Structure

Subunit Composition

Muscle-type ACh receptors were first isolated from electric tissue of electric eel (Electrophorus electricus) and electric ray (Torpedo spp.), which are the richest sources [9]. The receptors in Torpedo electric tissue and in fetal muscle are heteropentamers, with the subunit composition a2Py§ (Fig. 1A) [10]. In adult muscle, the receptor contains an e subunit in place of the y subunit and has a greater singlechannel conductance and a smaller mean single-channel-open time than fetal receptor [11]. The neuronal-type ACh receptors are also pentamers, but of one, two, or three types of subunits [4,5]. There are nine known neuronal a subunits (a2 to a10) and three known neuronal P subunits (P2 to P4); various combinations of a subunits and P subunits, when expressed heterologously, yield functional complexes with distinct functional properties. Some neuronal a subunits (e.g., a7) form functional homopentamers. Different regions of the peripheral and central nervous system express different combinations of the neuronal subunits.

Figure 1 Structure of the nicotinic acetylcholine receptors. (A) Schematic representation of the quaternary structure showing the arrangement of the sub-units in the muscle-type receptors, the location of the two ACh binding sites between an a and a y subunit and an a and a 8 subunit and opening to the periphery of the complex, and the axial cation-conducting channel (dashed lines). (B). The threading pattern of the subunits through the membrane.

Figure 1 Structure of the nicotinic acetylcholine receptors. (A) Schematic representation of the quaternary structure showing the arrangement of the sub-units in the muscle-type receptors, the location of the two ACh binding sites between an a and a y subunit and an a and a 8 subunit and opening to the periphery of the complex, and the axial cation-conducting channel (dashed lines). (B). The threading pattern of the subunits through the membrane.

Primary Structure

The subunits are 450 to 650 residues long. The aligned sequences are sufficiently similar that their common origin is certain; that is, they are all homologous [5,12,13]. One completely conserved characteristic of all of these sequences is a 15-residue loop closed by a disulfide bond between two Cys residues separated by 13 conserved residues. All sub-units also contain four hydrophobic segments (M1-M4) sufficiently long to form membrane-spanning a-helices. The subunits of the ionophoric receptors for y-aminobutyric acid, glycine, 5-hydroxytryptamine, and an invertebrate glutamate receptor are homologous to those of the nicotinic ACh receptors, and these subunits constitute a superfamily of Cys-loop receptors. Conservation of primary structure implies conservation of three-dimensional structure as well.

Secondary and Tertiary Structures

The topology of these subunits with respect to the membrane has been worked out in Torpedo ACh receptor

(Fig. 1B) [12]. The N-terminal half of each subunit is extracellular. Three membrane-spanning segments (M1, M2, M3) follow. A long cytoplasmic loop connects M3 to a fourth membrane-spanning segment (M4), and a short C-terminal tail is extracellular. Each subunit is glycosylated at one or more extracellular sites.

The structure of the extracellular domain of the ACh receptor can be inferred from the crystal structure of a water-soluble ACh binding protein secreted by snail glial cells [14]. The ACh binding protein is a homopentamer of a subunit that is homologous to the extracellular, N-terminal half of the ACh receptor subunits. The ACh binding protein subunit starts at its N terminus with two short helices; the remaining sequence forms ten P-strands and connecting loops arranged in a modified immunoglobulin fold. There is less certainty about the structures of the membrane-embedded and cytoplasmic domains of the receptor. Patterns of reactivity, infrared spec-troscopy, and prediction algorithms indicate that the membrane-spanning segments are largely, but not completely, a-helical [12,15].

Quaternary Structure

The overall shape of the ACh receptor is known from cryo-electron microscopy of two-dimensionally ordered Torpedo receptors in membrane [16] and from the X-ray structure of the ACh binding protein [14]. The receptor is a narrow-waisted cylinder, roughly 120 A in length, of which 65 A is extracellular, 30 A spans the lipid bilayer, and 25 A is intracellular. The extracellular domain is 80 A in diameter. The five subunits are arranged like thick barrel staves around an axial channel. The channel lumen is about 30 A in diameter in the extracellular domain and tapers to less than 10 A in the membrane domain. It is possible that access to the channel on the cytoplasmic side is through gaps between the cytoplasmic domains of neighboring subunits [16].

ACh Binding Sites

The ACh receptor a subunit was originally identified as contributing to the ACh binding site by its specific reaction with a radioactive affinity label, and the two a subunits correspond to the two ACh binding sites per receptor complex. The labeling was mapped to aCys192 and aCys193, and these adjacent Cys were shown to be disulfide-bonded to each other [12], a rare arrangement. Four aromatic residues, well separated in the sequence aTyr93, aTrp149, aTyr190, and aTyr198, were also affinity labeled [17]. These six residues in a are at the interface between the a subunit and a neighboring subunit. They form the principal side of the ACh binding site [14].

In the muscle-type receptor, the complementary side of the first ACh binding site is formed by the y (or e) subunit, and the complementary side of the second ACh binding site is formed by the 8 subunit. Affinity labeling pointed to yTrp53, yLeu109, yTyr111, yTyr117, and the aligned residues in the 8 subunit as contributing to these complementary sides [18].

Mutations of each of the residues from the principal and complementary sides of the site affect agonist binding, gating kinetics, or competitive antagonist binding [17,19]. The location of the ACh binding sites in the interface between subunits is consistent with the idea that binding of ACh promotes relative movement of the subunits that propagates through the bilayer to the gate [12,16].

In the homopentameric ACh binding protein, each of the five binding sites is formed in the interface between opposite sides of adjacent subunits [14]. All of the ACh binding site residues identified in the muscle-type receptor a subunit are on one side of the ACh binding protein subunit, and all of the binding site residues identified in muscle-type y and 5 subunits are on the other side of the ACh binding protein subunit and form the complementary side of the binding site. The ACh binding site is a cage of mostly aromatic residues, the door to which is formed by the adjacent disulfide-bonded cysteines. Based on the ACh binding protein structure, the ACh binding sites in the ACh receptors are about 30 A above the membrane, opening to the outside of the cylindrical extracellular domain.

Acetylcholine and other specific ligands of the ACh binding site contain at least one quaternary ammonium group or protonated tertiary ammonium group. The preponderance of aromatic residues lining the binding site cavity is consistent with n-cation interaction between aromatic rings and quaternary ammonium groups [20]. In addition, there are conserved negatively charged residues in y and 5 in the vicinity of the binding site that might move toward a bound ammonium group as part of the conformational change leading to channel opening [19].

Channel

The channel has three tasks. It must mitigate the high-energy barrier to the translocation of an ion from one polar aqueous phase to another, through a nonpolar lipid membrane; it must select among ions both by size and by charge; and it must open and close. The energy barrier is partly mitigated by the funnel shape of the channel [16] and by its water content. Only a short section (~ 6 A long) near the cytoplasmic end is narrow enough to force water and a cation to move in single file [21]. It is in this section, where the translocating cations contact the walls, that size selection occurs and where some of the residues determining charge selectivity reside [12,17]. The resting-state gate may also be in this section [22].

The channel-lining residues were identified by photola-beling with channel blockers, by the effects of mutations, and by the accessibility to small, charged sulfhydryl reagents of residues mutated to Cys [12,17]. The pseudo symmetry of the ACh structure, the sequence similarity among subunits, and the labeling within the channel indicate that in het-eropentameric complexes the five subunits make similar but not identical contributions to the channel lining. Toward the extracellular side of the membrane where the channel is relatively wide, residues from both M1 and M2 form the lining.

Toward the intracellular side of the membrane, where the channel is narrowest, the lining is formed just by M2 and by the residues immediately flanking the cytoplasmic end of M2 in the M1-M2 loop [23]. In the open state, the M2 residues that are exposed in the channel lie within a 100° sector of a helical wheel. By contrast, the M1 residues exposed in the channel do not conform to a regular secondary structure.

It is likely that aligned residues in the sequence are also in register in the channel lining, forming pentameric rings of similar residues exposed at different levels of the channel [17]. The functional roles of some of these rings are clear. Starting from the cytoplasmic end of the channel, in the predicted M1-M2 loop, a ring of four glutamates and one glut-amine (five glutamates in homopentameric receptor) strongly determines the magnitude of cation conductance [24] and the intrinsic negative electrostatic potential in the channel [25]. Three steps in the extracellular direction, a ring of four or five threonines and serines strongly determines selectivity among different cations ([26]; also see Chapter 36). These two rings are in the narrowest part of the channel. Rings of hydrophobic residues further in the extracellular direction stabilize the resting state compared to the open state [12].

The structure of the channel is different in the resting, open, and desensitized states. These differences are reflected in electron microscope images of ordered arrays of receptors in membrane [16] and in the binding of channel-blocking aromatic amines, the reactions of channel-lining residues with photolabels, and the reactivity of substituted cysteines with charged sulfhydryl reagents [22]. The reactivity differences and the underlying structural changes are widespread, beyond the immediate vicinity of the gate. One view based on the accessibility of substituted cysteines from the two sides of the membrane is that the resting-state gate is in the same narrow region at the cytoplasmic end of the channel that forms the selectivity filter [22,27]. Another view based on cryo-electron microscopy is that this gate is in the middle of the membrane-spanning portion of the channel [16]. These views could be reconciled if the narrow part of the channel formed by the cytoplasmic end of M2 and the M1-M2 loop led into an antechamber that extended into the plane of the bilayer.

The charge selectivity of a homopentameric ACh receptor [17], as well as of the 5HT3 receptor [28], is changed from cationic to anionic by a minimum of three changes in the M1-M2 loop and in M2. Two of the changes are in the narrowest region; the glutamate is changed to glutamine, eliminating the negative charge, and a proline is inserted just before it, lengthening this region by one residue. A third change is two-thirds the distance toward the extracellular end of M2, where a valine is changed to a threonine. The new residues match those in the anion-conducting glycine receptor. The reverse mutations in the glycine receptor change its selectivity from anionic to cationic [29]. The mutation of the ring of glutamates indicates an electrostatic contribution to charge selectivity, but the bases for the effects on charge selectivity of the other two mutations are not known.

Cytoplasmic Domain

The cytoplasmic domain of each subunit consists of a short loop between Ml and M2 and a long loop between M3 and M4. As discussed above, the M1-M2 loop is involved in selectivity and gating. The much larger M3-M4 loop is involved in subunit assembly [30] and in targeting [31], clustering [32], and anchoring [33,34] of the assembled receptor in the postsynaptic membrane. Phosphorylation of sites in this loop modifies the rate of desensitization and may regulate interactions of the receptor with cytoplasmic proteins [35,36].

References

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24. Konno, T., Busch, C., Von Kitzing, E., Imoto, K., Wang, F., Nakai, J., Mishina, M., Numa, S., and Sakmann, B. (1991). Rings of anionic amino acids as structural determinants of ion selectivity in the acetyl-choline receptor channel. Proc. Roy. Soc. London Ser. B Biol. Sci. 244, 69-79.

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