Influence of Lipids on nAChR Function

LGICs are membrane proteins that transiently open a pore through the lipid membrane in response to neurotransmitter binding. The nAChR is one of the best-understood members of this family, principally due to two factors that have aided in its characterization: (1) the rich source of nAChR present in the electric organ of some fishes (T. marmorata, T. californica and E. electricus) and (2) the presence of neurotoxins in snake venoms that bind specifically to the nAChR providing the means for assaying receptor binding and for affinity purification.

nAChRs are heteropentamers comprised of four different but highly homologous subunits designated as a, p, y, and 6 (Fig. 8.1; for reviews see the references in Karlin 2002). Each subunit contains an extracellular N-terminal domain (which includes the ACh (acetylcholine) binding sites), four hydrophobic transmembrane (TM) domains (M1-M4), and a small extracellular C-terminal domain. Several studies have provided convincing evidence that the TM2 domain segments from each subunit cluster around a central axis to form the ion channel pore, whereas TM1 TM3 and TM4 domains are in close proximity or exposed to the lipid interface (Miyazawa et al. 2003; Barrantes 2003).

These receptors are implicated in the propagation of electrical signals between the cells at the neural and neuromuscular synapse. Upon activation by agonist, nAChRs transiently open a cationic channel responsible for the initiation of post-

Achr Vesicles
Fig. 8.1. (a) Schematic representation of the quaternary structure showing the arrangement ofthe AChR subunits in reconstituted vesicles, (b) Cross-sectional slab through the pentamer at the middle ofthe membrane showing the four transmembrane segments (M1-M4) of each subunit. Based on Unwin (2003)

synaptic membrane depolarization. In the continued presence of agonist, the nAChR become refractory to the stimulus and the ionic current declines. This process, called desensitization, occurs because the fully liganded receptor eventually adopt a stable, high-affinity conformation that is not permeable to ions.

Extensive biochemical studies have demonstrated that the ability of the nAChR to support ion channel function requires the presence of specific lipids. In 1978 Epstein and Racker opened the way for more detailed studies of the influence of the lipid environment on the nAChR by measuring in a reproducible manner integrated flux responses specifically induced by cholinergic agonist in reconstituted systems. Since then, many experiments reconstituting nAChRs into artificial liposomes of defined composition have shown that the presence of certain lipids in the reconstituted samples, namely cholesterol and acidic phospholipids, are important in preserving the ability of this protein to exhibit an optimal cation channel activity (Gonzalez-Ros et al. 1980; Criado et al. 1984; Fong and McNamee 1986; Jones et al. 1988; Sunshine and McNamee 1992; Fernández et al. 1993). Such lipid effects on nAChR function are also known to be fully reversible. For instance, Mc-Namee's group used the re-reconstitution approach (reconstituting the protein twice, first in a lipid matrix that does not allow nAChR function, then in whole asolectin lipids) to demonstrate that an apparently "inactive" nAChR regains its function upon a second reconstitution into an appropriate lipid matrix (Jones et al. 1988). Rapid-kinetics stopped-flow studies have demonstrated that the presence of PA in the reconstituted membranes maintains an optimal nAChR cation channel activity. On the other hand, reconstitution into cholesterol/zwitterionic phospholipids, in the absence of anionic phospholipids, causes a loss in nAChR function (authors' submitted manuscript; Fig. 8.2). The lack of ion channel activity in samples containing PC as the only phospholipid present has been reported previously, using several different chemical species of synthetic PCs (Fong and McNamee 1986; Ochoa et al. 1989; Sunshine and McNamee 1992) or egg yolk PC (Fernández et al. 1993). It seems that this lipid stabilizes the nAChR in a non-responsive, desensitized state. Also, the need of cholesterol and negatively charged

Fig. 8.2. Representative stopped-flow

pyrene tetrasulfonate entrapped into reconstituted nAcChR vesicles by externally added Tl+. The figure shows Tl+ influx responses to 500 ^M carbamylcholine exhibited by reconstituted nAcChRvesicles madefrom different lipid mixtures traces corresponding to the rapid collisional quenching ofthe fluorescence on 1,3,6,8-

Fig. 8.2. Representative stopped-flow

phospholipids, particularly PA, to retain nAChR function upon reconstitution has been widely documented (Criado et al. 1984; Fong and McNamee 1986; Jones et al. 1988; Sunshine and McNamee 1992; Fernández et al. 1993). Similarly, preliminary data using the approach of transplanting the nAChR from reconstituted vesicles to the plasma membrane of live Xenopus oocytes (Morales et al. 1995; see below), show that microinjecting samples reconstituted in whole asolectin lipids (fully active samples) or in just egg phosphatitylcholine (inactive samples) produce comparable agonist-induced nAChR ion currents upon incorporation of the protein into the host cell membrane (manuscript in preparation).

These effects of specific lipids in nAChR function may be exerted through binding to specific sites of the protein or by modification of the physical properties of the bilayer. Previous results have demonstrated that membrane lipids interact differentially with nAChR. For example, sterol, PA and fatty acid spin labels have a relatively high affinity for nAChR compared with other spin labelled phospholipids (Ellena et al. 1983).

Additionally, several lines of evidence indicate a separate binding site for neutral lipids, namely non-annular sites. McNamee's group used the ability of brominated lipids to partially quench the intrinsic or modified fluorescence of the nAChR to monitor contacts with the surrounding lipid in reconstituted membranes. They found that quenching of PC was independent of and additive with that due to brominated cholesterol derivatives (Jones and McNamee 1988). These results argue strongly for independent binding sites for cholesterol and phospholipids.

Although cholesterol may affect the nAChR directly, it definitely has profound effects on the structure of the membrane environment, most notably in changes of membrane order or fluidity. In earlier studies both the agonist affinity and ion flux seemed to require an optimal fluidity (Fong and McNamee 1986). However, subsequent studies showed that while the ion flux activity of the nAChR was strongly influenced by lipid composition (Fernández-Ballester et al. 1994), there was no correlation with membrane fluidity as measured by steady state ani-sotropy of membrane probes (Shunshine and McNamee 1994). Measurements of membrane fluidity showed that cholesterol further ordered membranes containing PC and PA, but other sterols, like androstanol, did not; however, both neutral lipids supported similar ion fluxes. Thus neutral lipids do not exert their effect on the nAChR by changing bulk membrane order. Nevertheless, effects on bulk membrane order are sometimes different from those at the protein-lipid interface and it is possible that protein promotes the lateral segregation of specific lipids that allows an optimal packing density (daCostaet al. 2002; Poveda et al. 2002).

Careful delipidation experiments showed that a lipid/protein mole ratio approximately below 45 caused irreversible inactivation of the nAChR, consistent with the requirement of an annular shell of lipids around the periphery of the hydrophobic region (Jones et al. 1998). This requirement for a lipid annulus of 40-50 lipid molecules is supported by a variety of spectroscopic techniques establishing the presence of a lipid phase associated with the protein that differs from the bulk bilayer lipids in terms of molecular motion (Antollini et al. 1996). It is also in good agreement with theoretical predictions, which suggests the presence of a inner shell annulus of approximately 42-51 lipid molecules (Barrantes 1993).

The nature of the molecular species making up this dynamic annulus has not been wholly ascertained, although it seems obvious that both neutral and negatively charged lipids must play a role. It becomes clear that the nAChR annular lipids are important for its correct functional activity, but the precise mechanism by which these annular lipids affect the nAChR is not yet known.

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