Investigative Ophthalmology & Visual Science Cover Image for Volume 60, Issue 9
July 2019
Volume 60, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2019
Cone mitochondria shape cytosolic Ca2+ transients and recovery of the photoresponse
Author Affiliations & Notes
  • Rachel Hutto
    Biochemistry, University of Washington, Seattle, Washington, United States
  • Fatima Abbas
    John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States
  • Celia Bisbach
    Biochemistry, University of Washington, Seattle, Washington, United States
  • James Hurley
    Biochemistry, University of Washington, Seattle, Washington, United States
  • Frans Vinberg
    John A. Moran Eye Center, University of Utah, Salt Lake City, Utah, United States
  • Susan Brockerhoff
    Biochemistry, University of Washington, Seattle, Washington, United States
  • Footnotes
    Commercial Relationships   Rachel Hutto, None; Fatima Abbas, None; Celia Bisbach, None; James Hurley, None; Frans Vinberg, None; Susan Brockerhoff, None
  • Footnotes
    Support  PHS NRSA T32GM007270 from NIGMS, NEI EY026020, NEI EY028645, and P30EY001730.
Investigative Ophthalmology & Visual Science July 2019, Vol.60, 565. doi:
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      Rachel Hutto, Fatima Abbas, Celia Bisbach, James Hurley, Frans Vinberg, Susan Brockerhoff; Cone mitochondria shape cytosolic Ca2+ transients and recovery of the photoresponse. Invest. Ophthalmol. Vis. Sci. 2019;60(9):565.

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

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Abstract

Purpose : Photoreceptors rely on cytosolic Ca2+ clearance from the outer segment for proper recovery following light stimuli, and this is primarily accomplished by Na+/Ca2+,K+ exchangers (NCKX) channels on the plasma membrane. However, NCKX-deficient photoreceptors have demonstrated that an alternate, slower clearance pathway exists. Here we investigate whether mitochondria mediate Ca2+ clearance by analyzing zebrafish cones overexpressing or lacking the mitochondrial Ca2+ uniporter (MCU OE and KO).

Methods : We injected constructs of MCU cDNA tagged with T2A-RFP under control of a cone-specific promoter (gnat2) into zebrafish embryos to generate transgenic MCU OE lines. We generated global MCU KO fish using Crispr-Cas9 and confirmed KO with MCU antibody. We used Tg(gnat2:mito-GCaMP3) and Tg(gnat2:GCaMP3) zebrafish to report Ca2+. Data reported as mean ± SEM.
Electroretinogram recordings were performed ex vivo on isolated, dark-adapted zebrafish retinas supplemented with 40 µM DL-AP4 and 40 µM CNQX to isolate the photoreceptor component (a-wave). A 630 nm filter was used for the stimulus light to minimize rod responses.

Results : We found that overexpressing MCU approximately 100-fold in cones increases basal mitochondrial Ca2+ (394% ± 29 increase in [Ca2+] from WT). These cones have 2.3 ± 0.1 times faster clearance of a cytosolic Ca2+ bolus from the cell body than WT, and this is sensitive to the MCU inhibitor Ru360 (FigA,B). Furthermore, MCU OE dampens increases in intracellular Ca2+ (FigC). MCU OE cone light responses have faster recovery (Fig D). MCU OE fish have lower maximal cone responses, yet dim flash responses normalized to the maximal amplitude are larger (FigE,F). We are currently analyzing Ca2+ dynamics and the cone photoresponse in MCU KO fish.

Conclusions : Our results demonstrate that mitochondrial Ca2+ uptake can alter the cone photoresponse. Mitochondria in MCU OE cones clear Ca2+ faster from the cell body and can act as a sink for Ca2+ when it is cleared in response to light stimulation. Our work in MCU KO models will show the extent mitochondria in WT cones contribute to this response.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.

 

A. Ca2+ clearance in Tg(gnat2:GCaMP3) fish.
B. Decay constants of single exponential fits of data in A.
C. Peak change in cytosolic Ca2+ from data in A.
D. Isolated a-wave response of MCU OE and WT retinas.
E. Absolute amplitude of a-wave responses shown in D.
F. Normalized amplitude of a-wave responses (relative to max).

A. Ca2+ clearance in Tg(gnat2:GCaMP3) fish.
B. Decay constants of single exponential fits of data in A.
C. Peak change in cytosolic Ca2+ from data in A.
D. Isolated a-wave response of MCU OE and WT retinas.
E. Absolute amplitude of a-wave responses shown in D.
F. Normalized amplitude of a-wave responses (relative to max).

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