Date of Award

Summer 2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Neuroscience

First Advisor

Zhe Lu

Abstract

Cyclic nucleotide-gated (CNG) channels mediate the transduction of light signals to electrical signals in vertebrate photoreceptors. These channels are non-selective for cations and open upon cGMP binding. The intracellular cGMP concentration is elevated in darkness, and the current through CNG channels maintains the membrane of the rod photoreceptor at around -40 mV. When light enters the retina, it triggers a signal transduction cascade that decreases intracellular cGMP, and therefore CNG channels close. A reduction in CNG current hyperpolarizes the rod. Two molecular mechanisms are crucial for the proper physiological function of retinal CNG channels. First, block of these channels by physiological agents reduces membrane noise in rods. This feature enables rods to detect photons with high sensitivity. Second, CNG channels must be able to conduct current in response to the light-triggered changes in intracellular cGMP. In other words, they should open and close gradually in response to cGMP binding, not other stimuli such as voltage. This thesis addresses these two fundamental mechanisms of CNG channel function. We present the biophysical investigation of the mechanism underlying voltage sensitivity of ion channel block. That is, affinity of blocking agents for the pore of ion channels varies with membrane voltage. Two models have been postulated to explain this phenomenon. Here we use the retinal CNG channel (CNGA1) as a model to illustrate the analytical solutions and electrophysiological hallmarks of these two models. In addition, we show through a systematic mutagenesis study, that certain point mutations around the outer, narrow part of the channel close the channel at the relevant physiological voltages. However, these mutant channels can open in response to voltage at positive potentials only when bound by cGMP. This mutation-caused voltage gating reflects the inherent voltage gating capabilities of the wild-type channel, which must be suppressed to avoid their closure in a physiological setting. These results support the notion that the selectivity filter of the channel must be properly anchored to the surrounding protein structure in order for CNGA1 channels to achieve their physiological role.

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