| Cyclic nucleotide-gated (CNG) ion channels have important roles in visual and olfactory sensory transduction. These tetrameric proteins are activated by the direct binding of cyclic nucleotides to a carboxy-terminal binding domain. The native forms of CNG channels exist as heterotetramers of alpha and beta subunits. I am interested in the intersubunit interactions that underlie the tightly-regulated and unusual 3 CNGA1: 1 CNGB1 subunit ratio of retinal rod CNG channels. It has been demonstrated (Zhong et al., 2002) that a distal C-terminal fragment of the CNGA subunit from cone photoreceptors (CNGA3) forms trimers in solution. This CNGA3 fragment contains a predicted coiled-coil sequence that is conserved among all cloned CNGA subunits. In this thesis I test the hypothesis that the distal C-terminus of CNGA1 produces the 3 CNGA1: 1 CNGB1 subunit ratio by acting as a "trimerization domain". To test this hypothesis, in Chapter 2, I assessed the effects of CNGA1 distal C-terminal deletion on co-assembly with CNGB1 to produce functional heteromeric channels, using electrophysiology. I found that co-expression of distal C-terminal deleted CNGA1 with a CNGB1 subunit produced functional channels with marked elevation in the activation by the partial agonist cAMP, and altered block by the pharmacological agent l-cis-diltiazem. These results support a change in subunit stoichiometry away from 3 CNGA1: 1 CNGB1. To test if CNGA1 distal C-terminal deletion permits subunit ratios other than 3 CNGA1: 1 CNGB1, in Chapter 3 I used a FRET-based fluorescence imaging technique to assess if FRET between co-expressed mCitrine- and mCerulean genetic fusions of CNGB1 was enhanced when co-expressed with distal C-terminal-deleted CNGA1, relative to co-expression with full-length CNGA1. Enhanced FRET between fluorescent CNGB1 subunits was observed with co-expression with the CNGA1 deletion construct, indicating a change in subunit stoichiometry with this manipulation.;Chapter 4 describes a collaborative project with Kevin D. Black and Dr. William N. Zagotta. Using a combination of protein chemistry techniques, we show that distal C-terminal fragments of both CNGA1 and CNGA3 form trimers in solution. These results, in combination with the electrophysiology and FRET results of Chapters 2 and 3, support the importance of the CNGA1 distal C-terminal in regulating heteromeric channel stoichiometry by through its action as a "trimerization domain."... |
