Roger L. Papke - US grants

nicotinic receptors; membrane channels; neurophysiology; neurons; protein structure function; receptor expression; electrophysiology; chimeric proteins; receptor binding; Xenopus oocyte; transfection; voltage /patch clamp;

Recently 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 [l]. Nicotinic receptors are known to be involved in addictive processes[2], 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 [5]. 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. This proposal will 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 also 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 will express cloned nicotinic receptors in Xenopus oocytes, and with the study of chimeric and mutant subunits, we will identify 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 will be 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 will permit the disposition of inhibitory binding sites within the receptor complex to be evaluated. Our extensive preliminary 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 functional influences of these subunits in native receptors. The experiments in this proposal will provide important new insights into the nicotinic receptors of the brain in terms of their biophysical properties, their potential as therapeutic targets, and relationships between specific functional properties and drug sensitivity.

The alpha7 neuronal nicotinic acetylcholine receptor (AchR) subtype may be an important therapeutic target, with potential significance for the treatment of Alzheimer's disease, stroke, and schizophrenia. Activation of the alpha7 subtype has been shown to have cytoprotective effects and enhance synaptic transmission, which may underlie reported positive cognitive effects obtained with nicotinic agents. Our data indicate that alpha7 receptors have two contrasting modes of activation. Typically, studies have focused on the relatively large transient currents that can be stimulated by rapid application of high concentrations of agonist. However, in the continued presence of such high agonist concentrations, virtually all steady-state current is suppressed by the desensitization process, while at lower agonist concentrations a small amount of steady-state activation persists. Our preliminary data regarding the biophysics of the receptor and the cytoprotective effects of alpha7-selective agonists indicate that steady-state activation of this calcium permeable receptor subtype by low agonist concentrations may represent the functional modality of greatest therapeutic significance. We will therefore study the activation and desensitization properties of alpha7 receptors in detail, expanding our analysis of concentration/response function to include an analysis of the steady-state currents. We will conduct single-channel and whole-cell patch-clamp analysis, using the endogenous activators, Ach and choline, as well as the alpha7-selective partial agonist HMBA. We will test models of the biophysical properties of alpha7 receptors, towards the goal of improving therapeutic targeting of alpha7 receptors. HMBA is the active metabolite of DMXB (GTS-21), a drug just entering phase 2 clinical trials for Alzheimer's disease. DMXB itself is the prototype for a family of alpha7-selective anabaseine derivatives, many of which we have shown, like nicotine, have two phases of action, initially stimulating the receptor, then subsequently causing a long lasting inhibition. We will investigate the nature of this inhibitory activity and the molecular interactions which underlie it. We will characterize the desensitized states of the alpha7 receptor induced by Ach and choline, and determine whether the residual inhibition observed after the application of DMXB and similar agents represents accelerated desensitization or alternative forms of inhibition. In order to improve our ability to target alpha7 receptors for therapeutics, chimeras of the human and rat alpha7 receptors will be made. Then, through the use of agents which show selectivity for the activation or inhibition of the human and rat alpha7 wild-type receptors, it will be possible to identify structural elements of the proteins that regulate activation and desensitization.

[unreadable] DESCRIPTION (provided by applicant): The a7 neuronal nicotinic acetylcholine receptor is an important therapeutic target, relevant to Alzheimer's Disease, stroke, and schizophrenia. Understanding how this receptor responds to natural and experimental agonists is crucial for the therapeutic targeting of a7. We have shown that stimulation of a7 receptors by relatively low agonist concentrations can support an equilibration between activation and desensitization, resulting in a functional modality that we hypothesize to be the basis for therapeutic effects. In this continuation we will study how a7-selective agonists both activate this receptor and, in some cases, produce down-regulation of function through secondary inhibitory or desensitizing effects. Our experiments utilize structurally diverse a7-selective agonists including large molecules such as benzylidene anabaseines (BA) and indole tropanes (IT) and smaller probes for receptor selectivity such as choline and tropane. Our data suggest that BA compounds with residual inhibitory effects interact with hydrophobic residues on the margins of the ACh binding site. We will test that hypothesis through the use of site-directed mutants and new experimental agonists. We will develop novel compounds including additional indole-tropanes (IT) to evaluate as agonists and use as experimental probes. The prototype for the IT compounds is tropisetron, a 5HT3 receptor antagonist and a7-selective agonist. Our preliminary studies show that while the indole moiety in tropisetron has activity with 5HT3 receptors, the tropane group is, on its own, an a7 agonist. Indole conjugation appears to make tropisetron a more potent, though less efficacious, agonist for a7 than the small agonist tropinone. We hypothesize that, based either on intramolecular effects or specific interactions between the drug and amino acids in or near the agonist binding site, substitutions of the indole will modify the properties of IT agonists in ways that will be predictable based on our previous studies of BA compounds and models of the receptor binding site. The structurally diverse BA and IT agonists are much larger molecules than the endogenous agonists, ACh and choline, and are likely to have binding sites that include more points of contact on the receptor than the binding site of ACh. We will conduct scanning cysteine accessibility experiments to identify the binding sites for large and small molecular probes, utilizing both selective and nonselective agonists as well as competitive antagonists. We will define both the ligand and protein basis through which certain drugs achieve selectivity for a7 receptors. Taken together, our studies will define effective ways to activate a7 receptors with potentially therapeutic agents and will also provide insights into how new agents may be designed to selectively activate this receptor with minimized inhibitory side-effects due to receptor desensitization or the non-selective activation of other receptor subtypes. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The a7 nicotinic acetylcholine receptor is being energetically pursued as a drug target for diverse disorders, from Alzheimer's disease to septic shock. We have demonstrated that there are at least three distinct structural motifs which can be used to modify a core agonist structure, such as anabaseine or quinuclidine, to achieve a7 selectivity. For example, selectivity can be achieved through modification of the core agonist with the addition of a large hydrophobic side group such as a benzene ring. The precise chemical structure of the hydrophobic side group determines efficacy and potency, as well as another key feature, the ability to produce stable ion channel desensitization following a transient phase of ion channel activation. The desensitization is due to prolonged binding to the receptor, and the desensitizing properties of specific agents are likely to impact their therapeutic utility for specific indications. We show that drugs which desensitize and do not activate the receptor ion channel can still be effective at treating inflammatory diseases. We will use mammalian cells transfected with a7 alone, or in combination with pro-inflammatory cytokine receptors to test the hypothesis that drugs which induce stable desensitization of the a7 ion channel may still be effective at mediating ion channel independent signal transduction through the intracellular JAK/STAT pathway. We will also test the hypothesis that ion channel activation, in contrast, is essential for the enhancement of LTP, a memory-related process in the hippocampus. We have generated models for how the various a7-agonists dock in the ligand- binding domain of the a7 receptor and have identified amino acids which we hypothesize will have point-to- point interactions with substituents on the hydrophobic side groups of the a7-selective agonists. We will investigate the potential importance of hydrogen bonding and hydrophobic interactions on the binding, gating, and desensitizing properties of the specific receptor/ligand combinations. We will test our hypotheses with site-directed mutations, as well as with novel a7-selective ligands that will be restricted in their ability to form specific point-to-point interactions, for example, agents which are only able to be H-bond donors or acceptors. Wild-type and mutant receptors will be expressed in either Xenopus oocytes or transfected mammalian cells, and we will study ion channel properties by measuring both whole-cell and single-channel currents. We will use the Type 2 positive allosteric modulator PNU-120596 to measure the desensitizing properties of specific ligands and to overcome the intrinsically limited open probability of a7 receptors, making their single-channel currents more amenable to study. We will use tkP3BzPB, a novel highly selective a7 noncompetitive antagonist, to separate ion channel activation dependent and independent forms of signal transduction, and to further manipulate ion channel open probability. Together these studies will provide important advancements leading to the design of a7 agonists with optimized profiles of pharmacological properties for specific indications. PUBLIC HEALTH RELEVANCE: There are many types of nicotine receptors in the brain, and only some of them are related to why people become addicted to nicotine. One type of nicotine receptor that is not the cause of addiction is the alpha7-type receptor, and stimulation of this receptor combats conditions like schizophrenia, Alzheimer's disease, septic shock and other inflammatory diseases. We have identified drugs that will selectively stimulate alpha7 receptors in one of two different ways. One form of stimulation may help alleviate brain diseases;the other may help alleviate diseases like arthritis. We will use our new discoveries about how these drugs work to help make alpha7-stimulating drugs optimally designed to treat specific diseases.