Assessment of the functionality and stability of detergent purified nAChR from Torpedo using lipidic matrixes and macroscopic electrophysiology

Abstract

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 oocyte plasma 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.

Keywords: Detergents; Fluorescence recovery after photobleaching; Lipidic Cubic Phase; NAChR; Planar lipid bilayer; Two-electrode voltage clamp.

FIGURE 1

Macroscopic ion channel functional assay of crude membrane extracts from Tc nAChRs. Crude membranes extracted from Tc were microinjected into Xenopus laevis oocytes and macroscopic ACh evoked currents were measured at −70 mV using TEVC. a Representative response to a 5 sec application of 100 µM (represented by a bar) of oocytes injected with crude membrane extracts. b Representative response to a 5 sec application of 100 µM ACh (represented by a bar) following a 5 sec pre-application of 1 µM α-BTX.

FIGURE 2

Macroscopic ion channel functional assays of detergent solubilized and affinity purified nAChR-DCs. Responses were evoked by a 5 second application of 100 µM ACh (represented by bars) at −70 mV on oocytes injected with detergent solubilized purified nAChR-DCs. The following detergents were used a DDM; b OG; c sodium cholate; d FC-12; e FC-14; f FC-16; g LFC-12; h LFC-14 and I LFC-16. j Responses for all detergents 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; ***p<0.0001; **p<0.001.

FIGURE 3

Cholesterol analog detergents LCP-FRAP stability assay. Fractional fluorescence recovery and diffusion coefficient of each affinity purified nAChR using cholesterol analogue detergents. FRAP experiments of a sodium cholate, b BigChap and c CHAPSO and d CHAPS were recorded every five days for thirty 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 equation 1 for each fractional recovery of the triplicates. The mobile fraction e was obtained by averaging the last twenty points of the fractional recovery obtained in a,b,c. The diffusion coefficient f was calculated using equation 2

FIGURE 4

Phospholipid analog detergents lipidic matrix stability, LCP-FRAP assay. Fractional fluorescence recovery and diffusion coefficient of each affinity purified nAChR using phospholipid analog detergents of the Fos choline (FC) family. FRAP experiments of a FC-12, b FC-14 and c FC-16 were recorded every five days for thirty 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 equation 1 for each fractional recovery of the triplicates. The mobile fraction d was obtained by averaging the last twenty points of the fractional recovery obtained in a,b,c. The diffusion coefficient e was calculated using equation 2

FIGURE 5

Phospholipid analog detergents lipidic matrix stability, LCP-FRAP assay. Fractional fluorescence recovery and diffusion coefficient of each affinity purified nAChR using phospholipid analog detergents of the LysoFos choline (LFC) family. FRAP experiments of a LFC-12, b LFC-14 and c LFC-16 were recorded every five days for thirty 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 equation 1 for each fractional recovery of the triplicates. The mobile fraction d was obtained by averaging the last twenty points of the fractional recovery obtained in a,b,c. The diffusion coefficient e was calculated using equation 2