Having demonstrated the presence of nAChR subunit mRNA and protein in peripheral neuronal tissues, the next challenge was to demonstrate that these proteins were functional. The most convenient way of achieving this goal was to artificially express the protein products of the cloned subunits and measure the physiological responses to nAChR agonists. The Xenopus oocyte expression system has been widely used; for mammalian genes, mRNA was transcribed in vitro and injected into the oocyte, and for the avian genes, cDNA expression vectors with a promoter were used (Deneris et al., 1991, for review). The properties of cloned receptors have been reviewed by Sargent (1993), McGehee and Role (1995), and Lindstrom (1996). Growing concerns about potential differences between native receptors and those transiently expressed in oocytes led to the development of stable expression systems such as cell lines.
Demonstrations that cloned subunits could form functional receptors have been provided for most of the subunits isolated, except a6 and ß3. Mammalian a3ß2, a4ß2 (Boulter et al., 1987), mammalian a2ß2 (Wada et al., 1988), mammalian a2ß4, a3ß4, a4ß4 (Duvoisin et al., 1989), avian a4na4 (Ballivet et al., 1988), avian a3na4 (Couturier et al., 1990a), avian a7 (Couturier et al., 1990b), mammalian a7 (Seguela et al., 1993), a5 in conjunction with other a and ß subunits (Wang et al., 1996), and avian a8 (Gerzanich et al., 1994) form functional neuronal nAChRs in Xenopus oocytes. The most recent of the mammalian functional nicotinic receptors is composed entirely of homomeric a9 subunits (Elgoyhen et al., 1994).
Studies of the single channel properties of cloned receptors revealed differences in the conductance and kinetics when different subunits were expressed. For example, Papke et al. (1989) found that receptors assembled from a2ß2, a3ß2, or a4ß2 subunit pairs injected into Xenopus oocytes showed differing conductance states; Papke and Heinemann (1991) reported that a3ß4 expression in oocytes produced channels with increased probability of opening and increased bursting activity compared with channels formed from a3ß2 subunits. Thus, particular a and ß subunits can modulate or alter the physiological properties of neuronal nAChR channels. Indeed, Ramirez-Latorre et al. (1996) found that coexpression of a5 subunits with a4ß2 subunits in oocytes markedly increased the peak currents through nAChRs; Sivilotti et al. (1997) also found a change in conductance when a5 was coexpressed with a3ß4 in oocytes. Individual subunits can also contribute to the desensitization properties of the neuronal nAChR: comparison of a4na1 receptors with a3na1 receptors revealed that the latter showed stronger desensitization, indicating that a subunits can contribute to the desensitization properties of the nAChR (Gross et al., 1991), as can ß subunits because desensitization was more rapid in a3ß2 receptors than in a3ß4 receptors (Cachelin and Jaggi, 1991). Co-expression of a5 with a3ß2 or with a3ß4 increased the desensitization rates of both receptor types (Wang et al., 1996). Receptors assembled from a3ß4 (Verino et al., 1992), a7 (Couturier et al.,
1990b; Seguela et al., 1993), a8 (Gerzanich et al., 1994), and a9 (Elgoyhen et al., 1994) have all been reported to be permeable to Ca2+ ions.
Pharmacological studies with cloned receptors indicate that both a and ß sub-units contribute to agonist potency. Gross et al. (1991) found that a3na1 receptors were less sensitive to ACh than a4na1 receptors; differences in agonist sensitivity of a3 receptors were also found depending on whether a ß2 or ß4 subunit was coexpressed (Cachelin and Jaggi, 1991). Using different combinations of a2, a3, a4, ß2, and ß4, Luetje and Patrick (1991) found that each subunit combination gave distinct patterns of agonist sensitivity. Covernton et al. (1994) reported differing orders of agonist potency depending on whether a3 was expressed with ß2 or ß4. The a5 subunit was found to increase the potency of ACh and nicotine at receptors containing a3ß2 subunits (Wang et al., 1996) (Figure 4.6).
Thus, the diversity of subunits available to form functional neuronal nAChRs in peripheral neurons can potentially contribute to the diversity of the physiological and pharmacological properties of the receptors. But do native nAChRs exhibit the properties of the cloned receptors, as described in these studies? Colquhoun and colleagues have begun to compare the properties of cloned receptors with those of native receptors expressed by rat SCG neurons. An initial comparison of agonist potency indicated that nAChRs in rat SCG showed a similar, but not identical, agonist potency profile to a3ß4 receptors in oocytes (Covernton et al., 1994). Analysis of single-channel properties did not identify a channel of identical conductance to rat SCG nAChRs among a3ß4, a4ß4, or a3ß4a5 subunit combinations in oocytes (Sivilotti et al., 1997). Lewis et al. (1997), looking at single-channel conductance as well as agonist potency, found closer similarities between cloned (a3ß4) receptors expressed in a stable cell line and rat SCG nAChRs, than between a3ß4 receptors expressed in oocytes. The difficulties in comparing expressed receptors with native receptors might in part be explained by the fact that nAChR subunits in cells may combine with other subunits in the same superfamily but form different types of channels (Elsele et al., 1993), or they may be associated with other proteins that may modify their selectivity to agonists. Also, nAChR subunits may be at various states of phosphorylation, resulting in changes in many of their properties.
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