PAnAChR Interaction

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As stated above, nAChR binds preferentially anionic lipids, which are positive modulators of its function. Among them, PA seems to interact in a special fashion with this protein. In vitro studies with nAChR reconstituted in lipid vesicles of controlled composition show that PA is among those phopholipids that bind the protein with a higher affinity, and it is the most effective lipid in preserving nAChR function (Jones and McNamee 1988; Marsh and Barrantes 1978; Ellena et al. 1983; Esmann and Marsh 1985; Dreger et al. 1997), possibly through a stabilization of the resting versus the desensitized state of the protein (da Costa et al. 2002). On the other hand, as if a bidirectional coupling takes place, nAChR in a PA-containing membrane leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid-crystal phase-transition temperatures of the reconstituted lipid bilayers (da Costa et al. 2002; Wenz and Barrantes 2005). This strong interaction leads to the segregation of a PA-enriched domain from a complex mixture of lipids at determined lipid-to-protein ratios (Fig. 8.4; Poveda et al. 2002; Wenz and Barrantes 2005). However, nAChR has no detectable effect on the lateral distribution of lipids when PA is substituted by other zwitterionic or anionic phospholipids such as PC, PG or phosphatidylserine (PS) (Poveda et

Fig.8.4. (a, b) Representative temperature dependence of the infrared CD2 symmetric stretching vibration from perdeuterated phospholipids contained in reconstituted vesicles. Vesicles were prepared by detergent dialysis, in the absence (open symbols) or in the presence {filledsymbols) of nAChR protein, from identical amounts of lipid mixtures containing 25 mol% of cholesterol, 50 mol% of egg PC and 25 mol% of either d-DMPC (panel a) or d-DMPA (panel b). Protein-containing samples were prepared at a protein-to-phospholipid molar ratio of 1:3500. CaF2 windows were used in the spectrometer cell, (c) Differential scanning calorimetry studies on the effect of nAChR on lipid organization in reconstituted vesicles. The scans correspond to: pure DMPA (a), 25 mol% of cholesterol, 50 mol% of egg PC and 25 mol% of DMPA with (b) or without (c) nAChR. (d) Fluorescence anisotropy of the transparinaric acid (t-PnA) probe incorporated into vesicles composed by 25 mol% of cholesterol, 50 mol% of egg PC and 25 mol% of either DMPA in the absence {open symbols) or in the presence {filledsymbols) ofnAChR protein

Fig.8.4. (a, b) Representative temperature dependence of the infrared CD2 symmetric stretching vibration from perdeuterated phospholipids contained in reconstituted vesicles. Vesicles were prepared by detergent dialysis, in the absence (open symbols) or in the presence {filledsymbols) of nAChR protein, from identical amounts of lipid mixtures containing 25 mol% of cholesterol, 50 mol% of egg PC and 25 mol% of either d-DMPC (panel a) or d-DMPA (panel b). Protein-containing samples were prepared at a protein-to-phospholipid molar ratio of 1:3500. CaF2 windows were used in the spectrometer cell, (c) Differential scanning calorimetry studies on the effect of nAChR on lipid organization in reconstituted vesicles. The scans correspond to: pure DMPA (a), 25 mol% of cholesterol, 50 mol% of egg PC and 25 mol% of DMPA with (b) or without (c) nAChR. (d) Fluorescence anisotropy of the transparinaric acid (t-PnA) probe incorporated into vesicles composed by 25 mol% of cholesterol, 50 mol% of egg PC and 25 mol% of either DMPA in the absence {open symbols) or in the presence {filledsymbols) ofnAChR protein al. 2002; da Costa el al. 2004), although the segregation of a saturated PC from an unsaturated PC by the action of nAChR has recently been reported (Wenz and Barrantes 2005). In this case the authors suggest that the maintenance of this domain is predominantly due to lipid-lipid interactions opposite to that with PA, more stable and mainly maintained by protein-lipid interactions. The PA domain has been detected either through fluorescence, or FT-IR and DSC techniques, the latter sensitive to macroscopic events, indicating that macrodomains should be formed. In addition, from resonance energy transfer experiments it has been shown that these domains are located next to the protein (Poveda et al. 2002).

Membrane phospholipids, including those interacting with membrane proteins, diffuse very fast, around 108 cm2 s"1 (Ellena et al. 1983; East et al. 1985), so the ability of nAChR to change the lipid lateral distribution segregating PA around it will dramatically enhance its interaction with this phospholipid, hence explaining the strong modulating effect of PA on nAChR.

There are several publications describing the segregation of lipid domains by peptides or extrinsic proteins but only one for a membrane protein, rhodopsin (Polozova and Litman 2000). This kind of domain, whose organization is directed by a transmembrane protein, seems opposite to that of the so-called "rafts" (Simons and Ikonen 1997), where it is postulated that the physical properties of lipids are responsible for the segregation. However, a "clustering" model has been proposed that could be valid for the nAChR-PA interaction. In it, the action of certain proteins could cluster little initial rafts dispersed in the membrane (Harder et al. 1998). On the other hand, there are some examples dealing with a special selectivity of certain proteins for PA, such as that of the vesicular-stomatitis virus envelope-proteins (Luan el al. 1995). In spite of the low PA concentration in host membranes, these proteins interact specifically with PA giving cause for PA domains, which is an essential event for new viral particles to be formed. Moreover, PA domains have been proposed as scavengers of other essential biological anionic phospholipids such as PIP2 (Denisov et al. 1998), so the domain formation could configure an important regulation site in the membrane. PS is another anionic phospholipid present at high levels in membranes from the Torpedo electric organ (Gonzalez-Ros et al. 1982), so it could be a good candidate to enter the PA domain as reported in the stomatitis virus, where PS is only segregated when PA is present (Luan et al. 1995).

The fact that PA is the main component of the nAChR segregated domain (Poveda et al. 2002) opens a new possibility to regulate nAChR activity by activation of phospholipase D, as reported previously in other systems (Exton 1990; Billah and Anthes 1990). In addition, the activity of this enzyme can be regulated through the agonist binding to receptors coupled to G-proteins. In this way, addition of phospholipase D to nAChR-enriched vesicles results in an increase in protein function (Bhushan and McNamee 1993). Finally, PA levels can be increased by phosphorilation by diacylglycerol kinase of diacylglycerols resulting from phospholipase C hydrolysis of phosphatidylinositol.

Another important question about the segregation of the PA domain is related to the determinants in the protein and in PA responsible of this strong interaction that finally leads to domain segregation. Calorimetric studies and other experiments using proteases point to the transmembrane segments as the main structure responsible for domain segregation (Poveda et al. 2002). It has been proposed that some positive charged amino acid present at the end of the nAChR transmembrane helices, such as Arg-429 or His-408 at the M4, could be responsible for the stronger binding to anionic phospholipids, although there are no definite results (Blanton and Wang 1990, 1991; Blanton and Cohen 1992). On the other hand, the exact determinants in the PA molecule that could explain its strong interaction with nAChR are also unknown and only general properties of this phospholipid have been pointed out, such as its negative charge, its very small headgroup or its high capacity to form hydrogen bonds (Baenziger et al. 1999). Evidently, the negative charge is not the only factor, as salt screening or pH titration does not destabilize the domain. Moreover, other anionic lipids are not segregated by nAChR. At this point it is important to stress that PA has a higher pKa when the protein is present, so its anionic charge is diminished (Poveda et al. 2002). This could facilitate the formation of the PA domains since a lower repul sion and an enhanced attraction through hydrogen bonds between PA molecules would occur (Garidel et al. 1997), decreasing the electrostatic contribution to the free energy of the system (Denisov et al. 1998) so as to overcome the entropic effect that favours the homogeneous mixing oflipid components.

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