The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. In addition, the nAChR is also found in the electric organs of electric rays (e.g., the genus Torpedo). Cholesterol, which is a key lipid for maintaining the correct functionality of membrane proteins, has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs expressed in a model membrane with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P ratio in Xenopus laevis oocytes expressing the neuronal α7, α4β2, muscle-type, and Torpedo californica nAChRs in their macroscopic current responses. Using the two-electrode voltage clamp technique, it was found that the neuronal α7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle-type nAChR. The peak current versus C/P profiles during enrichment display different behaviors; α7 and Torpedo nAChRs display a hyperbolic decay with two clear components, whereas muscle-type and α4β2 nAChRs display simple monophasic decays with different slopes. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration produces a remarkable reduction in the macroscopic current responses of the neuronal α7 and Torpedo nAChRs functionality, whereas the muscle nAChR appears to be the most resistant to cholesterol inhibition among all four nAChR subtypes. Overall, the present study demonstrates differential profiles for cholesterol inhibition among the different types of nAChR to physiological cholesterol increments in the plasmatic membrane. This is the first study to report a cross-correlation analysis of cholesterol sensitivity among different nAChR subtypes in a model membrane.
The lipid-protein interface is an important domain of the acetylcholine receptor (AChR) that has recently garnered increasing relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the AChR. However, there is still a need to identify and gain insight into the mechanism through which lipid-protein interactions regulate AChR function and dynamics. In this study, we extend the Fourier Transform coupled Tryptophan Scanning Mutagenesis (FT-TrpScanM) approach to monitor the conformational changes experienced by the dM3 and dM4 transmembrane domains of the Torpedo californica AChR, and to identify which lipid-exposed positions on these domains are potentially linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptohan substitutions along the dM3 and dM4 transmembrane domains were characterized by two-electrode voltage clamp and 125 I-labeled a-bungarotoxin binding assays. The periodicity profiles and Fourier Transform spectra of these domains revealed a thinner-elongated helical structure for the closed-channel state and a thicker-shrunken helical structure for the open-channel state. The difference in oscillation patterns between the closed-and openchannel states shows a substantial conformational change along these domains as a consequence of channel activation. These results support the recently proposed spring model for the aM3 transmembrane domain of the Mus musculus AChR. Furthermore, the present data demonstrates that the lipid-protein interface of the AChR plays an important role in the propagation of the conformational wave needed for channel gating. Supported by NIH Grants 2RO1GM56371-12 and 2U54NS43011.
The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. The nAChR is also found in the electric organs of electric rays (e.g. Torpedo californica). Cholesterol is a key lipid for maintaining the correct functionality of membrane proteins and has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs when expressed in cell membranes with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P of Xenopus laevis oocytes expressing the neuronal α‐7, muscle‐type or Torpedo californica nAChRs in the functioning of the nAChR. Using the two‐electrode voltage clamp technique it was found that the neuronal ‐7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle‐type nAChR. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration renders a significant fraction of the neuronal ‐7 and Torpedo nAChRs “inactivated” whereas the muscle‐type nAChR tends to resist this functional inhibition. This work was supported by NIH grants RO1GM56371‐11, NCRR 1S0RR13705 and S06‐GM08102‐27 to JAL‐D. NDH‐R and CAB‐P were supported by the NIH‐MBRS RISE Grant R25GM61151.
The nicotinic acetylcholine receptor (nAChR), located in the cell membranes of neurons and muscle cells, mediates the transmission of nerve impulses across cholinergic synapses. The nAChR is also found in the electric organs of electric rays (e.g. Torpedo californica). Cholesterol is a key lipid for maintaining the correct functionality of membrane proteins and has been found to alter the nAChR function. We were thus interested to probe the changes in the functionality of different nAChRs when expressed in cell membranes with modified cholesterol to phospholipid ratios (C/P). In this study, we examined the effect of increasing the C/P of Xenopus laevis oocytes expressing the neuronal α‐7, muscle‐type or Torpedo californica nAChRs in the functioning of the nAChR. Using the two‐electrode voltage clamp technique it was found that the neuronal ‐7 and Torpedo nAChRs are significantly more sensitive to small increases in C/P than the muscle‐type nAChR. This study clearly illustrates that a physiologically relevant increase in membrane cholesterol concentration renders a significant fraction of the neuronal ‐7 and Torpedo nAChRs “inactivated” whereas the muscle‐type nAChR tends to resist this functional inhibition. This work was supported by NIH grants RO1GM56371‐11, NCRR 1S0RR13705 and S06‐GM08102‐27 to JAL‐D. NDH‐R and CAB‐P were supported by the NIH‐MBRS RISE Grant R25GM61151.
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