Structure-Function Studies of Nicotinic Acetylcholine Receptors Using Selective Agonists and Positive Allosteric Modulators

This dissertation primarily describes chemical-scale studies of nicotinic acetylcholine receptors (nAChRs) in order to better understand ligand-receptor selectivity and allosteric modulation influences during receptor activation. Electrophysiology coupled with canonical and non-canonical amino acids mutagenesis is used to probe subtle changes in receptor function.

The first half of this dissertation focuses on differential agonist selectivity of α4β2-containing nAChRs. The α4β2 nAChR can assemble in alternative stoichiometries as well as assemble with other accessory subunits. Chapter 2 identifies key structural residues that dictate binding and activation of three stoichiometry-dependent α4β2 receptor ligands: sazetidine-A, cytisine, and NS9283. These do not follow previously suggested hydrogen-bonding patterns of selectivity. Instead, three residues on the complementary subunit strongly influence binding ability of a ligand and receptor activation. Chapter 3 involves isolation of a α5α4β2 receptor-enriched population to test for a potential alternative agonist binding location at the α5 α4 interface. Results strongly suggest that agonist occupation of this site is not necessary for receptor activation and that the α5 subunit only incorporates at the accessory subunit location.

The second half of this dissertation seeks to identify residue interactions with positive allosteric modulators (PAMs) of the α7 nAChR. Chapter 4 focuses on methods development to study loss of potentiation of Type I PAMs, which indicate residues vital to propagation of PAM effects and/or binding. Chapter 5 investigates α7 receptor modulation by a Type II PAM (PNU 120596). These results show that PNU 120596 does not alter the agonist binding site, thus is relegated to influencing only the gating component of activation. From this, we were able to map a potential network of residues from the agonist binding site to the proposed PNU 120596 binding site that are essential for receptor potentiation.

Identification and characterization of the egg-laying hormone from the neurosecretary bag cells of Aplysia

This investigation has resulted in the chemical identification and isolation of the egg-laying hormone from Aplysia californica, Aplysia vaccaria, and Aplysia dactylomela. The hormone, which was originally identified as the Bag Cell-Specific protein (BCS protein) on polyacrylamide gels, is a polypeptide of molecular weight ≈ 6000, which is localized in the neurosecretory bag cells of the parietovisceral ganglion and the surrounding connective tissue sheath which contains the bag cell axons. All three species produce a hormone of similar molecular weight, but varying electrophoretic mobility as determined on polyacrylamide gels. As tested, the hormone is completely cross-reactive among the three species.

Although the bag cells of sexually immature animals contain the active hormone, sexual maturation of the animal results in a 10-fold increase in the BCS protein content of these neurons.

A seasonal variation in the BCS protein content was also observed, with 150 times more hormone contained in the bag cells of Aplysia californica in August than in January. This correlates well with the variation in the animals' ability to lay eggs throughout the year (Strumwasser et al., 1969). There are some indications that the receptivity of the animal to the available hormone also fluctuates during the year, being lower in winter than in swmner. The seasonal rhythm of the other species, Aplysia vaccaria and Aplysia dactylomela, has not been investigated.

A polyacrylamide gel electrophoresis analysis of water-soluble proteins in Aplysia californica revealed several other nerve-specific proteins. One of these is also located in the bag cell somas and stains turquoise with Amido Schwarz. The function of this protein has not been investigated.