Boundary lipids in the nicotinic acetylcholine receptor microenvironment.

The structural and functional properties of the nicotinic acetylcholine receptor (AChR), the archetype molecule in the superfamily of Cys-looped ligand-gated ion channels, are strongly dependent on the lipids in the vicinal microenvironment. The influence on receptor properties is mainly exerted by the AChR-vicinal ("shell" or "annular") lipids, which occur in the liquid-ordered phase as opposed to the more disordered and "fluid" bulk membrane lipids. Fluorescence studies from our laboratory have identified discrete sites for fatty acids, phospholipids, and cholesterol on the AChR protein, and electron-spin resonance spectroscopy has enabled the establishment of the stoichiometry and selectivity of the shell lipid for the AChR and the disclosure of lipid sites in the AChR transmembrane region. Experimental evidence supports the notion that the interface between the protein moiety and the adjacent lipid shell is the locus of a variety of pharmacologically relevant processes, including the action of steroids and other lipids. I surmise that the outermost ring of M4 helices constitutes the boundary interface, most suitable to convey the signals from the lipid microenvironment to the rest of the transmembrane region, and to the channel inner ring in particular.

Charged amino acid motifs flanking each extreme of the alphaM4 transmembrane domain are involved in assembly and cell-surface targeting of the muscle nicotinic acetylcholine receptor.

The alphaM4 transmembrane domain of the nicotinic acetylcholine receptor (AChR) is flanked by two basic amino acids (His(408) and Arg(429)) located at its cytoplasmic- and extracellular-facing extremes, respectively, at the level of the phospholipid polar head regions of the postsynaptic membrane. A series of single and double alphaM4 mutants (His(408)Ala, Arg(429)Ala, Arg(429)Glu, His(408)Ala/Arg(429)Ala, and His(408)Ala/Arg(429)Glu) of the adult muscle-type AChR were produced and coexpressed with wild-type beta, delta, and epsilon subunits as stable clones in a mammalian heterologous expression system (CHO-K1 cells). The mutants were studied by alpha-bungarotoxin ([(125)I]alpha-BTX) binding, fluorescence microscopy, and equilibrium sucrose gradient centrifugation. Cell-surface [(125)I]alpha-BTX binding diminished approximately 40% in His(408)Ala and as much as 95% in the Arg(429)Ala mutant. Reversing the amino acid charge (e.g., Arg(429)Glu) abolished cell-surface expression of AChR. Fluorescence microscopy disclosed that AChR was retained at the endoplasmic reticulum, with an enhanced occurrence of unassembled AChR species in the mutant clones. Centrifugation analysis confirmed the lack of fully assembled AChR pentamers in all mutants with the exception of His(408)Ala. We conclude that His(408) and Arg(429) in alphaM4 are involved in assembly and cell-surface targeting of muscle AChR. Arg(429) plays a more decisive role in these two processes, suggesting an asymmetric weight of the charged motifs at each extreme of the alpha subunit M4 transmembrane segment. (c) 2006 Wiley-Liss, Inc.

Cells defective in sphingolipids biosynthesis express low amounts of muscle nicotinic acetylcholine receptor.

The properties of the nicotinic acetylcholine receptor (AChR) are modulated by its lipid microenvironment. Studies of such modulation are hampered by the cell's homeostatic mechanisms that impede sustained modification of membrane lipid composition. We have devised a novel strategy to circumvent this problem and study the effect of changes in plasma membrane lipid composition on the functional properties of AChR. This approach is based on the stable transfection of AChR subunit cDNAs into cells defective in a specific lipid metabolic pathway. In the present work we illustrate this new strategy with the successful transfection of a temperature-sensitive Chinese hamster ovary (CHO) cell line, SPB-1, with the genes corresponding to the four adult mouse AChR subunits. The new clone, SPB-1/SPH, carries a mutation of the gene coding for serine palmitoyl transferase, the enzyme that catalyses the first step in sphingomyelin (Sph) biosynthesis. This defect causes a decrease of Sph de novo synthesis at non-permissive temperatures. The IC50 for inhibition of alpha-BTX binding with the agonist carbamoylcholine exhibited values of 3.6 and 2.7 microm in the wild-type and Sph-deficient cell lines, respectively. The corresponding IC50 values for the competitive antagonist D-tubocurarine (D-TC) were 2.8 and 3.4 microm, respectively. No differences in single-channel properties were observed between wild-type and mutant cell lines grown at the non-permissive, lipid defect-expressing temperature using the patch-clamp technique. Both cells exhibited two open times with mean values of 0.35 +/- 0.05 and 1.78 +/- 0.2 ms at 12 degrees C. Taken together, these results suggest that the AChR is expressed as the complete heteroligomer. However, only 10-20% of the total AChR synthesized reached the surface membrane in the mutant cell line and exhibited a higher metabolic turnover, with a half-life about 50% shorter than the wild-type cells. When control CHO-K1/A5 cells were treated with fumonisin B1, an inhibitor of sphingosine (sphinganine) N-acetyltransferase (ceramide synthase), a 45.5% decrease in cell surface AChR expression was observed. The results suggest that sphingomyelin deficiency conditions AChR targeting to the plasma membrane.

Mutations at lipid-exposed residues of the acetylcholine receptor affect its gating kinetics.

The firmest candidate among the transmembrane portions of the nicotinic acetylcholine receptor (AChR) to be in contact with the lipid bilayer is the fourth segment, M4. To explore the contribution of alphaM4 amino acid residues of mouse AChR to channel gating, we combined site-directed mutagenesis with single-channel recordings. Two residues in alphaM4, Cys418 and Thr422, were found to significantly affect gating kinetics when replaced by alanine. AChRs containing alphaC418A and alphaT422A subunits form channels characterized by a 3- and 5-fold reduction in the mean open time, respectively, suggesting an increase in the closing rate due to the mutations. The calculated changes in the energy barrier for the channel closing process show unequal and coupled contributions of both positions to channel gating. Single-channel recordings of hybrid wild-type alpha/alphaT422A AChR show that the closing rate depends on the number of alpha subunits mutated. Each substitution of threonine to alanine changes the energy barrier of the closing process by approximately 0.5 kcal/mol. Recordings of channels activated by high agonist concentration suggest that these mutations also impair channel opening. Both Cys418 and Thr422 have been postulated to be in contact with the lipid milieu and are highly conserved among species and subunits. Our results support the involvement of lipid-exposed residues in alphaM4 in AChR channel gating mechanism.

Apoptosis of retinal photoreceptors during development in vitro: protective effect of docosahexaenoic acid.

When rat retinal cells are cultured in a serum-free medium, the photoreceptor cells start dying after 7 days. The addition of docosahexaenoic acid (DHA) to the cultures prevents the selective death of photoreceptors. Here it is shown that, unlike other retinal neurons, photoreceptors die through an apoptotic pathway. Hallmarks of apoptosis, such as nuclear fragmentation and condensation and DNA cleavage forming a ladder pattern on an agarose gel, were observed. The timing and high selectivity of the triggering of photoreceptor cell apoptosis suggest the existence of a programmed cell death. Compared with other fatty acids, DHA not only was the most effective in promoting photoreceptor survival, but also the only one to decrease the number of apoptotic nuclei. The results suggest that DHA is important among the factors preventing apoptosis of photoreceptors in the developing retina. A limitation in the availability of this fatty acid might trigger apoptosis as a result of the failure to develop functional photoreceptor outer segments.