Ion channels in the nicotinic superfamily: nicotinic receptors and glycine receptors
Professor Lucia Sivilotti
|AJ Clark Professor of Pharmacology|
|Tel: 020 7679 3358|
Professor Lucia Sivilotti graduated in Pharmaceutical Chemistry from the University of Ferrara in Italy . After graduate work in Ferrara and Milan on the modulation of 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 Hospital Medical College in London. This project (on GABA receptors in the frog visual system) led to the description of what is now called the GABAC receptor and the award of a PhD in 1988. After a career break for family reasons, she undertook postdoctoral work at UCL, first with Clifford Woolf in the Anatomy Department, then with David Colquhoun in Pharmacology. From there, she joined The School of Pharmacy as a lecturer in Pharmacology in 1997, before moving back to UCL in 2003. She is a co-tutor on the UCL Graduate School course Understanding ion channel currents in terms of mechanisms and became a Professor of Pharmacology in 2008.
We work at understanding the functioning of receptors that mediate fast synaptic transmission, and focus on two classes of ion channels in the nicotinic superfamily, nicotinic and glycine receptors.
Different types of nicotinic receptors (which are all excitatory and activated by the transmitter acetylcholine) mediate the initiation of muscle contraction by the peripheral terminals of motor neurones in the spinal cord and the regulation of involuntary bodily functions, such as blood pressure at the level of autonomic ganglia. Yet other forms of nicotinic receptors are present in the brain and are affected by the nicotine in tobacco smoke. Glycine receptors are activated by the simplest amino acid, glycine, and are inhibitory, acting to dampen excessive neuronal activity, particularly in the lower levels of the central nervous system, such as the spinal cord. For instance, without glycine transmission there would be no coordination between muscles that have opposite effects on the movement of a limb. In man, inherited mutations in nicotinic or glycine receptors are known to produce neurological disease.
Our general aim is to understand in quantitative terms how a receptor functions as a one-molecule machine. This is only possible because, thanks to the techniques of patch clamp and single channel recording, the activity of a single receptor molecule can be seen in real time. This type of work is important not just for neuroscience (i.e. for understanding synaptic transmission), but also for pharmacology. Indeed, to this day concepts that are central to pharmacology and receptor theory, such as partial agonism and efficacy, are well understood (i.e. can be quantitatively formulated) only for receptors that belong to the ligand-gated ion channel class (largely because of past research in this department).
Some of the questions being investigated include:
- How do differences in the properties of the several types of receptors derive from differences in the sort of protein subunits that make them up (each of which is produced by a different gene)?
- How does the binding of the transmitter to the receptor result in channel opening and what portions of the subunits are important in this process?
- How many molecules of transmitter need to bind for the channel to open efficiently?
- How do disease mutations in the amino acid sequence of subunits impair the working of the receptor?
A combination of electrophysiological recording and molecular biology techniques are used in the laboratory in order to obtain in cell cultures or in Xenopus oocytes receptors that are similar to those in the brain of humans and animals. We then record the electrical currents they produce both in their normal form and in forms that have been mutated. While a range of electrophysiological recording techniques are in everyday use in the laboratory, a strong focus is on single channel recording and its interpretation.
What makes a drug efficacious? Partial agonists on the glycine receptor
In the classical view, receptor activation by an agonist requires a binding step followed by a conformational change. For a channel the conformation change results in the opening of the pore. A partial agonist is one that has poor efficacy at eliciting the second step. Thus, when a channel has a partial agonist bound it will stay open for less, possibly much less, than 100% of the time.
By direct fitting of activation mechanisms to single channel data, we have recently described a novel activation mechanism for the effect of glycine on its synaptic receptor. We were able to identify a conformation change to a ‘flipped’ conformation that is entered after binding but before the channel opens. We can measure all 14 rate constants for this mechanism. Flipping may correspond to the closure of the binding pocket, an effect local to the extracellular domain which then spreads to the transmembrane domains and results in the opening of the pore.
Glycine (the normal synaptic transmitter on these receptors) is a full agonist that can keep the channel open 96% of the time and is efficacious in producing both flipping and opening. Taurine on the other hand is a partial agonist and, even when it saturates the channel, it can keep it open only for 54% of the time. We have recently shown that this is because the early flipping reaction is disadvantaged when taurine is bound. This pattern may apply to all partial agonists in the superfamily. It is now important to extend our results to other partial agonists, as measuring the affinity of different agonists to the flipped, pre-activated state will give us information on the structure of this intermediate state. However, we are severely restricted in our work by the limited range of partial agonists available for this receptor and plan to design and synthesise novel structures.
Initial studies will consider analogues of glycine, by varying the substitution at the α-carbon. It has already been found that alanine and serine act as partial agonists, however unfortunately they are still too potent for this particularly study. We will therefore initially try minor modifications on this theme, such as N-methylated alanine (1) and N-methylated serine (2), along with other amino acids such as threonine (3) and valine (4). β-Amino acids such as taurine (5) will also be investigated, through substitutions on the nitrogen, and at the α and β positions (Figure 1).
In addition to carrying out this general exploration of glycine analogues for partial agonism, we will also take a molecular modelling approach. By comparing all the known agonists, a molecular modelling programme such as Cresset (for which we have a license in the department of chemistry) can predict the active conformation they are adopting. This will be interesting in testing the hypothesis put forward by Schmieden that glycine receptor-agonists adopt a cis-conformation of the ammonium and carboxylate groups, while antagonists adopt a trans conformation. The same programme can then be used to predict novel agonists of this receptor by comparing the electronic ‘outer skin’ with a commercial database. It is hoped that this approach could lead to the discovery of completely novel glycine receptor agonists and antagonists.
Each of the resulting compounds from these studies would be screened for agonist activity at a macroscopic level - the more promising compounds (i.e. those with intermediate efficacy) would be used for detailed single-channel analysis. We have all the facilities needed for characterising the different agonists by patch-clamp recording of recombinant ion channels expressed in mammalian cell lines or Xenopus oocytes, by single channel recording as well as by macroscopic recording of dose-response curves or fast concentration jumps.
 Lape, Colquhoun & Sivilotti, Nature doi:10.1038/nature07139
 Schmieden, V.; Betz, H. Mol. Pharm., 1995, 48, 919-927
 Cheeseright, T.; Mackey, M.; Rose, S.; Vinter, A. J. Chem. Inf. Model., 2006, 46, 665-676
- Lape, R., Colquhoun,D. & Sivilotti L.G. On the nature of partial agonism in the nicotinic superfamily. Nature http://dx.doi.org/10.1038/nature07139
- Plested, Groot-Kormelink, Colquhoun & Sivilotti (2007) Single channel study of the spasmodic mutation alpha1 A52S in recombinant rat glycine receptors. J.Physiol, 581, 51-73. see comment in J.Physiol.. 581,3
- Beato & Sivilotti (2007) Single-channels properties of glycine receptors of juvenile rat spinal motoneurones in vitro. J.Physiol, 580, 497-506
- Beato, Burzomato & Sivilotti (2007) The kinetic of inhibition of rat recombinant heteromeric α1β glycine receptors by the low affinity antagonist SR-95531. J.Physiol, 580: 171-179.
- Groot-Kormelink, Broadbent, Beato &. Sivilotti (2006) Constraining the expression of nicotinic acetylcholine receptors using pentameric constructs. Mol. Pharm., 69, 558-63; see comment in Mol Pharm. 69, 407-10
- Broadbent, Groot-Kormelink, Krashia, Harkness, Millar, Beato & Sivilotti, (2006) Incorporation of the beta3 subunit has a dominant negative effect on the function of recombinant central-type neuronal nicotinic receptors. Mol Pharm, 70, 1350-7.
- Burzomato, Beato, Groot-Kormelink, Colquhoun & Sivilotti (2004) Single-channel behavior of heteromeric alpha1beta glycine receptors: An attempt to detect a conformational change before the channel opens J.Neuroscience, 24, 10924-10940.
- Beato, Groot-Kormelink, Colquhoun & Sivilotti (2004)The activation mechanism of alpha1 homomeric glycine receptors J.Neuroscience, 24, 895-906
- Colquhoun & Sivilotti (2004) Function and structure in glycine receptors and some of their relatives. Trends Neurosci., 27, 337-344.
- Groot-Kormelink, Broadbent, Boorman &. Sivilotti (2004) Incomplete incorporation of tandem subunits into recombinant neuronal nicotinic receptors. J. Gen. Physiol. 123, 697-708.
- Burzomato, Groot-Kormelink, Sivilotti & Beato (2003) Stoichiometry of recombinant heteromeric glycine receptors revealed by a pore lining region point mutation. Receptor and Channels, 9, 353-361.
- Boorman, Beato, Groot-Kormelink, Broadbent &. Sivilotti (2003) The effects of beta3 subunit incorporation on the pharmacology and single channel properties of oocyte-expressed human alpha3beta4 neuronal nicotinic receptors. J.Biol.Chem., 278, 44033-44040.
- Beato, Groot-Kormelink, Colquhoun & Sivilotti (2002) Openings of the rat recombinant alpha1 homomeric glycine receptor as a function of the number of agonist molecules bound. J. Gen. Physiol., 119, 443-466.
- Groot-Kormelink, Beato, Finotti, Harvey & Sivilotti, (2002) Achieving optimal expression for single channel recording: a plasmid ratio approach to the expression of alpha1 glycine receptors in HEK293 cells, J. Neurosci. Methods, 113, 207-214
- Groot-Kormelink, Boorman & Sivilotti (2001) Formation of functional alpha3beta4alpha5 human neuronal nicotinic receptors in Xenopus oocytes: a reporter mutation approach, Br. J. Pharmacol. , 134, 789-796
- Boorman, Groot-Kormelink & Sivilotti (2000) Stoichiometry of human recombinant neuronal nicotinic receptors containing the beta3 subunit expressed in Xenopus oocytes. J.Physiol ., 529,565-577.