Function and structure in glycine receptors and some of their relatives

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Abstract

In the field of ligand-gated ion channels, recent developments, both in the knowledge of structure and in the measurement of function at the single-channel level, have allowed a sensible start to be made on understanding the relationship between structure and function in these proteins. In this review, the cases of glycine, nicotinic ACh and glutamate receptors are compared and contrasted, and problems such as how binding of agonist causes the channel to open, and why partial agonists are partial, are considered. Some observations, both structural and functional, suggest that more attention needs to be paid to conformational changes that occur before the channel opens. Such changes might account for the interaction found between subunits of the glycine receptor while it is still shut and, perhaps, the agonist-dependent structural changes seen in AMPA receptors. They might also complicate our understanding of the binding-gating problem.

Section snippets

Nicotinic ACh receptors

Nicotinic ACh receptors have five subunits (two α and three non-α) arranged quasi-symmetrically around the channel. Our knowledge of structure comes mainly from the electron microscopy work by Unwin and co-workers on Torpedo receptors 5, 6, and from the crystal structure of the Lymnea stagnalis ACh-binding protein 7, 8. The latter is a soluble pentamer of five identical subunits, each with 210 amino acids (less than half the 437 residues of the human α1 subunit), and with 20–24% sequence

The link between agonist binding and opening of the channel

The current structural knowledge of the transduction mechanism is restricted largely to the nicotinic receptor, and even in that case, it is still relatively speculative. Unwin's view is summarized in Box 1.

Crystallographic data give us only a static picture of the receptor. An ambitious functional approach to the problem of how the different protein domains move upon activation is analysis of ‘linear free energy relationships’ in nicotinic receptors 1, 17, 18, 19. These studies found that

Measurement of function: binding and gating

To understand how binding of agonist leads to opening of the channel, the first step must be to measure separately the initial binding of the agonist to the shut receptor, and the effectiveness of bound agonist in opening the channel. Making this distinction is the so-called ‘binding–gating problem’ [20]. In most cases, it can be solved only by single-channel methods because whole-cell measurements of agonist potency (EC50) cannot give us the separate physical constants for binding or gating.

Are the binding sites the same in the resting state?

In the (adult) nicotinic receptor, the subunits are arranged (anticlockwise) as αεαδβ, with binding sites at the αδ and αε interfaces (these are called the a and b sites in Figure 3). Good fits are obtained with mechanisms that assume that these two sites are different in the resting state. The extent of the difference in the affinity for ACh varies depending on species 27, 28, 32, 33, 34, 35, 36. A standard mechanism with two different sites (representing αδ and αε sites) is shown in Figure

Do the binding sites interact while the channel is still shut? Is there a conformation change before the channel opens?

In the scheme shown in Figure 3(a), the possibility arises that the affinity (in the shut state) for the second binding event (equilibrium constant K2=k2/k+2) might not be the same as that for the first binding event (equilibrium constant K1=k1/k+1), even though the sites are initially identical. The same possibility arises in Figure 3(b–d), although now there are separate values for the two different sites. If the second binding is tighter than the first (K1>K2), this is usually described,

Homomeric channels

The only homomeric ion channel to have been analyzed in detail by single-channel methods is the glycine α1 receptor 29, 30. It seems likely that this homomeric pentamer would be symmetrical, and therefore that the five binding sites would be identical in the resting state, although crystallographic evidence is thin because of the paucity of protein structures with no ligand bound. Indeed, it was found that mechanisms (analogous with those in Figure 3b–d) with initially different,

The nature of partial agonists

It was first suggested by del Castillo and Katz [45] that a partial agonist was one for which the gating equilibrium constant E=β/α is small, so the maximum possible response E/(1+E) (i.e. the maximum fraction of open channels) is well short of 1. This of course begs the question of what structural features determine the value of E, but we are a long way from being able to predict that from first principles. For most receptors, Katz's explanation is likely to be essentially right. For example,

Conclusions and future work

Progress is being made rapidly but there is a long way to go. There are still no high-resolution crystal structures of entire receptors in the shut and open conformations, so the field is still well behind the position that haemoglobin reached in 1960s. Large numbers of mutations have been made, but even those that have been analyzed in detail (some are reviewed in Ref. [2]) often do not make much sense in our present state of knowledge. We are a long way from being able to explain (much less

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