Cone mitochondria shape cytosolic Ca²⁺ transients and recovery of the photoresponse

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).