Physiol. Res. 53 (Suppl. 1): S103-S113, 2004 Activation and Modulation of Ligand-Gated Ion Channels J. KRŮŠEK, I. DITTERT, T. HENDRYCH, P. HNÍK, M. HORÁK, M. PETROVIC, M. SEDLÁČEK, K. SUŠÁNKOVÁ, L. SVOBODOVÁ, K. TOUŠOVÁ, E. UJEC, V. VLACHOVÁ, L. VYKLICKÝ, F. VYSKOČIL, L. VYKLICKÝ JR Department of Cellular Neurophysiology, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Received January 20, 2004 Accepted March 1, 2004 Summary Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs. Key words Acetylcholine receptor • GABA receptor • glutamate receptor • NMDA receptor • Vanilloid receptor • TRP receptor • Ionic channel Introduction and Ladislav Vyklický Sn. became founders of the laboratory. Their stays in the prominent laboratories The aim of this review is to outline the current abroad (Dept. of Biophysics, University College London, research interests of the members of the Department of Prof. B. Katz; Dept. of Physiology, Aberdeen, Prof. Cellular Neurophysiology of the Institute of Physiology Malcolm; Dept. of Physiology, University Gothenburg, ASCR celebrating its fiftieth anniversary of foundation. Prof. A. Lundberg) greatly influenced further scope of the The intellectual roots of the Department can be traced to Department http://www2.biomed.cas.cz/d331/index.html early sixties of the past century when an that soon became a part of the world scientific electrophysiological laboratory was founded as a community. The advent of new electrophysiological subdivision of the Department for the study of techniques, mostly the "patch clamp technique", neurotrophic functions headed by Prof. Ernest Gutmann. complemented with the original techniques for fast Three young scientists Drs. Radan Beránek, Pavel Hník application of drugs made it possible to study the PHYSIOLOGICAL RESEARCH ISSN 0862-8408 © 2004 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Fax +420 241 062 164 E-mail: [email protected] http://www.biomed.cas.cz/physiolres S104 Krůšek et al. Vol. 53 mechanisms of glutamate, acetylcholine and vanilloid affinities of both binding sites in the adult receptor (Akk receptor channel activation and modulation that and Auerbach 1996) enable to achieve theoretically the represent the major research projects of the Department at highest values of Hill coefficient (H = 2) to be attained. present. In the embryonic receptor with two different binding sites Ion channels are macromolecular protein pores (Blount and Merlie 1989), the Hill coefficient decreases in cell membranes. Although the precise evolutionary as the affinities of both sites differ (Fig. 1). past of different groups of ionic channels is not known, they are widespread in virtually all organisms. Ion g channels mediate many physiologically important processes e.g. receptor signaling, membrane potential 1.2 maintenance, accumulation and transduction of 1.0 electrochemical energy, and generation and spreading of action potential. Similarly to other macromolecules, they t n 0.8 αβεδ (adult) e can exist in various states e.g. resting, desensitized, r αβγδ (embryonic) r H = 1.98 inactivated, activated and different states induced by H = 1.01 cu 0.6 various allosteric modulators. ve i Individual classes of channels differ according to t a l 0.4 the stimulus required for the ion channel to undergo e R transition from the closed into the open state. Ligand- 0.2 gated ion channels represent a specific group that is activated by signal molecules and are involved in 0.0 chemical signaling. The role of chemically activated -6 -5 -4 -3 -2 10-7 10 10 10 10 10 channels is crucial in synaptic transmission in the nervous ACh concentration (M) system and understanding their function and pharmacology has been in the focus of the neuroscience Fig. 1. The degree of cooperation between nACh binding sites research for many years (for review see Hille 2001). underlies differences in the dose response relationship between the adult (filled circles) and embryonic (open circles) form of the Receptor channel activation receptor. Dose-response graph of relative membrane currents from COS cells clamped at –40 mV expressing adult (α2βεδ) and embryonic type of nACh receptor (α2βγδ). Experimental points Del Castillo and Katz (1957) were the first who were fitted by the Hill equation (I(C)=CH/(CH+KH) where C is agonist concentration, I(C) is the relative amplitude of membrane proposed a simple scheme to describe activation of current at concentration C, K is the apparent dissociation ligand-gated ion channels (Colquhoun and Sakmann constant for the agonist, and H is the Hill coefficient). Note a 1985, Ogden and Colquhoun 1983, 1985, Vyklický et al. steep curve with a Hill coefficient near to 2 for adult receptor and gradually rising curve with a Hill coefficient near to 1 for 1988). The scheme originally proposed for activation of embryonic receptor. Higher value of the Hill coefficient in the nicotinic acetylcholine (nACh) receptors is useful in adult receptor corresponds to more equivalent agonist binding sites than in the embryonic receptor. revealing fundamental principles of receptor channel activation and particularities of their biophysical properties that often have biological consequences. N-methyl-D-aspartate (NMDA) receptors are The muscle nACh receptor is activated from two ligand-gated channels that upon activation are permeable nonequivalent binding sites localized on the boundary for Ca2+ and monovalent cations. They are fundamental between α subunit and neighboring δ, γ or ε subunits (for for excitatory neurotransmission and normal CNS review see Arias 1997). It is still a matter of speculation function (for review see Kemp and McKernan 2002). whether the apparent cooperativity of cholinergic They share the same activation principles with other activation is inherent to a nonequivalence of binding sites ionotropic neurotransmitter receptors. Binding of the or to allosteric coupling of both binding sites. However, neurotransmitter – glutamate, increases the probability of the difference between the binding sites of embryonic and channel opening, however, this is not absolutely required adult receptors known from the literature could partially because the channel can open spontaneously even in the help to understand the observed difference in the Hill absence of glutamate (Tureček et al. 1997). Similar coefficient (Krůšek and Vyskočil 2003). Identical spontaneous openings were described for the nACh receptor channels (Jackson 1984, 1989, Jackson et al. 2004 Ligand-Gated Ion Channels S105 990). In this respect it is important to note that even in the within the homotetrameric channel complex (Vyklický et presence of saturating glutamate concentration, the al. 1999). However, the critical structural domains and probability of NMDA receptor channel opening is rather the mechanisms by which thermal stimuli translate into low, in the range of ~10 %, depending on the receptor TRPV1 channel gating were revealed recently (Vlachová subunit composition (Chen et al. 1999). This is in et al. 2003). contrast to the probability of opening of nACh receptors Noxious heat above ~43 °C, acidic pH (<6.8) or that is ~90 %. the irritant alkaloid capsaicin are required to open the A specific feature of NMDA receptor channel is TRPV1 channel and these stimuli act strongly that it is gated by two different agonists – the glutamate synergistically: capsaicin and acids lower the threshold (or NMDA; selective synthetic agonist) and glycine. for heat activation, while capsaicin-activated currents are Simultaneous binding of both agonists to specific augmented by heat or by lowering pH (Tominaga et al. recognition sites at the receptor is required for an increase 1998, Vlachová et al. 2001). Moreover, at room in the probability of channel opening (Benveniste et al. temperature and pH 7.3, TRPV1 behaves as a voltage- 1990, Johnson and Ascher 1987, Mayer et al. 1989a, gated outwardly rectifying channel since it can be Vyklický Jr. et al. 1990a). Although the binding sites for activated by depolarizing voltage steps in the absence of each agonist are localized at different subunits, the any agonist (Vlachová et al. 2003). The feature of presence of one agonist influences binding of the other. multimodality is well-characterized at the level of The binding of glutamate (or NMDA) to the receptor nociceptor, in particular, the nociceptive C fibers and it is reduces the affinity