Trends in Neurosciences
Function and structure in glycine receptors and some of their relatives
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=k−2/k+2) might not be the same as that for the first binding event (equilibrium constant K1=k−1/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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