July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
14-3-3 regulates the expression of HCN1
Author Affiliations & Notes
  • Sheila Baker
    Biochemistry, University of Iowa, Iowa City, Iowa, United States
  • Joseph Laird
    Biochemistry, University of Iowa, Iowa City, Iowa, United States
  • Shivangi Inamdar
    Biochemistry, University of Iowa, Iowa City, Iowa, United States
  • Colten Lankford
    Biochemistry, University of Iowa, Iowa City, Iowa, United States
  • Footnotes
    Commercial Relationships   Sheila Baker, None; Joseph Laird, None; Shivangi Inamdar, None; Colten Lankford, None
  • Footnotes
    Support  NIH Grant EY020542
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 2348. doi:
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    • Get Citation

      Sheila Baker, Joseph Laird, Shivangi Inamdar, Colten Lankford; 14-3-3 regulates the expression of HCN1. Invest. Ophthalmol. Vis. Sci. 2018;59(9):2348.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose : A key factor contributing to the temporal resolution of rods is the feedback current carried by hyperpolarization-activated and cyclic nucleotide-gated channel, family member 1 (HCN1). HCN1 is located in the inner segments of photoreceptors and opens in response to light-triggered hyperpolarization to carry an inward sodium/potassium current that shapes visual responses under medium and bright light conditions. HCN1 activity is primarily regulated by adjusting the concentration of channel at the plasma membrane, but how this is controlled is unknown. In this study the signaling adaptor 14-3-3 was investigated as a potential HCN1 regulator.

Methods : Expression of 14-3-3 in the mouse retina was analyzed by reverse-transcriptase digital droplet PCR and immunohistochemistry. Immunoprecipitation, thermal shift assays, isothermal titration calorimetry (ITC), and BioID proximity assays were used to analyze the interaction between 14-3-3 and HCN1. The half-life of HCN1 and mutant HCN1 incapable of binding to 14-3-3 was measured with a SNAP-tag based pulse-chase assay.

Results : Six of the seven 14-3-3 proteins were found to be expressed in the retina and YWHAE, coding for 14-3-3ε was enriched in photoreceptors where it localized to the inner segment. Several independent assays were used to confirm that HCN1 can bind to 14-3-3. Three potential 14-3-3 ligands within HCN1 were tested in peptide assays and only one site bound to 14-3-3 – amino acids 861-872 in the distal part of the intrinsically disordered C-terminal tail of HCN1. Using ITC, the Kd was calculated to be 15 μm, which is the typical Kd for a high affinity 14-3-3 ligand; phosphorylation of the peptide was required for 14-3-3 binding. A deletion mutant of HCN1 lacking the 14-3-3 binding site was found to be expressed at higher levels.

Conclusions : HCN1 was validated as a new client of the 14-3-3 family of signaling adaptors. In the retina HCN1 is enriched in photoreceptor inner segments along with 14-3-3ε. Phosphorylation-dependent recruitment of 14-3-3 to HCN1 negatively regulates expression of HCN1. This is opposite to the effect of the HCN channel accessory subunit, TRIP8b. Together, these findings suggests that dynamic modulation of the availability of HCN1 at the plasma membrane of photoreceptor inner segments could contribute to fine tuning the output of photoreceptors under different lighting conditions.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.


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