Define the functional role of membrane cholesterol in the regulation of the AChR.

 The overall goal of this project is to gain insight into the molecular basis of the cholesterol inhibition of AChR function.  We found that the Torpedo AChR is remarkably sensitive to small increase (12%) in the membrane cholesterol levels, whereas the muscle-type AChR remains almost insensitive. The macroscopic current parameters, single channel parameters, lateral diffusion coefficient and annular lipid composition of these two AChRs are being examined at physiological relevant levels of cholesterol content. These experiments will define the mechanism for cholesterol inactivation of these two nAChRs types.  The lateral diffusion of AChRs will be examined during the cholesterol enrichment using Fluorescence Recovery after Photobleaching (FRAP). These experiments will test the hypothesis that inhibition of nAChRs by cholesterol is due to clustering of nAChRs in a “non functional or silent” state.  Another objective of this aim is to evaluate the regulation of the novel aC418W mutation by cholesterol.  We found that the αC418W mutation preferentially accumulates in an apparent membrane microdomain in the oocyte surface membrane (Baez at al., 2006 submitted). After cholesterol depletion, a significant number of the αC418W mutants move from the “silent pool” to a functional pool of AChRs and display the normal αC418W ion channel kinetic. On the basis of these results, we hypothesize that the structure of the lipid-protein interface of the AChR regulates the manner in which receptors move between the silent and the functional surface pools.  These studies will provide novel information to define the molecular basis for the cholesterol regulation the AChR function. The physiological effects of cholesterol on the AChR function could have paramout implications if we consider that an increment of 12% cholesterol can deplete 40% of the nAChRs in the CNS. For instance, it has been noted that learning deficits in Alzheimer’s disease can be present even before any observable cell loss, suggesting that the initial disruption of cognitive function in this disease is not due to cell death, but rather due to the disruption of nAChR receptors and synaptic transmission. Along the same line, epidemiological evidence has suggested that high cholesterol is a risk factor for Alzheimer’s disease. Understanding the regulation of nAChR by cholesterol is of tremendous importance; however, this aspect of the nAChR has remained obscure.

 

 

 

 

Typical pattern of recovery of the fluorescently tagged AChR in the oocyte surface.  A) Confocal image of the oocyte membrane expressing the Torpedo AChR.  This panel illustrates two regions of interest (ROI) selected for 1) photobleaching and 2) reference.  Areas in black (no signal) are present due to normal invaginations in the oocyte membrane.  B) ROI 1 illustrates the usual fluorescence recovery pattern for the AChR as a function of time. Fluorescence recovery after photobleaching for both WT and  an a lipid-exposed mutation (LEM) Torpedo AChR. C) Partial recovery from photobleaching for WT Torpedo AChR (n=15; R2=0.83).  D)  Partial recovery after photobleaching for the LEM mutant Torpedo AChR (n=5; R2=0.82).

 

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