Roger L. PapkeRecently new advances have been made in our understanding of the functional roles which nicotinic acetylcholine receptors may play in the brain, and the cloning of the genes for these receptors has given us the tools to study the detailed molecular mechanism of these receptors  . Nicotinic receptors are known to be involved in addictive processes  and have been suggested to be affected in schizophrenia [3, 4] . They have also been shown to be important for cognitive processes and memory. Nicotinic agonists are being developed as therapeutics for the treatment of Alzheimer's dementia  . Along with an increased understanding of the functional roles that nicotinic receptors may play in brain function has come an appreciation for the multiple receptor subtypes that exist in the brain.
Characterization of ion channel structures associated with divalent-ion permeability and the specificity of use-dependent inhibitors.
In a project supported by the NIH, we are seeking to extend our understanding of how a crucial physiological property, the permeability to divalent ions, is regulated both on the level of receptor subtype (i.e. subunit combination) and in terms of the specific protein domains. Divalent ion permeability may be required for the neuronal plasticity associated with learning and memory and may also create a potential for excitotoxicity.
We express cloned nicotinic receptors in Xenopus oocytes, and with the study of chimeric and mutant subunits, we are identifying the molecular elements that regulate calcium permeability, as well as the molecular elements that are involved with use-dependent inhibition of the receptors. By evaluating the relationships between the elements that regulate divalent ion permeability and those which are associated with sensitivity to specific antagonists, it may be possible to define therapeutics which may target specific functionally important receptor subtypes, either to spare those receptors important for cognitive processes while targeting those involved in addictive processes, or to selectively block those which may put cells at risk of toxicity.
The analysis of receptor physiology and pharmacology is being carried out both at the level of whole-cell currents and in terms of a detailed study of single channel properties. The study of a related series of bifunctional inhibitors has permitted the disposition of inhibitory binding sites within the receptor complex to be evaluated (Francis and Papke 1996 in press; Francis et al., in preparation).
Our data has provided us with a strong basis for experimental design in terms of approaches and candidate sequences for important structural domains. We have evidence that divalent ion permeability is regulated by the gamma subunits of muscle receptors and the alpha5 subunits of neuronal receptors. This model will be directly evaluated with antisense knockout experiments directed at eliminating the functionalinfluences of these subunits in native receptors.
The regulation of calcium permeability is an important aspect of receptor function. It may become of great clinical usefulness to understand whether particular noncompetitive inhibitors may either target or spare receptors, based on that property. Calcium permeability can be associated with neuronal plasticity (e. g. learning), and when improperly regulated can lead to excitotoxicity. While in the past, therapeutic use of ganglionic blockers has been strictly limited to targets in the peripheral nervous system, more recently potential new therapeutic directions have been suggested which are relevant to this analysis. For example, noncompetitive inhibitors have been proposed as therapeutic approaches to alleviate nicotine addiction [2, 6-9] , and an understanding of the relationships between channel block by inhibitors and the natural process of ion permeation may permit agents to be developed which selectively target receptor sub-types involved with the addictive process, while sparing others, perhaps those that facilitate learning. Along that line, nicotinic channel activators are being developed as therapeutics for the treatment of Alzheimer's disease, and it has become clear that some of these compounds, as well as nicotine itself, have secondary effects as noncompetitive inhibitors  . Models for the therapeutic application of these compounds require first identifying the correct molecular targets, in terms of receptor subtypes, and then identifying the therapeutic activity, whether it may be the selective activation of one receptor subtype or the selective inhibition of another.
In addition to the relevance of these studies to potential new therapeutic applications, it should also be appreciated that the analysis of the two site model for prolonged inhibition by bifunctional compounds will lead to better understanding of the biophysical transitions of the channel. The experimental compounds which we have developed as probes of receptor structure have allowed us to clearly demonstrate effects that are strictly dependent on channel activation. Parallel studies of mutant receptors have identified the location of the inhibitory binding site, so that the analysis of site separation distances has provided new information about the disposition of protein subunits in the open state of the channel.
Second messenger modulation of nAChR Function
Nicotine's effects on human behavior are associated with the drug's activities on specific nicotinic acetylcholine receptors in the brain. This is linked to the phenomenon of nicotine addiction. While the immediate effect of nicotine is to activate receptors, chronic exposure to nicotine leads to the build up of large pools on non-activatable (inactive or desensitized) receptors. Prolonged receptor inactivation of this sort has been proposed to require post-translational modifications of the receptor protein. We are studying how neuronal nicotinic receptor function is regulated by second messenger systems, and potentially by feedback systems associated with the flow of calcium through the nicotinic receptors themselves. By studying cloned neuronal nicotinic receptor subunit genes expressed in Xenopus oocytes, using a conventional recording method (two-electrode voltage clamp, TEVC), we have generated preliminary data consistent with the regulation of receptor function by cAMP- and calcium-dependent second messengers. However, there are serious limitations to the use of TEVC for these studies, since it is difficult to measure or control the intracellular concentrations of ions and second messenger modulating factors. This is an especially crucial issue in regard to calcium. Calcium appears to play a role in controlling second messengers and therefore the functional state of the receptors. Calcium also directly activates chloride channels, and the activation of these channels can contaminate the nicotinic receptor responses.
We are currently developing a new method for recording the responses of nicotinic receptor subtypes expressed in Xenopus oocytes: the "cut-open oocyte Vaseline-gap voltage clamp" (COOVGVC) [11, 12] . With this method the oocyte is configured in a special series of chambers so that the apical surface is voltage clamped and exposed to the external bath solution, while the underside is either permeabilized or directly impaled and subsequently perfused. This method permits rapid and accurate voltage control over what is still a relatively large piece of membrane. By changing the solution passed through the perfusion pipette, there is rigorous control of intracellular ion concentrations and an ability to apply second messenger modulating factors to the interior surface of the membrane.
We will use this system to test the hypothesis that cAMP-dependent second messenger systems may be activated to up-regulate the function of neuronal nicotinic receptors, while calcium-dependent systems may down-regulate function, perhaps by regulating phosphatase activity. We will determine whether the calcium permeability of the receptors might produce a feedback mechanism through which acute activation leads to chronic inactivation. This may relate to the phenomenon seen in smokers, where there is a paradoxical up-regulation of nicotine binding sites which appear to be associated with nonfunctional nicotinic receptors. We will also determine the specificity of these effects for defined nicotinic receptor subtypes and test the hypothesis that the expression of a particular gene, alpha5, has a special role in regulating second messenger effects. Our preliminary data indicate that the expression of the alpha5 gene affects the calcium permeability of the receptor complex, which might in turn regulate a feedback mechanism.
Research activities and goals
- The primary focus of research conducted in the Papke laboratory is focused on obtaining an understanding of the functional significance of nicotinic acetylcholine receptors (nAChR) in the CNS, and how these receptors may be developed as therapeutic targets.
We study the nAChR of the brain both by expressing them artificially in Xenopus oocytes and transfected cells, and by identifying currents mediated by neuronal nAChR in fresh preparation of brain tissue. We conduct patch clamp recordings from brain slices and also acutely dissociated neurons. Large histaminergic neurons of the hypothalamus express alpha7-type nAChR at high levels and are useful for the biophysical characterization of these receptors.
We have also been interested in the role played by lithium conduction through specific neurotransmitter receptor subtypes in creating the therapeutic specificity of
lithium for the treatment of bipolar disorder.
I. Neuronal nAChR
Understanding the neuronal nicotinic alpha7 receptor subtype
The neuronal nicotinic alpha7 receptor subtype has a number of properties which suggest it may play unique roles in neuronal function, including cytoprotection and synaptic plasticity. These receptors have the highest calcium permeability of any ligand-gated ion channel and activation properties that make them extremely difficult to detect as mediators of synaptic activity in the CNS. However, alpha7 receptors have been implicated  as playing an important role in the modulation of synaptic function. Our work has identified these receptors as a likely target for new therapeutic agents shown to have positive behavior and cytoprotective effects (see below). We have recently identified choline as a selective activator of the receptors (Papke et al. 1996, in press Neuroscience Letters). Free choline concentrations in plasma have been reported to range from 10 to 40 ÁM and in cerebro-spinal fluid have been estimated to be 4 -12 ÁM  . While these are concentrations that are far lower than those used to activate large transient responses in oocytes, the exposure of alpha7-receptors to levels of circulating choline may result in a low level of tonic activity. The activation properties of alpha7-type receptors appear to be unique among nicotinic receptors in that, in the presence of high agonist concentrations, desensitization is rapid, yet a high affinity desensitized state is not observed. The macroscopic current stimulated by high agonist concentrations in oocytes and reported for putative alpha7-like receptors in hippocampal neurons and chick ciliary ganglion cells desensitizes during the application of agonist, yet small and sustained responses have been observed in response to low concentrations of agonist (2 ÁM nicotine or 10 ÁM ACh, in ciliary or hippocampal neurons respectively) [15, 16] . It was proposed that such low level activation is responsible for previously reported a-BTX sensitive increases in intracellular calcium  . In vivo extracellular choline concentrations have been shown to be increased following stoke, siezure and other forms of neurological trauma. Our results suggest that these increases in choline may work through alpha7 receptors to provide a form of cytoprotection mediated by small increases in intracellular calcium.
We have also recently obtained data supporting the re-evaluation of alpha7 receptor functional concentration-response relationships in the oocyte expression system. We can show that, due to the activation properties of the receptor, channels desensitize prior to the time when maximum agonist concentrations are achieved in the chamber. By synchronizing data acquisition with a method to evaluate the agonist solution exchange rate, we can estimate actual agonist concentrations coincident with maximal receptor activation. With this method we find that the EC50 for acetylcholine is at least 5-fold lower and that the cooperativity of activation is at least two time greater than previously reported (Papke et al., in preparation).
The characterization and development of therapeutic agents for Alzheimer's disease
Our laboratory has received support from three commercial agencies seeking to develop nicotinic receptor based therapeutics, Abbott Laboratories, R.J. Reynolds Research and Development, and Taiho Pharmaceutical.
Abbott Laboratories has supplied us with samples of its experimental drug ABT-418, and we have conducted an extensive characterization of its antagonist and antagonist properties for specific neuronal nicotinic receptor subtypes (Papke et al. submitted).
Recent experiments in our laboratory has indicated that one of experimental agonists developed by the R.J. Reynolds Research and Development Laboratories may provide a tool for defining the pharmacophore for channel inhibition by agonist (Watterson et al. 1996 Neuroscience abstract). Through the study of chimeric receptor subunits we have also identified the binding site for use-dependent inhibition by agonist.
With the support of Taiho Pharmaceutical, the compound GTS-21 was developed by Drs. Bill Kem and Ed Meyer at the University of Florida as a nicotinic agonist with great therapeutic potential for Alzheimer's disease. The Papke lab was instrumental in identifying this compound as an alpha7 selective agonist. With continuing support from Taiho and the NIH, we are participating in the further development of compounds with improved efficacy and selectivity. We have also recently begun defining a model for how these compounds may discriminate between human and rat receptor subtypes (Papke et al. 1996 Neuroscience abstract).
II. Neurotransmitter receptors and the therapeutic use of lithium salts
Within a very narrow range of concentrations, lithium attenuates the cycles of mania and depression in bipolar disorder. At similar concentrations, lithium is known to disrupt mechanisms of intracellular signal transduction, including signals mediated by phosphatidylinositol and cAMP. However, it is unclear whether the therapeutic efficacy of lithium is associated with these effects, in part because such effects should have global manifestations, while the therapeutic effects of lithium seem specific for bipolar disorder. Our experiments seek to evaluate the hypothesis that the specificity of lithium arises from the selective targeting of discrete synaptically active sites in the brain based on the differential uptake of lithium at those sites.
The characterization of lithium permeability through specific neurotransmitter receptor subtypes
Our experiments focus on the roles which different neurotransmitter receptors may play in lithium homeostasis. We express cDNAs for specific neurotransmitter-gated channels in Xenopus oocytes and use electrophysiology to measure lithium flow. Our preliminary data indicate that particular types of glutamate receptor channels (those containing the AMPA selective GluR2 subunit) favor the flow of lithium over other ions. Our data indicate that localized increases in intracellular lithium concentrations will occur at synapses where the receptors contain the GluR2 subunit. Lithium flow through neuronal nicotinic receptor channels may also target synaptic sites which may be distinct from those therapeutically important for the management of bipolar disorder and provide a potential mechanism for the reported memory impairment associated long term lithium treatment. Our experiments will evaluate in detail how GluR2 and other receptor subtypes can regulate lithium homeostasis in a model system that will both permit the direct measurement of lithium levels in chronic and acute experiments, and provide a means to evaluate how changes in intracellular lithium modulate phosphatidylinositol and cAMP mediated signal transduction. Ca2+-dependent chloride channels in the oocyte will be used as a reporter system for the function of phosphatidylinositol mediated signal transduction. The direct effects of lithium and gated-ion channel lithium flux on levels of inositolphosphate and the phosphatidylinositol intermediate CMP-PA will be measured in the oocyte. The effect of lithium on basal and metabotropic receptor-mediated changes in cAMP levels will also be measured. The role that receptor-gated lithium flux may play in the regulation of lithium homeostasis in mammalian cells expressing such receptors will also be directly evaluated.
The modeling of lithium microdomains as a mechanism for defeating coincidence detection in the CNS
The experiments described above will address the question of which neurotransmitter receptor subtypes may be directly involved with the therapeutic effects of lithium. We will seek to identify which receptors may be directly regulated by lithium either through anomalous mole fraction effects on channel conduction, or through competition between lithium and magnesium for channel regulatory sites. Our preliminary data indicate that the alpha7 nicotinic receptor subtype described above may in fact be directly inhibited by therapeutic concentrations of lithium.
Our experiments will also identify receptor subtypes such as the GluR2 (see above) which are most likely to permit local elevations of lithium in dendrites associated with synaptic activity. However, in order to apply our fundamental characterization of lithium flux through specific receptor subtypes, it will be necessary to model how the macroscopic currents recorded in whole oocytes will change the microenvironment of synapses. A large degree of synaptic plasticity is believed to arise from coincidence detection at active synapses, such that depolarization is coupled to calcium signals which locally activate second messenger systems, resulting in the stimulation of long term changes in synapse properties. Our working hypothesis is that at sites where synaptic receptors have high lithium permeability, micro-domains of elevated lithium will inhibit those second messenger systems and defeat the reinforcing effects of coincidence detection and thereby modulate manic behavior. We are developing mathematical models to relate the macroscopic properties of lithium flux through specific receptor subtypes in oocytes, the modulation of lithium homeostasis in that system, and the microenvironment of synapses.