The application proposes to examine the physiology, pharmacology and kinetics of GABAc receptors on retinal neurons and GABA-rho subunits expressed in Xenopus oocytes. In the first specific aim, the applicant will investigate the five subunits of GABA-rho, which he has cloned from white perch retina. He will examine their electrophysiological and pharmacological properties. He plans to compare the GABA dose-response curves among the five subunits and the pharmacology of antagonists such as I4AA and picrotoxin. In the different subunits, the applicant proposes to compare the kinetics of both the onset and offset of the GABA response using outside-out patch recordings extracted from oocytes. After deciphering these biophysical properties of the individual subunits, he plans to study GABAc receptors expressed in white perch retinal neurons. There will be particular emphasis on bipolar cells and the sensitivity and pharmacology of these cells will be compared to H4 horizontal cells. The applicant will also localize various subunits of the GABAc receptor in white perch retina using in situ hybridization and RT-PCR techniques. The rationale is that a different mix of GABAc receptor subunits in each type of neuron may produce distinct response properties. In a second specific aim, the applicant proposes to study the molecular structure of the GABAc receptor. The rationale is that although the GABAc receptor subunits seem similar to each other, the pharmacology and kinetics are different. Since the amino acid sequence of the subunits are very similar, it may be possible to identify just a few amino acids that account for these differences. The applicant will use site-directed mutagenesis and construction of chimeras to investigate structure/function relationships among these subunits. In the third and final specific aim, the applicant will investigate the modulation of GABAc receptors by second messengers. Based on sequencing data, the applicant has identified cAMP-dependent protein kinase (PKA), protein kinase C (PKC), and calmodulin (CaM)-II kinase consensus sites on the putative cytoplasmic domain of all of these receptor subunits. He will, therefore, investigate the role of phosphorylation in the modulation of each of the five subunits.
Showing the most recent 10 out of 26 publications
