Oocyte as a Cell Model for the Study of Lipid Protein Interactions

Most of the studies dealing with the functional and structural dependence of nAChR on its surrounding lipids have been carried out on model membrane systems to avoid the complexity of the cell membrane and to prevent the changes that a single variable can make in the whole system. Though these model systems are useful, providing a reductionism approach, we must develop novel methods allowing the study of the lipid-protein interaction in native cell membranes in order to confirm the results obtained in artificial systems.

One of the putative cell models for these studies is the Xenopus oocyte. These cells have been widely used for the biophysical characterization of many ion channels, neurotransmitter receptors and transporters, thanks to their ease of use, amenability for electrophysiological recordings and their capability to translate efficiently and faithfully exogenous mRNAs (Miledi et al. 1989; Soreq and Seidman 1992; Miller and Zhou 2000). Though Xenopus oocytes are capable of making a large number of post-translational modifications of the proteins coded by exogenous mRNA (such as acetylation, glycosylation or phosphorylation), and to assemble oligomeric receptor/channel complexes, they cannot always match the processing carried out by the cells that natively express them. Almost certainly, this is the reason for the failed or altered function of some foreign proteins expressed in oocytes. So, for instance, Torpedo nAChRs expressed in oocytes display an altered pattern of glycosylation (Buller and White 1990) and neuronal nAChRs do not exhibit the properties of native receptors, likely because oocytes fail to assemble their different subunits correctly (Sivilotti et al. 1997). Besides, there are specific lipid requirements of membrane proteins, which might constitutes a handicap for heterologous expression in a functional form (Opekarova and Tanner 2003). To overcome this handicap, nAChRs, and other membrane proteins, have been functionally transplanted to the Xenopus oocyte membrane by intracellular injection of plasma membranes (Marsal et al. 1995; Aleu et al. 1997; Sanna et al. 1998; Miledi et al. 2002; Palma et al. 2003; Miledi et al. 2004) or proteoliposomes bearing the purified protein (Morales et al. 1995; Le Caherec et al. 1996; Ivorra et al. 2002).

Oocyte injection of proteoliposomes bearing a purified protein, instead of fragments of cellular membranes, has several advantages:

(i) It allows the study of single molecular entities.

(ii) The transplanted protein does not need to be one of the most abundant in the cellular membrane, although the presence of a large amount of protein simplifies its purification.

(iii) It permits to study the influence that the lipid composition of the reconstitution matrix has on the functional properties of the transplanted protein.

This last point has special relevance, since many proteins are, and need to be, surrounded by specific lipids to develop their full functional activity (see above). Therefore, microtransplantation of purified proteins into the Xenopus oocyte membrane arises as an excellent way to unravel lipid-protein interactions, since it allows the insertion of proteins with specific lipids bound to them. Moreover, using this approach it is possible not only to change the ratio of different phospholipids surrounding the protein, to determine their functional relevance, but also the length of the acyl chains, to induce local changes in bilayer thickness and elasticity that might also be important for the protein activity (Martinac and Hamill 2002; Lundbaek et al. 2004).

Furthermore, an additional advantage of using Xenopus oocytes as the cell model for functional and biophysical studies of heterologous proteins is that their membrane lipid composition is well known (Caldironi et al. 1996; Stith et al. 2000) and can be, at least partially, customized. For instance, the cholesterol content in the oocyte membrane can be easily modified, inducing not only changes in bilayer stiffness but also in the functional activity of different proteins, including nAChRs (see above). The normal cholesterol/phospholipid (C/P) molar ratio in the Xenopus oocyte membrane (about 0.5) can be almost duplicated by incubating the cells in a solution containing cholesterol-enriched liposomes, whereas a significant decrease in this ratio is obtained by incubating them with methyl-P-cyclodextrin (Santiago et al. 2001). Likewise, the content of other specific lipid molecules can be modify either by oocyte incubation with lipid-defined liposomes or by activating specific pathways of lipid metabolism. It should be noticed that some lipids are charged molecules and hence certain changes in the lipid composition around some proteins, mainly ion channels, might affect their function by an electrostatic mechanism. So, it is well known that the ion channel biophysical properties can be modulated by fixed charges present in the protein itself or by charged molecules in its surroundings, specially phospholipids. This is because a charged surface in the neighbourhood of the ion channel influences the concentration of ions at the channel mouth and consequently its conductance (Latorre et al. 1992; Anzai et al. 1994).

Interestingly, the PA modulation of nAChR observed on in vitro systems has been corroborated in vivo using the Xenopus oocyte model (Morales et al. in preparation; Fig. 8.5). In these experiments, purified nAChRs reconstituted in either PA:PC:Chol (25:50:25 molar ratio), PC:Chol (75:25 molar ratio), or soybean lipids, are injected in oocytes, where they are efficiently inserted in the plasma membrane. Then, the functional activity, and properties, of the transplanted nAChRs are assessed using the voltage clamp technique. The amplitude of the acetylcholine (ACh)-elicited currents in the injected oocytes depended on the reconstitu-

tion matrix used. The ACh-current was higher when the nAChR was reconstituted in PA than when it was reconstituted either in soybean or PC lipids, which were very similar each other (see Fig. 8.5). This effect was not due to the different fusion efficiency of the different proteoliposomes to the oocyte membrane. It is worth noting that when nAChRs are reconstituted with those lipid mixtures in vitro, the activity is higher for soybean lipids than for the PA-mixture while no activity is found in PC:Chol mixtures. The fact that in the cell membrane the nAChR in PC:Chol reversibly recovers its function suggests that the system is sufficiently dynamic to allow the injected lipid around nAChR to be exchanged for the own oocyte membrane lipids. On the other hand, when reconstituted nAChR in PA is injected into oocytes, larger Ah currents were elicited suggesting that nAChR binds PA tightly, impeding its free exchange with other bulk membrane lipids and leading to the formation of a PA-rich domain segregated around the protein. The permanent interaction with PA, a positive modulator, would result in enhanced protein activity. An interesting observation that supports this hypothesis is the fact that, as nAChR is purified from the Torpedo electric organ, the PA content of the lipids which accompanies the protein is progressively increased from 0.51.6% up to 2.2-2.9% (Gonzalez-Ros et al. 1982).

Fig.8.5. Bar diagram showing the amplitude of the peak ACh (100 |jM) currents (lAch) elicited in oocytes previously injected with nAChRs reconstituted in asolectin (R-Aso, open bar), a mixture of PC (75%) and cholesterol (25%, R-PC+Cho, hatched bar) or a mixture of PA (25%), PC (50%), and cholesterol (25%, R-PA+PC+Cho, crossed bar). Values were normalized to the amplitude of the currents obtained in the R-Aso group. The inset shows a representative record of the lAch recorded in the R-Aso group. The arrow indicates the measurement of lAch and the bar indicates the ACh application time. In all experiments the membrane potential was held at -60 mV. The number of observations is given in brackets. Asterisks indicate significant differences with the R-Aso group (p < 0.01)

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Many more studies are needed on in vivo models to fully understand the functional modulation of membrane proteins by their surrounding lipids, but undoubtedly these are the first steps in this direction.

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