Baker, Sheila1
1Department of Biochemistry and Molecular Biology, University of Iowa, Iowa City, IA
Purpose: KCNV2 retinopathy is an enigmatic congenital retinal degeneration affecting children. It is a form of early-onset macular degeneration with aberrant electrical signaling that is diagnosed using ERG. Mutations in the gene KCNV2 were identified as the cause of the disease at the turn of the century. KCNV2 encodes a voltage-gated ion channel, Kv8.2. Kv8.2 only forms functional channels by assembling with Kv2.1. The heteromeric Kv2.1/Kv8.2 channel regulates photoreceptor membrane potential. We developed mice lacking Kv8.2 expression to investigate the molecular mechanisms of Kv2.1/Kv8.2 function and to model KCNV2 retinopathy.
Methods: We generated Kv8.2 KO mice using CRISPR. Kv8.2 KO mice were bred to a mouse strain with an all-cone retina (NRL KO, RPE65 R91W+/-) to generate Conefull: Kv8.2 KO. Retina function was analyzed using ERG. Retina structure was analyzed using OCT, histology, and transmission electron microscopy. Recombinant Kv2.1-T1 domain was purified from E. coli and the X-ray crystal structure solved by molecular replacement. AlphaFold2 was used to create a hetero-tetrameric Kv2.1/Kv8.2-T1 model.
Results: A series of ERG tests demonstrate that the Kv8.2 KO mice phenocopy the features of KCNV2 retinopathy. Rod-driven signaling is characterized by a reduced amplitude and square a-wave, the b-wave is delayed and has a supernormal amplitude, and the c-wave amplitude is reduced. Cone-driven signaling elicited by flickering light in both Kv8.2 KO and Conefull: Kv8.2 KO is reduced. Kv8.2 KO mice undergo a mild retinal degeneration, 30% loss at 10 months of age, but no loss of cones, measured out to 1 year of age. Degeneration in Conefull mice is accelerated in Conefull:Kv8.2 KO. Most missense mutations associated with KCNV2 retinopathy are found in the T1 tetramerization domain which indicates that channel assembly is particularly vulnerable. To investigate the molecular mechanism of channel assembly we were able to solve the X-ray crystal structure of the Kv2.1-T1 domain and modeled in a Kv8.2-T1 subunit. This revealed that two features adding stability to the Kv2.1-T1/T1 interface are inter-
Conclusions: Kv8.2 KO mice phenocopy the aberrant electrical signaling characteristic of KCNV2 retinopathy. The defective cone signaling is independent of rods, but the presence of rods protects cones from degenerating. Additional molecular studies are needed to learn how assembly of Kv8.2 with Kv2.1 is regulated. Meanwhile, our mouse models could be used for pre-clinical testing of gene or drug therapies for KCNV2 retinopathy.
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