Antonius M. VanDongen - US grants

DESCRIPTION The long term objective of this project is to elucidate the molecular basis of ion channel behavior. A universal property of ion channels is that they open and close stochastically. Both ligand- and voltage-gated channels share a common architecture of subunits or domains arranged around a central pore. The pore-forming regions of K channels and glutamate receptors seem to be structurally conserved, suggesting that fundamental channel functions like permeation and gating may share a molecular basis. Open/close transitions usually involve a coordinated movement of all subunits contributing to the pore. However, this concerted behavior seems to break down in partially activated K channels, giving rise to subconductance levels. In this project the role of individual subunits in shaping channel behavior will be further evaluated using the drk1 K channel and the NMDA receptor as model systems. Specific aims are: (i) To determine the role of individual subunits in permeation and gating; (ii) To determine how ion selectivity is affected by subunit composition; (iii) To determine subunit-subunit interactions in channel activation. For each model system, mutations are available in which activation, permeation, or selectivity are severely compromised. Significance: understanding channel behavior at the molecular level will help establish a molecular basis for disorders in which excitability and neurotransmission are impaired. The ability to explain fundamental aspects of ion channel function in terms of molecular structure is also a prerequisite for rational drug design.

DESCRIPTION: (Applicant's Abstract) The long term objective of this research is to understand the molecular basis of nicotine addiction, associated with the usage of tobacco. Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels that are the primary central target of nicotine. A diverse family of nAChRs is expressed in the central nervous system, where their pharmacology and physiological roles are only partially understood. The availability of recombinant neuronal nAChRs subunits now makes it possible to characterize the interaction of nicotine and acetylcholine with specific nicotinic receptor subtypes. Nicotine has a dual agonist-antagonist action because it can desensitize its receptor. Preliminary data show that central nAChRs receptor subunits vary dramatically in their desensitization properties and pharmacology. In this project we will characterize the pharmacology and physiology of five recombinant nAChRs expressed in Xenopus oocytes. Receptors will be functionally characterized using whole cell voltage clamp and single channel patch clamp techniques. Special attention will be given to the antagonist mecamylamine (MECA), which facilitates smoking abstinence in nicotine patch therapy. The specific aims of this project are: (i) to define the pharmacological profiles of the specific recombinant receptors, (ii) to determine the potency of nicotine as an agonist, during prolonged drug application, (iii) to characterize the role of subunits in desensitization by acetylcholine and nicotine, and (iv) to characterize the physiological interaction of acetylcholine, nicotine and mecamylamine. These experiments will establish a molecular basis for the pharmacology and physiology of specific neuronal nAChRs. The role of individual subunits in determining the dual pharmaco-logical action of nicotine will be defined. The results will be important for understanding both the development of tobacco dependence during smoking, as well as its reversion during smoking cessation therapy.

The long term goal of this project is to define the molecular basis of agonist affinity and efficacy in the NMDA receptor, a ligand-gated ion channel that belongs to the glutamate receptor family. Activation of NMDA receptors requires binding of two co-agonists, glycine and L-glutamate, to receptor domains in the in the NR1 and NR2 subunits. Occupancy by both agonists initiates a series of molecular events that culminates in opening of the associated ion channel. The objective of this proposal is to identify specific molecular determinants of the interaction of agonists with the NMDA receptor. The recently published crystal structure of the ligand binding domains of a related glutamate receptor (GluR2) predicts which amino acids are in direct contact with the agonists. Preliminary data from our lab suggest the existence of transduction elements in the glycine binding pocket and a highly conserved region in the M3 transmembrane segment. Therefore, the following specific aims are proposed: (1) To identify amino acid residues that determine agonsist affinity and efficacy. Site-directed mutagenesis has identified many amino acid residues whose mutation caused shifts in the agonist dose-response curves. However, such shifts in agonist sensitivity cannot be interpreted unambiguously. A new approach will therefore be used which can distinguish between mutations that affect agonist affinity or efficacy. By using of cysteine-substitution mutagenesis and thiol-specific modifying reagents, the same population of channels can be studied before and after modification. Full and partial agonists will be employed to unequivocally interpret alterations in efficacy and affinity. Parallel experiments using the GluR2 receptor will be used to confirm the structural assignments. (2) To test the hypothesis that the M3 segment is a transduction segment coupling ligand binding to channel opening. The M3 transmembrane segment of glutamate receptors contains a strictly conserved amino acid sequence. Cysteine substitutions in this region identified a residue for which thiol modification results in constitutively active NMDA receptors. Since this modification requires the presence of agonists, it was hypothesized that M3 undergoes a conformational change upon receptor activation and that thiol modification locks the receptor in the active state. These studies will result in a detailed molecular picture of the dynamic change in structure that accompany activation of the NMDA receptor.

structural biology; NMDA receptors; potassium channel; protein structure function; gene mutation; membrane potentials; membrane permeability; Xenopus oocyte; voltage /patch clamp; Xenopus; polymerase chain reaction; site directed mutagenesis;