Molecular Neurobiology and Genetics: Review Investigation of Neural Function and Dysfunction

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Neuron, Vol. 20, 427–444, March, 1998, Copyright 1998 by Cell Press Molecular Neurobiology and Genetics: Review Investigation of Neural Function and Dysfunction

Tim Green, Stephen F. Heinemann,† of related proteins, to defined genetic manipulation to and Jim F. Gusella*† create transgenic mouse “knockouts,” molecular neuro- Molecular Neurobiology Laboratory biology has underpinned many of our recent insights The Salk Institute for Biological Studies into how the brain functions. A consequence of these La Jolla, California 92037 developments is the identification and study of genes *Molecular Neurogenetics Unit that cause neurological disease, revealing the role that Massachusetts General Hospital affected genes play in normal nervous system function Charlestown, Massachusetts 02129 and increasing the chances for treatment and cure. Much work in the field of neurobiology has focused on the study of the synapse and membrane excitability, since it is generally believed that the regulation of these During the last 60 years, our understanding of the brain processes underlies much of higher brain function, in- has advanced at a spectacular rate due to the efforts of cluding memory, learning, and cognition. Synaptic trans- researchers trained in physics, mathematics, anatomy, mission is mediated by neurotransmitter molecules re- physiology, biochemistry, medicine, and most recently leased from the presynaptic cell, which activate two genetics and molecular biology. The differing perspec- classes of receptors. One is the G protein–coupled re- tives provided by these diverse specializations have ceptor class, in which ligand binding leads to activation provided insights into brain function that might have of G proteins and which usually operates in time frames been missed if neurobiology had been a more homoge- of seconds. The activated G proteins act as second neous discipline. Neuroscience is now an integral part messengers to regulate the metabolism of the cell, as of modern biology, thanks in part to the unifying effects well as by directly affecting channel proteins. These of the newest entrants to the field, geneticists and mo- “slow” G protein–coupled receptors play a role in modu- lecular biologists. This review will focus on some of the lating synaptic transmission, cell excitability, and sen- advances which have resulted from the application of sory systems. The other class of receptors is the neuro- molecular–genetic techniques to the study of the brain. transmitter- or ligand-gated ion channels, which on The human brain is estimated to have about a hundred ligand binding undergo a fast conformational change to billion nerve cells, two million miles of axons, and a open an integral pore within a time frame of 1–100 ms. million billion synapses, making it the most complex The majority of synaptic transmission in the brain is structure, natural or artificial, on earth. The advantage mediated by these “fast” receptors. and power of the genetic approach is grounded in the Following the old adage to write about what you know, fact that the information to build the brain is coded in and because of space limitations, we will restrict de- the genome, a much simpler structure than the brain tailed discussion to these “fast” receptors. The history itself. Mammals are thought to have about one hundred of the use of molecular techniques in neurobiology is in thousand genes, only a subset of which are expressed many ways encapsulated in the efforts to understand in the brain, and all of which will soon be sequenced on these channels. We present here a personal historical completion of the genome project a few years from now. view of the cloning of two neurotransmitter receptors: In addition, a steady stream of ingenious techniques are the first receptor type cloned, the nicotinic acetylcholine being developed to manipulate and analyze the genome receptor; and the cloning of the major excitatory recep- with ever increasing ease and power. tor class in the mammalian brain, the glutamate-gated Molecular cloning has made it possible to identify the ion channel receptor (GluR). In addition we discuss the genes and determine the primary structure of many of impact, advantages, and limitations of the molecular the proteins that mediate synaptic function and mem- approach in the investigation of channel structure and brane excitability. Site-directed mutagenesis and func- function and the mutations that have been discovered tional studies in heterologous expression systems have to cause brain dysfunction and disease. begun to elucidate how proteins such as ion channels work at the molecular level. High resolution structures of domains from channels and receptors are being ob- Ion Channel Cloning: an Historical Perspective tained for the first time, while structures for whole recep- Cloning of the Acetylcholine-Gated Ion tor molecules are hopefully not far behind. These struc- Channel Receptor tures will make it possible, at long last, to understand The contribution of cDNA cloning to the determination the mechanisms underlying channel gating and ion flow of primary structure is both obvious and overwhelming. at the atomic level. Most recently, the technology of The first receptor cDNAs cloned were four subunits of homologous recombination or “gene targeting” has the nicotinic receptor (nAChR), which is activated by the made it possible to create mutations in specific mouse neurotransmitter acetylcholine as well as nicotine, one genes and in defined brain regions, enabling the dissec- of the most widely consumed drugs. Cloning was made tion of the role of individual proteins in brain function. possible by the serendipitous evolution of two systems. From the initial use of gene cloning to identify families The first was the electric organ of Torpedo electric fish, a modified muscle that uses the nicotinic receptor ex- pressed at high levels to generate a large current to † To whom correspondence should be addressed. paralyze its prey. The second was the evolution of snake Neuron 428

Figure 1. Timelines of Cloning and Topological Models (A) The dates when the first members of each main subfamily were cloned are indicated for four of the main neuronal ion channel families. The asterisk denotes the publication of the first 55 amino acids of Torpedo nAChR subunit protein sequences (Raftery et al., 1980). (B) The topological models are placed to indicate the approximate dates that they were first proposed, where space allows. Membrane domains are shown as either rectangles for transmembrane domains or loops for reentrant domains. Regions implicated in the pore region are colored green, and the lipid bilayer is indicated in gray. In all cases, the extracellular space is to the top. References for topological models, reading left to right: nAChR-type: first and fourth (Claudio et al., 1983; Devillers-Thiery et al., 1983; Noda et al., 1983), second (Finer- Moore and Stroud, 1984), and third (Ratnam et al., 1986); voltage-gated: first (Noda et al., 1984), 8-crossing (data not shown; Greenblatt et al., 1985; Guy and Seetharamulu, 1986), second (Noda et al., 1986), and third (Guy and Conti, 1990); ATP-gated: first (Brake et al., 1994) and second (Rassendren et al., 1997); iGluR (ionotropic glutamate receptor): first (Hollmann et al., 1989), second (Seeburg, 1993), and third (Wo and Oswald, 1994; Hollmann et al., 1994; Bennett and Dingledine, 1995). peptide neurotoxins, which bind with high affinity and group at Stanford. At around the same time, Shosaku specificity to nicotinic receptors in the electric organ. Numa and colleagues in Japan and Jean-Pierre Chan- In the early 1970s, a number of laboratories built affinity geux and colleagues in France used the newly available columns with these neurotoxins and purified nicotinic protein sequence to clone the nicotinic receptor cDNAs. receptor from the electric organ of fishin milligram quan- Two years after the work was first started, cDNA se- tities. The availability of large quantities of nicotinic re- quences for all four constituent Torpedo nicotinic recep- ceptor made it possible to produce specific antibodies tor subunits were reported by these laboratories in the and to determine N-terminal protein sequence of the space of six months (Ballivet et al., 1982; Noda et al., nicotinic receptor subunits (Devillers-Thiery et al., 1979; 1982, 1983; Claudio et al., 1983; Devillers-Thiery et al., Raftery et al., 1980; reviewed by Karlin and Akabas, 1983; Figure 1A). This presaged a flood of nicotinic re- 1995). The availability of large qutities of purified recep- ceptor cloning by homology screening, including cDNAs tor also enabled the unambiguous determination of sub- coding for the related mammalian muscle nicotinic ace- unit stoichiometry, something that still eludes us for tylcholine receptor expressed at the neuromuscular many of the other receptors, and revealed the first in- junction. One unanticipated discovery was the finding stance of what has turned out to be a common theme that during the development of the neuromuscular junc- amongst ion channels: receptors made up of a number tion there is a switch in the composition of the structure of distinct but homologous subunits (Raftery et al., of the nicotinic receptor due to the substitution of the 1980). gamma subunit with the epsilon subunit, conferring new The work to isolate the cDNAs for the nicotinic recep- biophysical properties to the receptor (Mishina et al., tor began in the early 1980s. In the laboratories of Steve 1986). Heinemann and Jim Patrick at the Salk institute, Marc Perhaps one of the most important findings coming Ballivet started a project using immunological methods from the cloning of the nicotinic receptor has been the and cloning vectors newly developed by Paul Berg’s discovery of a large family of related genes that code Review: Green, Heinemann, and Gusella 429

for neuronally expressed nicotinic receptors. Using low kainate, or NMDA (Hollmann and Heinemann, 1994; stringency homology screening, the laboratories of Sprengel and Seeburg, 1995). The characterization of Boulter, Patrick, and Heinemann at the Salk Institute recombinant glutamate receptors has revealed a num- cloned the first nicotinic receptor subunit expressed in ber of unexpected properties. The first was that non- the brain, ␣3 (Boulter et al., 1986). Further screening NMDA receptor subtypes have a high permeability to led to the discovery of a large family of about a dozen calcium ions and that this is regulated by the subunit nicotinic receptor genes, expressed in almost every composition of the receptor (Hollmann et al., 1991). This region of the mammalian brain. In addition, these sub- has important implications, since it is well established units have been demonstrated to assemble in a com- that glutamate receptors are involved in the two best- binatorial manner, increasing the potential molecular studied examples of synaptic plasticity, long-term po- diversity of native nicotinic receptors. One of these sub- tentiation (LTP) and long term depression (LTD), which units, ␣7 (Couturier et al., 1990; Schoepfer et al., 1990), are thought to play a role in the formation of memory has proved extremely useful in molecular studies as (Bliss and Collingridge, 1993). The elevation of intracellu- a “model” nicotinic receptor, since it forms functional lar calcium is thought to be the signal that is essential homomeric channels. for initiating LTP and LTD, evidence that the regulation The cloning of large numbers of nicotinic receptor of calcium permeability is a crucial process for normal subunits was unexpected because there was, and still brain function (reviewed by Nicoll et al., 1988; Malenka is, little evidence for synaptic transmission mediated and Nicoll, 1990). The finding that excess calcium can by nicotinic acetylcholine receptors in the mammalian lead to neuronal cell death also suggests that the regula- brain. While synaptic transmission has not been demon- tion of calcium permeability plays an important role in strated, there is strong evidence that functional nicotinic maintaining the health of the brain (Choi, 1994). acetylcholine receptors are present in many areas of Permeability to calcium ions in non-NMDA receptors the brain, including the medial habenula, the interpedun- was found to be largely determined by a single amino cular nucleus, the retina, the lateral and medial genicu- acid in the ion channel. Receptor subunits containing late, and the neocortex. Working at the Salk Institute, the neutral amino acid glutamine have high calcium per- Ana Elgoyhen discovered the latest nicotinic subunit, meability, while subunits containing the basic residue ␣9, using a genomic screen. This subunit most likely arginine have low calcium permeability (Hume et al., forms a postsynaptic receptor mediating the cholinergic 1991; Verdoorn et al., 1991). As might have been ex- efferent input to hair cells of the inner ear (Elgoyhen et pected, the highly calcium-permeable NMDA receptors al., 1994). Further confirming the functional importance have the neutral amino acid asparagine at this position. of nicotinic receptors, a nonopioid analgesic was re- In AMPA receptors, permeability to calcium is deter- cently described that acted through selective modula- mined by the presence or absence of the GluR2 subunit. tion of neuronal nicotinic receptors (Bannon et al., 1998). Seeburg’s group made the discovery that in GluR2 the Mice with null or “knockout” mutations in two nicotinic residue at the Q/R site was determined by RNA editing receptor subunit genes, ␣7 and ␤2, have been reported (Orr-Urteger et al., 1997; Picciotto etal., 1998). Both mice (Sommer et al., 1991). The GluR2 gene codes for a gluta- are viable and have altered physiological responses to mine at the Q/R site, while virtually all GluR2 mRNA is nicotine in specific areas of the brain. Most significantly, edited to the codon for arginine, resulting in low calcium the mice lacking the ␤2 subunit have altered avoidance permeability. Investigation of the mechanism of editing learning and behavioral responses to nicotine (Picciotto revealed that the RNA editing process is dependent on et al., 1998). We expect that with further such character- intronic sequences 3Ј to the edited site (Higuchi et al., ization of the nicotinic receptors, the data will help to 1993; Egebjerg et al., 1994). The GluR5 and GluR6 kai- explain the powerful and diverse consequences of nico- nate receptors have now also been shown to be regu- tine consumption, including nicotine addiction. lated by RNA editing at the Q/R site (Kohler et al., 1993). Cloning of the Glutamate-Gated Ion For some glutamate receptor subtypes, the efficiency Channel Receptor of editing has been shown to vary during development In 1988, Michael Hollmann came to the Salk Institute and between different brain regions (Sommer et al., 1991; and began a project to clone the glutamate receptor by Paschen and Djuricic, 1994). Recently, a similar form of looking for functional expression from pooled cDNAs in RNA editing has been found in a G protein–coupled Xenopus oocytes. This “expression cloning” method, receptor activated by serotonin, the 5-HT2C receptor first developed by Shigetada Nakanishi’s group in Tokyo (Burns et al., 1997), making RNA editing an increasingly (Masu et al., 1987), was necessitated by the absence important modulator of brain receptor function. of high affinity ligands, which precluded methods rely- In common with virtually every other field of biological ing on the initial isolation of protein that had been research, transgenic mice are now being generated with used previously to clone neurotransmitter receptors and targeted mutations to glutamate receptor genes. Muta- channels. The successful cloning of the first glutamate- tions to the GluR2 gene have provided interesting addi- activated channel receptor, GluR1 (Hollmann et al., tions to the debate over the role of editing in receptor 1989), has been followed by the isolation of 17 related function. Mice lacking the GluR2 gene are viable, and genes in mammals, including subunits for the NMDA express calcium-permeable AMPA receptors (Jia et al., receptor, the first of which was cloned by Shigetada 1996). In contrast, when the intronic sequence control- Nakanishi’s group in Kyoto by expression cloning (Mori- ling editing was removed and replaced with a selec- yoshi et al., 1991). Most of the proteins encoded by tion marker sequence, the mice died as heterozygotes these genes have been shown to participate in func- (Brusa et al., 1995). The explanation for this difference tional glutamate receptors selective for either AMPA, awaits the arrival of transgenic mice with ever more Neuron 430

subtle mutations. The generation of transgenic mice is Ultimately, the question of how many sequences remain also beginning to illuminate the function of kainate re- to be discovered will be answered by another scion of ceptors, which is poorly understood in part due to the molecular biology, the comprehensive genome sequenc- lack of specific drugs. A newly described knockout of ing project. the kainate-selective GluR6 subunit exhibits reduced locomotion, altered synaptic physiology in the hippo- Ligand-Gated Channel Receptors: campus, and reduced susceptibility to kainate-induced Structures in Flux seizures (Mulle et al., 1998). Analysis of Primary and Secondary Structure Unsurprisingly, given the supposed role of NMDA re- Despite early (and continued) expectations that three- ceptors in a wide range of brain processes, null mutants dimensional structure would routinely be modeled from for the NR1 subunit are homozygous lethal (see Ebralidz primary sequence alone, little has been learned from et al., 1996; McHugh et al., 1996; Tsien et al., 1996). In deduced primary sequences in isolation. Rather, pattern work that perhaps foreshadows the future power and matching has been used to identify functionally impor- elegance of transgenic techniques, Tonegawa and col- tant domains. Initial topological models were based on leagues produced a mouse lacking the NR1 subunit only hydropathy plots, used to identify those regions most in the CA1 region of the hippocampus. The mutation is likely to be in the membrane (Kyte and Doolittle, 1982). only induced after birth, avoiding the complication of Figure 1B shows the development of these topological developmental artifacts. Analysis of these mice pro- models for four neuronal channel families. Modifications vided genetic evidence for the importance of both the were made to these models over time to fit emerging NMDA receptor and the CA1 region in LTP and spatial (and sometimes contradictory) data. It is also apparent learning (Ebralidz et al., 1996; McHugh et al., 1996; Tsien that models were heavily influenced, often erroneously, et al., 1996). This experiment provides the strongest by what was known of the topologies of the other chan- evidence so far that the most extensively studied form nel families at the time new subunits were cloned. These of synaptic plasticity, LTP, mediates some forms of models therefore demonstrate both the great strength learning. Null mutations have also been made for the of molecular biology, the generation of hypotheses and other NMDA subunits. Mice lacking NR2B die around provision of the tools to test them, and its great weak- birth, while NR2A and NR2C null mutants display vari- ness, taking the resulting models rather too seriously. ous, less severe, neuronal deficits (Ebralidz et al., 1996). Which of the current incarnations (Figure 2) will prove In work that provides an intriguing insight into the mech- to be correct remains to be seen, but a recurring limita- anisms underlying thesephenotypes, C-terminal trunca- tion of these models has been the difficulty in detecting tions of these same subunits also gave the same pheno- short membrane domains. An ␣ helix must be 25–30 types as the null mutants (Sprengel et al., 1998). In a final amino acids to cross the bilayer, but ␤ strands or other unexpected twist, mice lacking the “orphan” glutamate extended conformations require only 7–8 residues to receptor subunit ␦2, which has not yet been demon- cross, and reentrant loops can be any length and are strated to form part of a functional glutamate receptor, not necessarily hydrophobic. The general assumption had deficits in motor coordination, synapse formation, has been that the membrane domains are ␣ helices. and LTD (Kashiwabuchi et al., 1995). Taken together, However, in the 9 A˚ structure of the T. marmorata these results demonstrate the potential for transgenic nAChR, only the pore-lining domain has the electron techniques to confirm the expected, reveal the unex- density expected of an ␣ helix (Unwin, 1993b). This sug- pected, and perhaps ultimately help provide a complete gests that the other three hydrophobic domains may genetic dissection of brain function. instead cross multiple times as ␤ strands (Figure 2A). Ion Channel Cloning: Too Much of a Good Thing? The presence of ␤ structure is supported by spectro- Following the cloning of the nicotinic receptors, cDNAs scopic measurements of nAChR membrane domains for other receptors and channels were soon identified, (Go¨ rne-Tschlenokow et al., 1994), as well as data sug- using either protein sequence, homology screens, or gesting at least part of M1 has a nonhelical conformation functional expression. Beyond the four receptor families (Akabas and Karlin, 1995). To what extent the normally shown in Figure 1, a large number of cation channels rectangular representations of transmembrane domains (both voltage-gated and “open channel”) have also been in topological models are in reality zig-zag crossings identified and cloned, many having new and unantici- (with the intriguing potential for small segments to ex- pated structures (Figure 2; see also Armstrong and Hille, tend beyond the bilayer), will have to await high resolu- 1998, for a review of voltage-gated channels [this issue tion structures. of Neuron]). In addition, for many of the ion channels, Tertiary and Quaternary Structure numerous subunit subtypes have been identified, pro- The three dimensional (3-D) structure of synaptic ion viding, at least in theory, the potential for tremendous channels is perhaps the area in which molecular neuro- functional, temporal, and spatial diversity. The possible biology has had the least impact, but can be expected combinations appear far in excess of what is required to make the most significant contribution in the future. to explain the known functional diversity, much of which Beyond topological models,what we knowabout synap- is in any case derived from artificial pharmacological tic ion channel structure is essentially limited to the distinctions. This suggests a subtlety of function that Torpedo nAChR structure (Figure 3). This structure was may require additional analytical tools to characterize. determined by cryo–electron microscopy (cryo-EM) us- The rate of discovery of new receptor genes has inevita- ing tubular crystals grown directly from membranes iso- bly slowed, and the recent cloning of the GABAB receptor lated from the electric organ of Torpedo electric fish marks the identification and sequencing of the last (Unwin, 1993b). In this structure, the subunits are ar- known major receptor class (Kaupmann et al., 1997). ranged in a ring around the central ion channel, and Review: Green, Heinemann, and Gusella 431

Figure 2. A Topological Menagerie of Mammalian Neuronal Channels Domains are colored as for Figure 1. Except for the nicotinic receptor, no structure is implied for the transmembrane domains (rectangles). The agonists for the three ligand-gated channels are shown in red. (A) Nicotinic acetylcholine receptor family. Family includes both anion- and cation-selective channels. Ligand binds in the N-terminal domain, and all subunits have a conserved 13 amino acid disulfide loop. M1, M3, and M4 are thought to cross as ␤ strands, and M2 as an ␣ helix (see enlarged region). The channel is formed by the M2 segment from five homologous subunits (see inset). (B) Ionotropic glutamate receptor family. Channels are permeable to Naϩ,Kϩ, and Ca2ϩ. Ligand binds to two regions, one before M1 and the other between M3 and M4. The pore is formed by the reentrant loop M2, and current evidence suggests the channel is a pentamer. (C) P2X ATP-gated receptor. An ATP binding motif is present in the M1–M2 loop. Residues in M2 are involved in the pore (Rassendren et al., 1997), but the stoichiometry is unknown. (D) Voltage-gated ion channels (Armstrong and Hille, 1998). Channels highly selective for either Naϩ,Kϩ,orCa2ϩ. Voltage changes are detected by S4. The reentrant loop H5 contributes to the pore. Channels have 4-fold symmetry (inset), from either four internal repeats (Naϩ and Ca2ϩ) or four subunits (Kϩ). (E) Inward rectifying Kϩ channels. Channels are tetrameric (Yang et al., 1995), with two putative membrane domains and a reentrant loop. There is some evidence of an alternative topology with four membrane domains and no loop (Schwalbe et al., 1997). (F) Open rectifying Kϩ channels (Leonoudakis et al., 1998). Channels activate instantaneously, independent of voltage. Membrane domains and loops identified by hydropathy analysis and homology. Pore suggested to be formed by reentrant loops, although there is no experimental evidence for this. each contributes homologous sequences to the pore. see also Armstrong and Hille, 1998). The picture is less The same basic arrangement is generally assumed to clear for ionotropic glutamate receptors. The size of apply to the other neuronal ion channels, although such the native receptor complex determined by biochemical assumptions should be made with caution: for example, techniques is consistent with either four or five subunits the aquaporin water channels appear by low resolution (Brose et al., 1993). Electrophysiological analysis of EM to have a central pore, but the water is fluxed inde- mixed wild-type/mutant receptor populations supports pendently within each of the four subunits (Li et al., a pentameric structure (Ferrer-Montiel and Montal, 1996; 1997). Premkumar and Auerbach, 1997), although this is far The muscle nAChR is a pentamer composed of four from universally accepted and is the subject of active different subunits in the stoichiometry ␣2␤␥␦ or ␣2␤⑀␦ in research. The stoichiometry and arrangement of the the case of adult muscle. The other members of this ATP-gated and open rectifying channels shown in Figure family (see Figure 1A) are also pentamers, demonstrated 2 is currently unknown. either directly using EM (Nayeem et al., 1994; Boess et Further progress on 3-D structure determination has al., 1995), or indirectly using biochemical or electrophys- been limited, primarily due to the difficulties encoun- iological techniques (Langosch et al., 1990; Cooper et tered when working with membrane proteins. While het- al., 1991). In contrast, the voltage-gated ion channels erologous expression of soluble proteins has fueled the have 4-fold symmetry, either from the four internal re- enormous expansion in structure determination by X-ray peats in Naϩ and Ca2ϩ channels, or by the association crystallography and nuclear magnetic resonance (NMR) of four subunits as for Kϩ channels (Figures 2D and 2E; spectroscopy, membrane protein expression remains Neuron 432

subunits is to use molecular cloning techniques for the expression of functional subdomains for crystallization. This technique is being used increasingly to study solu- ble proteins that have proved refractile to expression or crystallization. This approach, however, requires accu- rate identification of functional domains so that indepen- dently folding units can be stably expressed. Not least because of the uncertainties in subunit topology, identi- fication of such domains in ion channels has been slow. This now appears to be changing, as independent functional domains from nACh receptors, glutamate re- ceptors, and voltage-gated channels are now being identified and expressed (e.g., Kuusinen et al., 1995; Kim et al., 1997; West et al., 1997). Indeed, the first domain structures are starting to appear. The structure of the well-characterized inactivation domain from an

N-type KV channel has been determined by NMR (Antz et al., 1997), and the structure of the tetramerization

domain from Shaker KV has just been determined by X-ray crystallography (Kreusch et al., 1998). This struc- ture reveals an unexpectedly narrow channel at the cen- ter of the tetramer. If this constriction indeed forms a cytoplasmic vestibule, the entryway to the pore is not only quite far from the presumed selectivity filter but is also only wide enough for dehydrated ions to pass. One prediction of the earlier biophysical studies is that at

some point the ions must be stripped of H2O as they pass through the channel (Armstrong and Hille, 1998). Further important functional insights will undoubtedly emerge as more structures are determined. It is also hoped that the next few years will yield a more reliable system for the recombinant expression of integral mem- brane proteins, an advance that would help reveal the structures of these proteins in the same way gene clon- ing has revealed their sequences over the last 15 years.

Channel Function: a Molecular Perspective Both ligand- and voltage-gated ion channels share a common functional paradigm. Detection of neurotrans- mitters or voltage changes is directly coupled, through the process of gating, to the opening of the permeation pathway. For several of these channels, significant prog- ress has been made in identifying the domains responsi- ble both for detection or ligand binding and ion perme- ation. In large measure, this progress has been due to the application of molecular biology, through the gener- ation of chimeric proteins and single-site mutants. Again, we will concentrate here on work relating to the Figure 3. Three-Dimensional Representation of the Torpedo Nico- ligand-gated channels; the voltage-gated channels are tinic Acetylcholine Receptor discussed in detail elsewhere in this issue (Armstrong The image is based on data at 9 A˚ resolution obtained using cryo– electron microscopy (Unwin, 1993b). The receptor is viewed from and Hille, 1998). the side (top) and from above (bottom). The top two-thirds of the Ligand-Gated Ion Channel Agonist Binding Sites -A˚ ) are located synaptically, followed by the mem- Of the three families of neurotransmitter-gated ion chan 70ف) receptor brane-spanning region (colored band) and the smaller cytoplasmic nels (Figures 2A–2C), the agonist binding sites for the domain. The subunits are located with 5-fold symmetry around the acetylcholine and glutamate receptors are best charac- central pore, visible in the bottom view. Reproduced from Unwin, terized. In nACh and related receptors, the agonist bind- 1993a, with permission. ing site is located in the large N-terminal domain before the first transmembrane domain. A combination of pho- problematic. Indeed, the membrane proteins for which toaffinity labeling and site-directed mutagenesis has led structures have been determined can all be purified in to the identification of regions involved in the agonist relatively large quantities from natural sources. One al- binding sites of the ion channel receptors for acetylcho- ternative to determining the structure of intact channel line (Changeux et al., 1992), glycine (Schmieden et al., Review: Green, Heinemann, and Gusella 433

1992; Rajendra et al., 1995) and GABA (Buhr et al., 1996; changes, but using current techniques it should soon Wingrove et al., 1997). In all these receptors, several be possible to identify the molecular changes underlying noncontiguous regions have been identified as being this theoretical framework. Part of the difficulty of study- important for ligand binding. The acetylcholine binding ing gating is that it is part of the same equilibrium with site in the Torpedo nicotinic receptor appears from the ligand binding, makingthe twoevents difficult to dissect. EM structure to be contained within the ␣ subunit, half- Individual residues might affect this equilibrium, and way up the extracellular domain (Unwin, 1993b). This therefore control gating, by stabilizing or destabilizing contrasts somewhat with data from photoaffinity label- the open or closed states or the transition between the ing suggesting the binding site might be located in the two. Intuitively, such residues would be expected to interface between subunits (Dunn et al., 1993) and evi- have interacting complements, which might be identi- dence that mutations within the non-␣ subunits affect fied by rescue of nonfunctional mutants. Such an ap- agonist binding. However, without a structure it is diffi- proach will have to await the generation of putative cult to distinguish between mutations that are directly gating mutants. To complicate the issue further, these within the binding site and those that perturb its struc- channels also have at least one state (desensitized) in ture indirectly. addition to open and closed. In the continued presence Regions comprising the agonist binding site of iono- of agonist, many receptors enter a closed, unresponsive tropic glutamate receptors have also been identified, in state. As with gating, the underlying mechanism is work that serves as a testament to the power of molecu- poorly understood, although two studies have now iden- lar techniques. This work has been guided by the obser- tified regions in the N-terminal domain mediating one vation, made when the first NMDA receptor subunit was type of desensitization in the NMDA receptor (Krupp et cloned, that part of the sequence of glutamate receptor al., 1998; Villarroel et al., 1998). More such studies will subunits is similar to that of the bacterial glutamine be needed, as it is likely that the first structures deter- periplasmic binding protein (GlnH). This similarity ex- mined will be of dissected subdomains, missing the tends across two noncontiguous domains of the recep- linkages crucial to understanding gating and desensiti- zation. A complete description of how channels open tor, one just before the first membrane domain M1 and and close remains one of the biggest challenges facing the second between M3 and M4 (by convention count- the field. ing the reentrant loop as M2), although it is very weak. The Ion Channel At the time, these two regions were thought to be on The pores of the neurotransmitter-gated channels are opposite sides of the membrane, but changes to the often visualized as constrictions whose size and charge topological models later placed them both on the out- properties determine channel selectivity. Despite their side (Figure 2B). different topographical architectures, the ion pores in The importance of thesedomains in agonist activation nicotinic and glutamate channels appear functionally of GluRs was first demonstrated with chimeras, con- similar. Both receptor classes have wide vestibules at structed betweenAMPA- and kainate-selective subunits each end of the channel, narrowing to a central selectiv- (Stern-Bach et al., 1994). This work showed that these ity filter of similar size (Bormann et al., 1987; Villarroel domains (termed S1 and S2) were together responsible et al., 1995). In molecular terms, a single region has for agonist selectivity. Based on this work, Kuusinen et generally been labeled as the pore-lining domain, for al. (1995) joined the GluR2 subunit S1 and S2 domains example M2 in nAChRs and the reentrant loops in the via a short linker sequence and expressed the construct voltage-gated channels and glutamatereceptors (Figure in insect cells. This fusion was both soluble and bound 2). While this is convenient, and these regions have been 3 to [ H]AMPA and other glutamate receptor ligands with elegantly demonstrated to form part of the permeation the expected affinities, demonstrating the sufficiency of pathway, it is probably an oversimplification. For the these domains for ligand binding. Furthermore, changes acetylcholine receptor, the architecture of the ion chan- to residues in both S1 and S2 have been found to affect nel has been investigated using a combination of photo- receptor function or ligand binding affinities, consistent affinity labeling, subunit chimeras, and single-site mu- with a role for these domains in agonist activation (e.g., tants. The second transmembrane domain (M2), was Uchino et al., 1992; Kuryatov et al., 1994; Paas et al., first identified as the pore region using a chimera be- 1996; Laube et al., 1997; Swanson et al., 1997). This tween the Torpedo and bovine nAChR ␦ subunits. The work has spawned numerous models of the S1/S2 bind- chimeras had the conductance properties correspond- ingsite, based on structures determinedfor the periplas- ing to the ␦ subunit M2 region they contained (Imoto mic binding proteins. Given the level of sequence simi- et al., 1986). Further experiments using open channel larity, the utility of these models probably lies more in blockers as photoaffinity labels identified residues inter- providing testable hypotheses than for rational drug de- acting with the pore (reviewed by Unwin, 1993a), and sign. In the long term, the ability to express the agonist rings of positive charges at each end of the channel binding domain as a soluble polypeptide will probably were demonstrated to affect ion flux using stepwise have the greatest impact if and when it leads to a 3-D mutagenesis (Imoto et al., 1988). structure. The most comprehensive studies are now being done Coupling of Binding to Channel Opening using the technique of cysteine-scanning mutagenesis. The mechanisms by which agonist binding is translated This involves the application of molecular biological and into channel opening is the least understood aspect of chemical techniques, by carrying out systematic muta- ion channel function, both for the ligand- and voltage- tion of single residues to cysteines and using two cyste- gated channels. Gating has long been described as a ine-labeling reagents having different permeability prop- “black-box” process involving allosteric conformational erties. This method provides a powerful technique to Neuron 434

probe the pore region (Akabas et al., 1992; Akabas et techniques, it became possible to determine the chro- al., 1994). In this way, pore-lining segments have now mosomal location of a genetic defect. This is accom- been identified in a wide range of channel families, and plished by tracking the inheritance of naturally occurring use of this technique should allow the identification of sequence variations (polymorphisms) in human DNA as most channel-lining domains. The results from this work they are transmitted through the generations of families are also often used as evidence of the secondary struc- with an inherited disorder (Gusella, 1986). By identifying ture of the regions studied. This aspect is less convinc- statistically significant coinheritance (“genetic linkage”) ing, however, given the necessary assumption that the of a DNA marker with the genetic defect, it is possible cysteine is not disturbing the structure and the likelihood to infer the presence of the disease gene in the same that even small distortions in structural elements will chromosomal vicinity as the polymorphic DNA sequence. affect the observed labeling pattern. The chromosomal assignment of the disease gene then Beyond the Ion Channel offers the possibility of identifying the culprit gene on It is known that the ligand-gated receptors can be linked the basis of its location in the genome without any prior to proteins inside the cell. This was first demonstrated knowledge of the nature of its protein product. This for the nicotinic receptor expressed in the electric organ strategy, which has become known as positional or loca- and muscle (see Scotlet al., 1993). Recent work has tion cloning, is applicable to any inherited disorder but demonstrated that glycine (Kirsch et al., 1991) and gluta- is particularly important for nervous system disorders mate receptors are also capable of binding to a diversity in which the functional complexity of the target tissue of cytoplasmic proteins (reviewed by Sheng, 1996; Ken- hampers the direct identification of defective proteins nedy, 1997). Mary Kennedy and her collaborators found (Gusella, 1989; Collins, 1990). that the NMDA receptor is associated with the postsyn- Mapping the Huntington’s Disease Gene aptic density (PSD) (Moon et al., 1994; reviewed by The first major success with this strategy came with Kennedy, 1997), which triggered a search for proteins the discovery of a DNA marker linked to Huntington’s that associate with glutamate receptors. Using the two- disease (HD), a then-enigmatic autosomal dominant dis- order that was best known as the affliction that felled hybrid genetic screen, Kennedy and Seeburg’s groups folk singer Woody Guthrie (Martin and Gusella, 1986). collaborated to demonstrate binding of the NMDA NR2 HD typically has its onset in mid-life (mean, 40 years)as ف -subunit to the PDZ domain of the PSD-95 protein, ex subtle, insidious, adventitious movements and gradually plaining the previous finding that the NMDA receptor is progresses to full-blown chorea that consumes all parts found in the PSD (Kornau et al., 1995). Morgan Sheng of the body. In addition to the writhing, dance-like move- and his colleagues demonstrated that the PSD-95 pro- ment disorder, HD also produces psychiatric changes tein can cause clustering of NMDA receptors and Shaker 15years afterف and cognitive decline with death ensuing potassium channels and in a nice experiment showed onset. The clinical signs are paralleled by a characteris- that the clusters are disrupted in a Drosophila PSD- tic pattern of progressive neuropathology that devas- 95 null mutant (Tejedor et al., 1997). Huganir and his tates the caudate nucleus and causes extensive neu- colleagues discovered a protein they termed GRIP, ronal loss throughout the basal ganglia and cortex. In which binds through PDZ domains to the AMPA receptor 1983, we used two large HD families, one American and GluR2 subunit (Dong et al., 1997). There is increasing one Venezuelan, to map the HD gene to chromosome evidence that these receptor-associated proteins are 4, accomplishing the first step in our goal to identify the involved in a number of functions such as clustering nature of this genetic defect (Gusella et al., 1983, 1984). and localizing the receptor to specific regions in the cell, Mapping Other Neurogenetic Disorders regulating receptor function, and catalyzing posttransla- The success in HD demonstrated the power of the ge- tional modifications of receptors. The discovery that netic linkage strategy and set off a torrent of similar ligand-gated channel receptors associate with cyto- studies in a wide variety of inherited disorders, including plasmic proteins raises the possibility that the binding many nervous system diseases. Within the next decade, of neurotransmitter to the receptor may directly activate linked DNA markers were found for such neurogenetic a signal transduction pathway within the cell, in addition disorders as Alzheimer’s disease, Lou Gehrig’s disease to gating or opening the receptor channel and catalyzing (amyotrophic lateral sclerosis or ALS), neurofibromatosis ion flow through the membrane. This possibility is sup- 1 and 2, ataxia telangiectasia, and torsion dystonia (Ta- ported by the recent report that AMPA receptors can ble 1). Ultimately, the ability to attack genetic disease activate G proteins in the absence of an ion flow (Wang with this approach was a major impetus for the initiation et al., 1997b). of the Human Genome Project to sequence the entire genome and to identify all its genes. Indeed, the early An Alternative Molecular Biological Approach days of the Human Genome Project were aimed at satu- to Nervous System Function rating the human chromosomes with DNA markers by Neurologic Disease Genes—the Indirect Approach identifying and mapping highly polymorphic stretches While cDNA cloning based on protein sequence and of dinucleotides and other short repeats in human DNA functional characteristics was crucial for identifying to facilitate the positional cloning strategy. Disease gene genes of known importance for brain function, such as isolation was also a driving force for the development ligand-gated ion channel receptors, a valuable approach of new physical mapping and large-insert cloning tech- for identifying unknown mutant genes that cause neuro- nologies. logic dysfunction also emerged in the 1980s. By merging Cloning Based on Map Location the tenets of Mendelian genetics with the extensive vari- When the HD locus was mapped, the molecular biologi- ation in human DNA detectable with molecular biological cal tools for cloning and assessing several million base Review: Green, Heinemann, and Gusella 435

Table 1. Representative Autosomal Neurogenetic Defects Identified from Genetic Linkage Mapping

Year Year Omim Neurogenetic Disorder Linked Cloned Numbera Protein Product

Alzheimer’s disease 1992 1995 104311 presenilin 1 Alzheimer’s disease 1995 1995 600759 presenilin 2 Amyotrophic lateral sclerosis 1991 1993 105400 superoxide dismutase 1 Ataxia telangiectasia 1988 1995 208900 novel, similar to phosphatidylinositol 3Ј kinase and yeast rad3 Nonsyndromic deafness 1 1992 1997 124900 Drosophila diaphanous homolog Dentatorubro-pallidoluysian 1994 1994 125370 atrophin 1, novel, CAG repeat Atrophy Dystonia with diurnal variation 1993 1994 128230 GTP cyclohydrolase 1 Dystonia 1989 1997 128100 torsin A, novel ATP binding protein Friedreich ataxia 1988 1996 229300 frataxin, novel Hereditary multi-infarct 1993 1996 125310 Drosophila Notch3 homolog Dementia Huntington disease 1983 1993 143100 huntingtin, novel, CAG repeat Infantile neuronal ceroid 1990 1995 256730 palmitoyl-protein thioesterase Lipofuscinosis Juvenile neuronal ceroid 1989 1995 204200 novel Lipofuscinosis Myotonic dystrophy 1981b 1992 160900 myotonin kinase Neurofibromatosis 1 1987 1990 162200 neurofibromin, GAP-related domain Neurofibromatosis 2 1987 1993 101000 merlin, related to cytoskeletal ERMs Niemann-Pick C 1993 1997 257220 novel, cholesterol trafficking Progressive myoclonic epilepsy 1991 1996 254800 cystatin B Spinal muscular atrophy 1990 1995 253300 novel Spinocerebellar ataxia 1 1976b 1993 164400 ataxin 1, novel, CAG repeat Spinocerebellar ataxia 2 1993 1996 183090 ataxin 2, novel, CAG repeat Spinocerebellar ataxia 3 1993 1994 109150 ataxin 3, novel, CAG repeat Spinocerebellar ataxia 7 1995 1997 164500 novel, CAG repeat Stargardt’s disease 1992 1997 248200 novel, ABC transporter Wilson disease 1985b 1993 277900 Cu2ϩ transporting ATPase a Full clinical descriptions and references can be obtained from Online Mendelian Inheritance in Man on the World-Wide Web at http:// www.ncbi.nlm.nih.gov/Omim. b Genetic linkage was achieved first with expressed protein markers rather than DNA markers.

pairs of DNA surrounding a linked genetic marker to find shared by the greatest proportion of HD patients, as the an unknown disease gene had not been developed. As site of the defect. In 1993, a decade after the HD locus the technology advanced, the search for the HD gene was mapped, the disease gene was identified as a novel within the terminal 4p16.3 cytogenetic band applied 4p16.3 gene with a very unexpected mutation (Hunting- such novel technologies as pulsed-field gel electropho- ton’s Disease Collaborative Research Group, 1993). resis, chromosome jumping libraries, CpG island librar- The HD Defect ies, NotI-end clone libraries, cosmid contig construc- More often than not, when a gene is identified by posi- tion, yeast artificial chromosomes, phage P1 clones, tional cloning the nature of the defect provides new site-directed chemical cleavage of chromosomal DNA, insight, identifying a previously unsuspected or un- radiation hybrid mapping, cDNA selection, exon trap- known protein. A primary example of this came with the ping, and many others (Gusella and MacDonald, 1993). 1993 discovery that one form of Lou Gehrig’s disease Other neurogenetic disorders succumbed to this ap- (amyotrophic lateral sclerosis) is caused by mutations proach and a number of interesting and often surprising altering superoxide dismutase 1, an enzyme long stud- disease genes were found (Table 1). However, HD ied but never implicated in the pathogenesis of motor proved difficult because of uncertainty in pinpointing its neurons (Rosen et al., 1993). In HD, both the target precise location near the tip of the chromosome 4 short protein and the nature of the mutationcame as a surprise arm. Ultimately, a 2 Mb candidate region was defined (Gusella et al., 1993; Huntington’s Disease Collaborative and entirely cloned (Bates et al., 1992; Baxendale et al., Research Group, 1993; Gusella and MacDonald, 1995; 1993), but identification of the HD gene within this large Sharp and Ross, 1996). HD is due to an expanded, unsta- segment depended on an additional genetic strategy ble CAG trinucleotide repeat in a gene encoding a large kDa dubbed huntingtin. On normal 350ف that has subsequently proved increasingly valuable in novel protein of tracking down disease genes. We used highly polymor- chromosomes, the CAG repeat is polymorphic, dis- phic DNA markers to identify a common pattern of alleles playing 10–35 triplets, and is inherited in a Mendelian (or haplotype) that implicated about one-third of seem- fashion, whereas HD chromosomes possess a repeat ingly unrelated HD patients from around the world as of Ͼ35 triplets that has a mutation rate approaching 1 having a common ancestor (MacDonald et al., 1992). as it changes in length in most meioses, with a bias This haplotype analysis ultimately targeted a segment toward size increases. The CAG repeat is within the kb, the region whose marker alleles were huntingtin coding sequence, 17 codons from the amino 150ف of only Neuron 436

terminus, where it designates a run of consecutive gluta- mine residues immediately upstream from a proline-rich region. The remaining 97% of the protein is not similar to any others that have been reported, except for a number of HEAT motifs, domains of unknown function named for proteins in which they were first identified by computational analysis (Huntingtin-Elongation factor 3-A subunit of protein phosphatase 2A-TOR1) (Andrade and Bork, 1995). Huntingtin is expressed widely in both the nervous system and in other tissues, but its normal physiological role remains unknown. Mouse knockout and knock-in experiments have shown that total lack of huntingtin causes embryonic lethality at gastrulation (Duyao et al., 1995; Nasir et al., 1995; Zeitlin et al., 1995), whereas reduced levels of huntingtin support further development but result in abnormal neurogenesis (White et al., 1997). The expanded polyglutamine segment of huntingtin, when introduced into the homologous mouse protein, does not impair these developmental functions (White et al., 1997). Thus, the human HD mutation ap- pears to act by a “gain-of-function” mechanism. Interestingly, HD was the second of a growing list of neurodegenerative disorders found to be caused by a lengthened polyglutamine segment in unrelated, but widely expressed, proteins. The first disorder of this type was spinal bulbar muscular atrophy, caused by a CAG repeat expansion in the androgen receptor gene (Brooks and Fischbeck, 1995). Subsequently, expanded CAG repeats have been found in dentatorubropallidoluy- sian atrophy and spinocerebellar ataxias 1, 2, 3, 6, and 7 (Koshy and Zoghbi, 1997; Gusella et al., 1997). In each disease, a lengthened run of glutamines alters a protein, producing a distinctive pattern of neuronal cell loss, with an age of onset of clinical symptoms that varies inversely with the number of CAG repeats (Figure 4). Thus, from the exercise of positional cloning, a common mecha- nism for neurodegenerative disease has emerged that appears to have two components: (1) a neuronal toxicity determined by the number of consecutive glutamines and (2) a specificity of neuronal loss determined by the protein context in which the polyglutamine is presented to the cell. The polyglutamine segment in mutant huntingtin alters Figure 4. Increasing CAG Length Decreases Age at Onset in Poly- the physical properties of the protein, disproportionately glutamine Disorders delaying its migration on SDS-PAGE gels and dramati- Age at onset of neurologic symptoms is plotted against CAG repeat length for spinocerebellar ataxia 2 (SCA2), spinocerebellar ataxia 1 cally increasing its reactivity with certain monoclonal (SCA1), Huntington’s disease (HD), dentatorubropallidoluysian atro- antibodies (Persichetti et al., 1995; Trottier et al., 1995a, phy (DRPLA), and spinocerebellar ataxia 3 (also Machado-Joseph 1995b). A truncated amino terminus of the abnormal disease [SCA3]) based on a compilation of published data (Gusella protein will aggregate in vitro and in vivo in transgenic et al., 1997). mice (Davies et al., 1997; Scherzinger et al., 1997). The altered physical properties of mutant huntingtin are also reflected in the HD postmortem brain with the recent protein causes a specific disruption of neuronal function observation of dystrophic neurites and intranuclear that triggers a cell death program of which aggregate and cytoplasmic huntingtin immunoreactive inclusions formation is a by-product. The latter possibility is more (DiFiglia et al., 1997). Similar results have been observed attractive in explaining the specificity of neuronal loss for the mutant versions of the other neurodegenerative despite widespread expression of the culprit proteins polyglutamine disorders (Davies et al., 1998; Ross, 1997). and raises the question as to whether this specifity lies in However, it remains uncertain whether these aggregates the normalrole of huntingtin and the other polyglutamine are a cause or a consequence of pathogenesis. The next disease proteins in the function of the adult brain. few years will see the delineation of whether the altered More Complex Disorders: Alzheimer’s Disease physical properties of these proteins lead directly to the and the Psychiatric Disorders development of aggregates that are toxic to the neurons, As the genetic linkage approach has become more pow- or alternatively, whether the physical change in each erful with a dense map of highly polymorphic markers, Review: Green, Heinemann, and Gusella 437

increasing attention is being paid to disorders that may The ligand-gated ion channels offer similar potential be caused by more than one disease gene or by the for displaying genetic variation that either causes or interaction of multiple genes. The strategy has met with modifies human disease. Table 2 lists the genetic loci great success in Alzheimer’s disease (AD), where three encoding ligand-gated ion channel receptor subunits in different disease genes and one risk factor locus have human that have been either identified or inferred by been identified (Goate, 1997; Hutton and Hardy, 1997; work in other species. Their cytogenetic map locations Kim and Tanzi, 1997). AD involves deposition in the brain indicate that these receptor genes are spread through- of extracellular amyloid, prompted by a 42 amino acid out the human chromosomes, with some notable clus- peptide cleaved from a much larger protein, and devel- ters of genes, such as the GABAA receptor genes on opment of intraneuronal neurofibrillary tangles. Muta- chromosomes 4p, 5q, and 15q, that probably reflect tions in the amyloid protein precursor gene as well as radiations from a common ancestor by duplication. alterations in two homologous genes encoding preseni- Startling Mutations in the Glycine Receptor lins 1 and 2 can increase the production of amyloido- The first disorder associated with a mutation in a neuro- genic peptide and cause early-onset AD. In addition, transmitter receptor was found, not surprisingly, by po- the E4 allele of the apolipoprotein E gene is a significant sitional cloning. Hyperekplexia (Startle disease; Kok dis- risk factor for the development of late onset AD. Addi- ease) is characterized by muscle rigidity of nervous tional genetic factors in AD are actively being sought. system origin, particularly in neonates, and by an exag- A similar approach to psychiatric disorders, most no- gerated startle response to unexpected auditory or tac- tably bipolar affective disorder and schizophrenia, has tile stimuli. The underlying genetic defect was first met with much greater difficulty, perhaps because of mapped to chromosome 5q by genetic linkage using more problematic phenotypic characterization and greater DNA markers (Ryan et al., 1992a). As the symptoms of heterogeneity of genetic contributions. Various reports hyperekplexia are markedly reduced by clonazepam, of genetic linkage and association in each of these dis- this implicated the GABAA receptor gene clusterat 5q33– eases have not yet produced a clearcut pathogenic lo- 5q35. However, more detailed genetic linkage studies with physically mapped DNA markers excluded these cus. However, as a result of global cDNA sequencing genes, leaving the GLRA1 glycine receptor gene as the analyses, there are now a substantial number of discrete prime candidate (Ryan et al., 1992b). Initially, Arg271 genes available for analysis as candidates. This re- was identified as the target of missense alterations in source suggests that a hybrid strategy that utilizes in- four families with autosomal dominant hyperekplexia formation from genetic transmission to assess individ- (Shiang et al., 1993). Substituting uncharged amino ual candidate loci (e.g., the transmission-disequilibrium acids (Leu or Gln) for the charged residue appears to test) can effectively complement genome-wide linkage reduce the agonist sensitivity of the receptor (Rajendra searches to place the hope of major progress in under- et al., 1994). Mutations affecting other codons have sub- standing behavioral disorders on the horizon. A major sequently been identified in other families (Shiang et al., class of candidates for such studies is the collection of 1995; Elmslie et al., 1996; Milani et al., 1996; Seri et al., genes encoding ligand-gated ion channel receptors. 1997). The homology of human chromosome 5q and mouse Chr 11 led to the discovery that recessive mutations Ligand-Gated Ion Channel Receptor Genes in the mouse homolog of GLRA1 underlie two mouse in Disease neurologic mutants, spasmodic and oscillator. spas- Genomic Distribution of Ion Channel modic displays a phenotype similar to hyperekplexia Receptor Genes and is also caused by a missense mutation (Ryan et al., The crucial role of ion channels in the operation of the 1994). oscillator has a more severe phenotype, including brain and neuromusculature, the high degree of se- progressive tremor and muscle spasms leading to death quence conservation of ion channel proteins, and the by 3 weeksof age. It is causedby a frameshift-producing capacity to specifically alter ion channel function all deletion predictedto eliminate the final cytoplasmic loop support the view that naturally occurring mutations in and transmembrane domain (Buckwalter et al., 1994). ion channel genes could underlie a variety of human Interestingly, recessive forms of hyperekplexia have disorders. This was first demonstrated for the muscle now been found in human, including both missense and ϩ voltage-gated Na channel ␣ subunit, which coinherits null mutations (Rees et al., 1994; Brune et al., 1996). The in family studies with hyperkalemic periodic paralysis, a lack of lethality of the latter indicates that the loss of disorder involving episodic muscle weakness associated functional glycine receptors can be compensated to a with increased extracellular potassium (Fontaine et al., greater degree in human than in mouse. No mutations 1990). Subsequently, it was recognized that different in other glycine receptor genes have yet been found in missense alterations in this gene can produce a variety human. However, the discovery of a mutation in the of other symptoms, including cold-induced muscle stiff- homolog of GLRB in the spastic mouse suggests that ness (paramyotonia congenita) and fluctuating myoto- these genes should be scanned for alterations in pa- nia. The individual clinical signs in a given patient pre- tients with hyperekplexia-like symptoms without GLRA1 sumably reflect differential effects of the specific mutations (Kingsmore et al., 1994; Koch et al., 1996). mutation on the kinetics of Naϩ channel function. This Defective Brain and Muscle general finding has subsequently been demonstrated Acetylcholine Receptors for a number of other muscle and neuronal voltage- Another brain neurotransmitter-associated defect has gated ion channels in genetic disorders of both human been found in the CHRNA4 nicotinic acetylcholine re- and mouse (Bulman, 1997). ceptor gene on chromosome 20. A null mutation at this Neuron 438

Table 2. Ligand-Gated Ion Channel Receptor Genes

Receptor Human Map Mouse Map Human Gene Symbol Subunits Locationa Locationa Human Disorder Mouse Disorderb

Nicotinic Cholinergic Receptor

CHRNA1 ␣1 2q24–q32 2 Congenital myasthenic syndrome CHRNA2 ␣2 8p21–q21 14 CHRNA3 ␣3 15q24 9 CHRNA4 ␣4 20q13.2–q13.3 2 Nocturnal frontal lobe epilepsy CHRNA5 ␣5 15q24 9 CHRNA6 ␣6UNUN CHRNA7 ␣7 15q14 7 CHRNA8 ␣8UNUN CHRNA9 ␣9UNUN CHRNB1 ␤1 17p12–p11 11 Congenital myasthenic syndrome CHRNB2 ␤2 1p21 3 CHRNB3 ␤3 8p11.2 UN CHRNB4 ␤4 15q24 9 CHRNG ␥ 2q21.1–q21.3 1 CHRND ␦ 2q33–q34 1 CHRNE ⑀ 17p13–p12 11 Congenital myasthenic syndrome Serotonin Receptor

HTR3 5-HT-3 11q23.1–q23.2 UN

GABAA Receptor

GABRA1 ␣1 5q33–q35 11 GABRA2 ␣2 4p13–p12 5 GABRA3 ␣3 Xq28 X GABRA4 ␣4 4p14–q12 7 GABRA5 ␣5 15q11.2–q12 7 GABRA6 ␣6 5q33–q35 11 GABRB1 ␤1 4p13–p12 5 GABRB2 ␤2 5q33–q35 UN GABRB3 ␤3 15q11.2–q12 7 cleft palate 1 GABRG1 ␥1 4p14–q12 UN GABRG2 ␥2 5q33–q35 11 GABRG3 ␥3 15q11.2–q12 7 GABRD ␦ 1p UN GABRE ⑀ Xq28 UN GABRR1 ␳1 6q14–q21 4 GABRR2 ␳2 6q14–q12 4 GABRP ␲ UN UN Glycine Receptor GLRA1 ␣1 5q32 11 Hyperekplexia spasmodic, oscillator GLRA2 ␣2 Xp22.1–p21.3 X GLRA3 ␣3UN 8 GLRA4 ␣4UNX GLRB ␤ 4q32 3 spastic Ionotropic Glutamate Receptor

GRIA1 AMPA 1 5q33 11 (GluR1) GRIA2 AMPA 2 4q32–q33 3 (GluR2) GRIA3 AMPA 3 Xq25–q26 X (GluR3) GRIA4 AMPA 4 11q22–q23 9 (GluR4) GRID1 ␦1UNUN GRID2 ␦2UN 6 lurcher continued Review: Green, Heinemann, and Gusella 439

Table 2. continued

Receptor Human Map Mouse Map Human Gene Symbol Subunits Locationa Locationa Human Disorder Mouse Disorderb

GRIK1 kainate 1 21q22.1–q22.2 16 (GluR5) GRIK2 kainate 2 6q16.3–q21 10 HD modifier (GluR6) GRIK3 kainate 3 1p34–p33 4 (GluR7) GRIK4 kainate 4 11q23 9 GRIK5 kainate 5 19q13.2 7 GRIN1 NMDA 1 9q34.3 2 GRIN2A NMDA 2A 16p13 16 GRIN2B NMDA 2B 12p12 6 GRIN2C NMDA 2C 17q25 11 GRIN2D NMDA 2D UN 7

P2X ATP Gated Receptor

P2RX1 1 17p UN P2RX2 2UNUN P2RX3 3 11q12 2 P2RX4 4 12q24 UN P2RX5 5UNUN P2RX6 6UNUN P2RX7 7 12q24 UN

Gene locus, mapping and phenotype details were compiled from combining the information available in the public databases on the World- Wide Web: Online Mendelian Inheritance in Man (OMIM: www.ncbi.nlm.nih.gov/Omim), the Genome Database (GDB; gdbwww.gdb.org/gdb), Mouse Genome Informatics (MGI; www.informatics.jax.org/index.html), and GenBank (www.ncbi.nlm.nih.gov). a UN ϭ Unknown. b Excludes targeted (knockout) mutations. locus was originally implicated in benign familial neona- opening in the presence of acetylcholine, and acetylcho- tal convulsions (Beck et al., 1994), but the subsequent line-independent channel opening. These changes com- discovery of missense mutations in a nearby voltage- promise normal transmission at the neuromuscular junc- gated Kϩ channel gene has raised doubts about this tion and lead to endplate myopathy, presumably by finding (Biervert et al., 1998; Singh et al., 1998). However, cationic overload. Interestingly, a recessive myasthenic alteration of CHRNA4 does cause a separate disorder, syndrome involving endplate acetylcholine receptor de- autosomal dominant nocturnal frontal-lobe epilepsy, a ficiency is caused by CHRNE mutations associated with partial epilepsy typified by brief, violent, sleep-associ- decreased subunit production, decreased affinity for ated seizures (Steinlein et al., 1995, 1997). A missense acetylcholine, diminished acetylcholine-induced chan- mutation and a codon insertion, both affecting M2 of nel opening, and/or suppression of desensitization (En- the ␣4 subunit, each can cause this phenotype, although gel et al., 1996b; Ohno et al., 1997). In this case, overall it is not clear whether this effect is due to reduced dysfunction of the neuromuscular junction is mitigated function or hyperactivity of the receptor (Forman et al., by continued expression of the fetal CHNRG gene. 1996). Disease Candidates in Waiting Another route to identifying the genetic basis of inher- No inherited human disorder has been connected with ited disorders is direct sequencing of reasonable candi- a definite mutation in a GABAA receptor gene, although date genes without prior linkage mapping of the genetic inactivation of the GABAB3 homolog in mouse produces defect. Among the ligand-gated ion channels, this has a developmental deficit leading to cleft palate (Culiat et been most successful in the congenital myasthenic syn- al., 1995). Similarly, no pathogenic mutation has yet been dromes, in which the endplate acetylcholine receptor is demonstrated in a human glutamate receptor gene, al- an obvious candidate. In the autosomal dominant slow though mouse knockout mutations of these genes cause channel myasthenic syndrome, a variety of missense subtle to severe neurologic symptoms. Interestingly, po- mutations have been found in three different subunit sitional cloning revealed recently that the lurcher mouse, genes, CHNRA1, CHNRB1, and CHRNE (Gomez and which displays ataxia due to loss of cerebellar Purkinje Gammack, 1995; Ohno et al., 1995; Sine et al., 1995; cells during postnatal development, is caused by a mis- Engel et al., 1996a; Gomez et al., 1996; Croxen et al., sense mutation in the GRID2 homolog (Zuo et al., 1997). 1997; Milone et al., 1997; Wang et al., 1997a). These The heterozygous substitution of Thr in place of a highly mutations provide a natural structure–function experi- conserved Ala residue in transmembrane domain 3 of ment, in which different alterations have variously been the delta2 glutamate receptor leads to a large constitu- implicated in producing increased affinity for acetylcho- tive inward current, providing a genetic model for excito- line, decreased rate of acetylcholine dissociation, en- toxic cell death in the nervous system. Mutations in hanced steady-state desensitization, prolonged channel glutamate receptor genes may ultimately be found to Neuron 440

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