahring,
Mouse strain-specific changes in nicotinic receptor expression with age.
2005,
Pubmed
Guzmán,
Tryptophan scanning mutagenesis in the alphaM3 transmembrane domain of the Torpedo californica acetylcholine receptor: functional and structural implications.
2003,
Pubmed
,
Xenbase
Hertling-Jaweed,
Rapid preparation of the nicotinic acetylcholine receptor for crystallization in detergent solution.
1988,
Pubmed
Ivorra,
Protein orientation affects the efficiency of functional protein transplantation into the xenopus oocyte membrane.
2002,
Pubmed
,
Xenbase
Jensen,
Lipids do influence protein function-the hydrophobic matching hypothesis revisited.
2004,
Pubmed
Lasalde,
Tryptophan substitutions at the lipid-exposed transmembrane segment M4 of Torpedo californica acetylcholine receptor govern channel gating.
1996,
Pubmed
,
Xenbase
Lee,
Mutations in the M4 domain of Torpedo californica acetylcholine receptor dramatically alter ion channel function.
1994,
Pubmed
,
Xenbase
Lee,
How lipids and proteins interact in a membrane: a molecular approach.
2005,
Pubmed
Marsal,
Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.
1995,
Pubmed
,
Xenbase
Marsh,
Energetics of hydrophobic matching in lipid-protein interactions.
2008,
Pubmed
Matar-Merheb,
Structuring detergents for extracting and stabilizing functional membrane proteins.
2011,
Pubmed
Mitra,
Three-dimensional structure of the nicotinic acetylcholine receptor and location of the major associated 43-kD cytoskeletal protein, determined at 22 A by low dose electron microscopy and x-ray diffraction to 12.5 A.
1989,
Pubmed
Miyazawa,
Structure and gating mechanism of the acetylcholine receptor pore.
2003,
Pubmed
Morales,
Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.
1995,
Pubmed
,
Xenbase
Müller,
Congenital myasthenic syndromes: spotlight on genetic defects of neuromuscular transmission.
2007,
Pubmed
Nelson,
Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers.
1980,
Pubmed
Nollert,
Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases.
2001,
Pubmed
Ortiz-Acevedo,
Tryptophan scanning mutagenesis of the gammaM4 transmembrane domain of the acetylcholine receptor from Torpedo californica.
2004,
Pubmed
,
Xenbase
Ortiz-Miranda,
Mutations in the M4 domain of the Torpedo californica nicotinic acetylcholine receptor alter channel opening and closing.
1997,
Pubmed
,
Xenbase
Padilla-Morales,
Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology.
2016,
Pubmed
,
Xenbase
Padilla-Morales,
Effects of lipid-analog detergent solubilization on the functionality and lipidic cubic phase mobility of the Torpedo californica nicotinic acetylcholine receptor.
2011,
Pubmed
Privé,
Detergents for the stabilization and crystallization of membrane proteins.
2007,
Pubmed
Pucadyil,
Confocal fluorescence recovery after photobleaching of green fluorescent protein in solution.
2006,
Pubmed
Quik,
Multiple roles for nicotine in Parkinson's disease.
2009,
Pubmed
Rodríguez Cruz,
Congenital myopathies with secondary neuromuscular transmission defects; a case report and review of the literature.
2014,
Pubmed
Santiago,
Probing the effects of membrane cholesterol in the Torpedo californica acetylcholine receptor and the novel lipid-exposed mutation alpha C418W in Xenopus oocytes.
2001,
Pubmed
,
Xenbase
Santiago,
Tryptophan scanning mutagenesis in the TM3 domain of the Torpedo californica acetylcholine receptor beta subunit reveals an alpha-helical structure.
2004,
Pubmed
,
Xenbase
Schürholz,
Functional reconstitution of the nicotinic acetylcholine receptor by CHAPS dialysis depends on the concentrations of salt, lipid, and protein.
1992,
Pubmed
Seddon,
Membrane proteins, lipids and detergents: not just a soap opera.
2004,
Pubmed
Sunshine,
Lipid modulation of nicotinic acetylcholine receptor function: the role of neutral and negatively charged lipids.
1992,
Pubmed
Tamamizu,
Functional effects of periodic tryptophan substitutions in the alpha M4 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.
2000,
Pubmed
,
Xenbase
Unwin,
Refined structure of the nicotinic acetylcholine receptor at 4A resolution.
2005,
Pubmed
Wallace,
Monoolein lipid phases as incorporation and enrichment materials for membrane protein crystallization.
2011,
Pubmed
