What single-channel analysis tells us of the activation mechanism of ligand-gated channels: the case of the glycine receptor

Abstract

Glycine receptors are, in several ways, the member of the nicotinic superfamily that is best-suited for single-channel recording. That means that they are ideal for testing ideas about how activation proceeds in a ligand-gated ion channel from the binding of the agonist to the opening of the channel. This review describes the quantitative characterization by single-channel analysis of a novel activation mechanism for the glycine receptor. The favourable properties of the glycine receptor allowed the first detection of a conformation change that follows the binding of the agonist but precedes the opening of the channel. We used the term 'flipping' to describe this pre-opening conformational change. The 'flipped' state has a binding affinity higher than the resting state, but lower than the open state. This increased affinity presumably reflects a structural change near the agonist binding site, possibly the 'capping' of the C-loop. The significance of the 'flip' activation mechanism goes beyond understanding the behaviour and the structure-function relation of glycine channels, as this mechanism can be applied also to other members of the superfamily, such as the muscle nicotinic receptor. The 'flip' mechanism has thrown light on the question of why partial agonists are not efficacious at keeping the channel open, a question that is fundamental to rational drug design. In both muscle nicotinic and glycine receptors, partial agonists are as good as full agonists at opening the channel once flipping has occurred, but are not as effective as full agonists in eliciting this early conformational change.

Lucia Sivilotti (University College London, UK) qualified as a Pharmaceutical Chemist at the University of Ferrara in Italy. After postgraduate work in Ferrara and Milan on transmitter release in the CNS, she was awarded travelling fellowships by the Royal Society and the Italian Ministry of Education to work at St Bartholomew's College in London. Her project there (on field potentials recorded from the in vitro frog visual system) led to the characterization of the GABA_C receptor and the award of a PhD in 1988. From her days as a postdoc in David Colquhoun's lab in the Pharmacology Department at University College London, the focus of her research has been on channels in the nicotinic superfamily and how they work as single molecules.

Figure 1

A diagrammatic example of how an activation mechanism is fitted to sets of single-channel data with the HJCFIT program.

Figure 2

The extended version of a Jones & Westbrook-type mechanism (Jones & Westbrook, 1995) used to fit the single-channel activity of glycine heteromeric receptors. Up to three molecules of agonist A can bind to the resting receptor R (middle row, black). After binding glycine, the receptor can open (AR* states, bottom row, red) or desensitise (AD states, top row, green). The equilibrium constants are K (equilibrium dissociation constant), E (the equilibrium constants for the conformational change from resting to open, ratio between the opening and the closing rate) and D (the equilibrium constants for the conformational change from resting to desensitised, ratio between the rate of entry and that of exit from the desensitised states); numbers in small print shown by the arrows of the scheme are the values of the rates (or rate constants; units are s⁻¹ or s⁻¹m⁻¹, as appropriate) obtained by Burzomato et al. (2004).

Figure 3

The ‘flip’ mechanism (Burzomato et al. 2004) used to fit the single-channel activity of glycine heteromeric receptors. R denotes the resting conformation (black, top row), F flipped (dark red, middle row, see text) and F* open (red, bottom row). The equilibrium constants are K_R and K_F (equilibrium dissociation constant for the resting states or the flipped states, respectively), F (the equilibrium constants for the conformational change from resting to flipped, ratio between the rate of entry and that of exit to the flipped states) and E (the equilibrium constants for the conformational change from flipped to open, ratio between the opening and the closing rates); the numbers in small print shown by the arrows of the scheme are the values of the rates (or rate constants; units are s⁻¹ or s⁻¹m⁻¹, as appropriate) obtained by Burzomato et al. 2004).

Figure 4

Energy diagram and activation mechanism for glycine heteromeric receptors fully bound to the full agonist glycine (red) or to the partial agonist taurine (blue). In the energy diagram (A), the main difference between the two agonists is in the first step, the transition from resting to flipped. This is downhill for glycine, but uphill for taurine. The calculations used a frequency factor of 10⁻⁷ s⁻¹ (Andersen, 1999) and the lines are shifted vertically so they meet at the open state. B, the activation mechanism shows the values for the rates (numbers near the arrows, expressed in s⁻¹) and the equilibrium constants F₃ and E₃ for flipping and gating in the receptor saturated by glycine or taurine (abbreviated as Gly and Tau; values from Burzomato et al. 2004 and Lape et al. 2008).