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Title: Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment

Abstract

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis ofmore » in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.« less

Authors:
 [1];  [2];  [2];  [3];  [3];  [2];  [2];  [4];  [2];  [3];  [2];  [1];  [2];
  1. Temple Univ., Philadelphia, PA (United States). Dept. of Biology, College of Science and Technology
  2. Univ. of Pennsylvania, Philadelphia, PA (United States). Dept. of Clinical Studies-Philadelphia, School of Veterinary Medicine
  3. Temple Univ., Philadelphia, PA (United States). Inst. for Computational Molecular Science
  4. Veterinary Vision (United Kingdom)
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Oak Ridge Leadership Computing Facility (OLCF)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1565391
Resource Type:
Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 9; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Science & Technology - Other Topics

Citation Formats

Tanaka, Naoto, Dutrow, Emily V., Miyadera, Keiko, Delemotte, Lucie, MacDermaid, Christopher M., Reinstein, Shelby L., Crumley, William R., Dixon, Christopher J., Casal, Margret L., Klein, Michael L., Aguirre, Gustavo D., Tanaka, Jacqueline C., Guziewicz, Karina E., and Khanna, Hemant. Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment. United States: N. p., 2015. Web. doi:10.1371/journal.pone.0138943.
Tanaka, Naoto, Dutrow, Emily V., Miyadera, Keiko, Delemotte, Lucie, MacDermaid, Christopher M., Reinstein, Shelby L., Crumley, William R., Dixon, Christopher J., Casal, Margret L., Klein, Michael L., Aguirre, Gustavo D., Tanaka, Jacqueline C., Guziewicz, Karina E., & Khanna, Hemant. Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment. United States. doi:10.1371/journal.pone.0138943.
Tanaka, Naoto, Dutrow, Emily V., Miyadera, Keiko, Delemotte, Lucie, MacDermaid, Christopher M., Reinstein, Shelby L., Crumley, William R., Dixon, Christopher J., Casal, Margret L., Klein, Michael L., Aguirre, Gustavo D., Tanaka, Jacqueline C., Guziewicz, Karina E., and Khanna, Hemant. Fri . "Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment". United States. doi:10.1371/journal.pone.0138943. https://www.osti.gov/servlets/purl/1565391.
@article{osti_1565391,
title = {Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment},
author = {Tanaka, Naoto and Dutrow, Emily V. and Miyadera, Keiko and Delemotte, Lucie and MacDermaid, Christopher M. and Reinstein, Shelby L. and Crumley, William R. and Dixon, Christopher J. and Casal, Margret L. and Klein, Michael L. and Aguirre, Gustavo D. and Tanaka, Jacqueline C. and Guziewicz, Karina E. and Khanna, Hemant},
abstractNote = {Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.},
doi = {10.1371/journal.pone.0138943},
journal = {PLoS ONE},
number = 9,
volume = 10,
place = {United States},
year = {2015},
month = {9}
}

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