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Data Brief
2015 Dec 25;6:433-7. doi: 10.1016/j.dib.2015.12.010.
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Functionality and stability data 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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The presented data provides additional information about the assessment of affinity purified nicotinic acetylcholine receptor (nAChR) rich membrane solubilized with long chain (16 saturated carbons) lysophospholipid with glycerol headgroup (LFG-16). The assessment of stability and functionality of solubilized membrane protein is a critical step prior to further crystallization trails. One of the key factors for this task is the appropriate choice of a detergent that can support nAChR activity and stability comparable to the crude membranes. The stability of the nAChR-LFG-16 complex incorporated into lipid cubic phase (LCP) was monitored for a period of 30 days by means of fluorescence recovery after photobleaching (FRAP) and the functionality was evaluated after its incorporation into Xenopus oocyte by means of the two electrode voltage clamp technique.
Fig. 1. Structure of the phospholipid analog detergents 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LFG-16) used for the solubilization nicotinic acetylcholine receptor from Torpedo californica electric organ, using the phospholipid analog detergent 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LFG-16).
Fig. 2. Phospholipid analog detergents lipidic matrix stability, LCP-FRAP assay. Fractional fluorescence recovery and diffusion coefficient of each affinity purified nAChR using the phospholipid analog detergent 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1′-rac-glycerol) (LFG-16). FRAP experiments were recorded every five days for 30 days. All fluorescence recovery experiments were performed in triplicates, averaging five recoveries on different areas of the lipidic matrix with the nAChR incorporated. The fractional recovery was calculated using equationF(t)=[ft−f0f∞−f0] where f(t) is the corrected fluorescence intensity of the bleached spot, f0 is the corrected fluorescence intensity of the bleached spot in the 600 msec after bleaching, and f∞ is the average of corrected fluorescence intensity in the five pre-bleached images.
Fig. 3. Macroscopic ion channel functional assay of LFG-16 solubilized and affinity purified nAChR-DCs. Responses were evoked by a 5 second application of 100 μM ACh (represented by bars) at â70 mV on Xenopus oocytes injected with LFG-16 solubilized purified nAChR-DCs. Responses were normalized to the respective crude membranes used for solubilization plotted as mean ±SEM and compared using an unpaired t-test in Graph Pad Prism 6.
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