Cyclic nucleotide-gated (CNG) ion channels play a key role at several stages of signal processing in the retina. In photoreceptor outer segments, they detect and signal the drop in cGMP concentration resulting from the absorption of light by rhodopsin; in inner segments on the cone photoreceptors they modulate transmitter release onto the bipolar cells; and in one-bipolar cells they are responsible for the inhibitory response to glutamate. CNG channels are highly specialized for their role in signal processing. The long-term goal of our research is to understand the molecular mechanisms that underlie their specializations. cGMP activates the channel by binding directly to a cyclic nucleotide-binding domain in the carboxyl-terminal region of each channel subunit. cAMP is a very poor activator of these channels. This cyclic nucleotide selectivity arises from a conformational change in the cyclic nucleotide-binding site associated with opening of the ion conducting pore. Furthermore, these channels are modulated by Ca/2+- calmodulin and phosphorylation. This modulation is thought to involve a protein-protein interaction between the amino-terminal region of each subunit and the cyclic nucleotide-binding domain. In this proposal we will examine the molecular mechanism underlying the conformation change in the cyclic nucleotide-binding site and how it is regulated by this protein-protein interaction. The channels will be studied electrophysiologically by exogenously expressing the cDNA clones in Xenopus oocytes, and biochemically by expressing the amino terminal region and cyclic nucleotide-binding domain of the channel as fusion proteins in bacteria. We will probe the conformation change in the cyclic nucleotide-binding site with single-channel recording, site- directed mutagenesis, and state-dependent cysteine modification. In addition, using protein interaction assays, we will examine the structural determinants for the interaction between the amino-terminal region and the cyclic nucleotide-binding domain and how they account for modulation of the channels by Ca/2+-calmodulin and phosphorylation. These experiments should provide insight into the role of CNG channels in signal transduction.
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