1. Alteration of membrane domain compositions by Protein nAChRs Jigesh Patel, Robert Fisher, Dr. Juyang Huang
Unwin, Nigel. "Refined Structure of the nAChR at 4 Å resolution." JMB, 2004:
Unwin, Nigel. "Refined Structure of the nAChR at 4 Å resolution." JMB, 2004:
Buboltz, Jeffrey T, and Gerald W Feigenson. "A novel strategy for the preparation of liposomes: rapid solvent exchange." BBA-Biomembranes, 1999:
Hassan-Zadeh, E, F Hussain, and J Huang. "Gramicidin Peptides Alter Global Lipid Compositions and Bilayer Thicknesses of Coexisting Liquid-Ordered and Liquid-Disordered Membrane Domains." Langmuir, 2017:
Unwin, Nigel. "Refined Structure of the nAChR at 4 Å resolution." JMB, 2004:
Zhao, Jiang, et al. "Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol." BBA-Biomembranes, 2007:
Buboltz, Jeffrey T, and Gerald W Feigenson. "A novel strategy for the preparation of liposomes: rapid solvent exchange." BBA-Biomembranes, 1999:
Hassan-Zadeh, E, F Hussain, and J Huang. "Gramicidin Peptides Alter Global Lipid Compositions and Bilayer Thicknesses of Coexisting Liquid-Ordered and Liquid-Disordered Membrane Domains." Langmuir, 2017:
Unwin, Nigel. "Refined Structure of the nAChR at 4 Å resolution." JMB, 2004:
Zhao, Jiang, et al. "Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol." BBA-Biomembranes, 2007:
Abstract
In this study, the effects of adding membrane protein nAChRs to DOPC/DSPC/cholesterol lipid bilayers containing coexisting phases are investigated. This work is the first study of its kind that investigates the effect of ion-transmitter nAChRs on Lo+Ld phase boundaries. The modification of phase boundary by nAChRs is determined using fluorescence microscopy on Giant Unilamellar Vesicles (GUVs). After phase boundaries are determined, thermodynamic tie-lines and protein's partition coefficients will be measured. Those data will allow us to precisely determine the exact concentrations of nAChRs in various cell membrane domains. Accurate measurement of the perturbations of the phase boundaries by the protein could serve as an important means to quantitatively understand the universal behavior of a range of membrane proteins.
nAChRs
The nicotinic ACh receptor (Fig. 3) is fairly large channel protein consisting of 2200 amino acids, weighs approximately 290 kDa and spans ~160 Å across the cell membrane. It is mainly found at the nerve-muscle synapse, where it mediates fast chemical transmission of electrical signal in response to ACh released into the synaptic cleft. A B
Figure 2 A) Damp-film method B) Electroformation of GUVs
Determining the Phase Boundary of Lo + Ld Coexisting Region
The liposomes were produced using the RSE procedure (Fig. 1). The GUVs were then prepared using the damp-film method (Fig. 2A) followed by electroformation (Fig 2B). Fluorescence images and videos of GUVs were captured using CCD camera on an inverted microscope with a 40× objective. All images and videos were captured at 25 °C. The mole fraction of nAChR in all samples was fixed. The overall lipid composition of a sample is specified by two parameters: the R value [R = χDSPC/(χDSPC + χDOPC)] and cholesterol mole fraction χC. In order to simplify the comparison of results with and without nAChR, the protein is not included in lipid composition calculation.
Figure 3 Nicotinic acetylcholine receptors (nAChRs)
Conclusion
In DOPC/DSPC/Cholesterol ternary mixtures containing coexisting Lo+Ld phases, nAChRs alters the phase boundary and thermodynamic tie-lines. Our study shows that trans-membrane proteins play a significant role in the controlling of the lipid composition of membrane domains. Membrane domain size, composition, order, and protein partition behavior are important to biomembrane function, membrane protein activity, disease development and diagnosis, and drug design. Our studies advances our knowledge about fundamental protein-lipid interactions. A
References:
Buboltz, Jeffrey T, and Gerald W Feigenson. "A novel strategy for the preparation of liposomes: rapid solvent exchange." BBA-Biomembranes, 1999:
Hassan-Zadeh, E, F Hussain, and J Huang. "Gramicidin Peptides Alter Global Lipid Compositions and Bilayer Thicknesses of Coexisting Liquid-Ordered and Liquid-Disordered Membrane Domains." Langmuir, 2017:
Unwin, Nigel. "Refined Structure of the nAChR at 4 Å resolution." JMB, 2004:
Zhao, Jiang, et al. "Phase studies of model biomembranes: Complex behavior of DSPC/DOPC/Cholesterol." BBA-Biomembranes, 2007:
Figure 1 RSE apparatus and cross-sectional schematic
Figure 4 Ternary phase diagrams of DOPC/DSPC/Cholesterol with nAChRs (the dashed line) and without the protein (the solid line). Blue dots: GUVs having coexisting Lo+Ld phases; red dots: GUVs having only one phase.