There is a fundamental gap in knowledge regarding how mutations in the genes encoding cyclic nucleotide- gated (CNG) ion channels can produce achromatopsia, cone dystrophy and macular degeneration in hu- mans. Our long-term objective is to understand the mechanisms controlling the activity of these channels and the pathophysiology of retinal diseases associated with CNG channel mutations. The core objectives of this application are to determine the cellular mechanisms responsible for the effect of cone CNG channel gating or trafficking mutations on cell viability, and the structural features critical for control of channels by phosphoinositides. Recently, we have functionally characterized several disease-associated mutations in the CNGA3 and CNGB3 subunits of cone CNG channels and discovered dramatic effects on channel gat- ing, regulation and/or trafficking, but the cellular consequences of these defects have not been determined. The central hypothesis is that gain-of-function mutations in cone CNG channels lead to photoreceptor death via enhanced or uncontrolled channel activity, disturbance of intracellular calcium (Ca2+) homeostasis and subsequent Ca2+-dependent apoptosis. Conversely, trafficking defects are expected to impair cell viability via endoplasmic reticulum (ER) stress. The rationale for the proposed research is that developing an under- standing of photoreceptor dysfunction and loss associated with abnormal CNG channel activity will provide insight into possible treatments for several related cone dystrophies. Guided by strong preliminary data, we will address these issues by pursuing two specific aims: (1) identify the connection between disease asso- ciated functional changes in cone CNG channels and the cellular mechanisms leading to photoreceptor dys- function and death;and (2) determine the mechanisms and interactions underlying the ability of CNGB3 subunits to confer sensitivity to channel control by phosphoinositides. These studies will utilize molecular and cellular manipulations, biochemical approaches and/or electrophysiological studies of human CNG channels expressed in cone photoreceptor derived 661W cells or Xenopus oocytes, and as transgenes in zebrafish cone photoreceptors. The proposed research is innovative in that informative in vitro studies will be extended to transgenic expression of mutant CNG channels in vivo. Overall, the proposed work is signif- icant because it is expected to enhance our understanding of the mechanisms that lead to retinal degenera- tion and blindness, and to provide insight into potential approaches for prevention of photoreceptor loss.
The proposed research has relevance to public health, because completion of these studies will provide important insight for preventing and treating vision loss. Our work is focused on understanding vision at the molecular and cellular levels and how mutations in genes coding for proteins critical for vision can lead to dysfunction and retinal degeneration. We study ion channel proteins that are responsible for generating electrical signals ultimately interpreted by the brain as visual information. The major goal for this new project is to elucidate specific mechanisms linking pathogenic changes in channel function or control to cell death.
|Dai, Gucan; Sherpa, Tshering; Varnum, Michael D (2014) Alternative splicing governs cone cyclic nucleotide-gated (CNG) channel sensitivity to regulation by phosphoinositides. J Biol Chem 289:13680-90|
|Dai, Gucan; Peng, Changhong; Liu, Chunming et al. (2013) Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides. J Gen Physiol 141:413-30|
|Dai, Gucan; Varnum, Michael D (2013) CNGA3 achromatopsia-associated mutation potentiates the phosphoinositide sensitivity of cone photoreceptor CNG channels by altering intersubunit interactions. Am J Physiol Cell Physiol 305:C147-59|
|Meighan, Starla E; Meighan, Peter C; Rich, Elizabeth D et al. (2013) Cyclic nucleotide-gated channel subunit glycosylation regulates matrix metalloproteinase-dependent changes in channel gating. Biochemistry 52:8352-62|
|Liu, Chunming; Sherpa, Tshering; Varnum, Michael D (2013) Disease-associated mutations in CNGB3 promote cytotoxicity in photoreceptor-derived cells. Mol Vis 19:1268-81|
|Meighan, Peter C; Meighan, Starla E; Rich, Elizabeth D et al. (2012) Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels. Channels (Austin) 6:181-96|
|Duricka, Deborah L; Brown, R Lane; Varnum, Michael D (2012) Defective trafficking of cone photoreceptor CNG channels induces the unfolded protein response and ER-stress-associated cell death. Biochem J 441:685-96|
|Yamamoto, B J; Elias, P D; Masino, J A et al. (2010) The angiotensin IV analog Nle-Tyr-Leu-psi-(CH2-NH2)3-4-His-Pro-Phe (norleual) can act as a hepatocyte growth factor/c-Met inhibitor. J Pharmacol Exp Ther 333:161-73|
|Bright, Scott R; Rich, Elizabeth D; Varnum, Michael D (2007) Regulation of human cone cyclic nucleotide-gated channels by endogenous phospholipids and exogenously applied phosphatidylinositol 3,4,5-trisphosphate. Mol Pharmacol 71:176-83|
|Brady, James D; Rich, Elizabeth D; Martens, Jeffrey R et al. (2006) Interplay between PIP3 and calmodulin regulation of olfactory cyclic nucleotide-gated channels. Proc Natl Acad Sci U S A 103:15635-40|
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