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Advisory Committee Chair

Steven J Pittler

Advisory Committee Members

Kent T Keyser

Timothy W Kraft

Alecia K Gross

Shu-Zhen Wang

Document Type

Dissertation

Date of Award

2010

Degree Name by School

Doctor of Philosophy (PhD) School of Optometry

Abstract

The cGMP-gated cation channel (CNG) in the rod photoreceptors is an essential component of phototransduction, and may have a role in maintaining structural integrity of the rod outer segment (ROS). The ß-subunit of the CNG channel (CNGB1) in the rod photoreceptors is comprised of an N-terminal glutamic acid-rich region (GARP') and a C-terminal channel-like region. Additionally, two soluble proteins GARP1 and GARP2 are alternatively spliced from the CNGB1 gene. Although GARPs are known to interact with other proteins in the rod photoreceptors, their role is currently unknown. To determine the role of GARP2 in the rod photoreceptors, we generated transgenic mice expressing GARP2 (GARP2 tg). Our results show that GARP2 alone could not rescue the structural phenotype of the CNGB1 knockout, suggesting that more than one GARP-region containing protein or the ß-subunit of the CNG channel is necessary, and that GARP2 by itself is not sufficient to promote proper ROS structural integrity. GARP2 was expressed three-fold over the wild type in the GARP2 tg and it was correctly localized to the rim region of the ROS. Other ROS proteins examined were also properly localized and their expression levels were unchanged, except for the channel α-subunit, which was slightly elevated. Abnormally elongated membranes were occasionally observed in the outer segments of the GARP2 tg, which were also shorter than wild type rods. ERG analysis revealed that at 1 month, the average maximum rod a-wave responses were decreased 33%, which may be attributed to a reduced ROS length. The half-maximum rod sensitivity was unchanged in the GARP2 tg mice, indicating normal rod function. The parameter S derived from a fit to the rod phototransduction model was increased more than 50%, indicating an increase in phototransduction gain. Our findings suggest that GARP2 has a dual function in ROS: a structural role in proper rod outer segment disk morphogenesis and maintenance of structural integrity, and a functional role in regulating the activation phase of phototransduction.

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