Calcium uptake by mitochondria is required to maintain distinct Ca²⁺ pools in cone photoreceptors

Abstract

Purpose : Ca²⁺ regulates distinct cellular processes in cone photoreceptors (PRs) from photoresponse recovery in the outer segment (OS) to neurotransmission in the synapse. Mitochondria are located directly between the OS and the cell body and are known to buffer Ca²⁺ in other cell types. We hypothesize that mitochondria buffer Ca²⁺ within cone PRs to compartmentalize Ca²⁺ signaling.

Methods : Adult zebrafish retinas expressing a genetically encoded fluorescent Ca²⁺ sensor in the cone PR cytosol (GCaMP3) or mitochondria (mitoGCaMP3), were dissected, flat-mounted, stained with BODIPY, and sliced into 400-µm sections. Slices were transferred to an imaging chamber. Slices were perfused with Ringer’s solution, then treated with either increased extracellular Ca²⁺, 25 μM sildenafil, 10 mM KCl, or with 10 μM Ru360. GCaMP fluorescence was monitored by confocal microscopy.

Results : We found that synaptic and cell body Ca²⁺ levels can be selectively increased when the cells are depolarized with KCl, whereas OS Ca²⁺ levels remain unchanged. Conversely, OS Ca²⁺ can be dramatically increased using a Pde6 inhibitor sildenafil. Treatment with sildenafil for 30 minutes causes Ca²⁺ to accumulate in the OS, while Ca²⁺ pools in the cell body and synapse are low. These findings indicate that Ca²⁺ diffusion between PR compartments is controlled. To determine if mitochondria maintain distinct Ca²⁺ pools, we monitored mitochondrial Ca²⁺ levels using mitoGCaMP3. Raising Ca²⁺ either in the OS or cell body causes an apparent rise in mitochondrial Ca²⁺. Treatment of slices with Ru360, an inhibitor of the mitochondrial calcium uniporter (MCU), abolishes compartmentalization, implicating mitochondria in the segregation of Ca²⁺ pools.

Conclusions : PRs use Ca²⁺ to modulate signaling processes in the OS, cell body, and synapse. Here we show that Ca²⁺ is compartmentalized between OS and cell body in PRs, and this gradient is maintained by mitochondrial Ca²⁺ buffering via the MCU. The influence of OS Ca²⁺ and cell body Ca²⁺ on mitochondrial function are not yet known. Understanding how Ca²⁺ homeostasis is regulated in PRs will provide an understanding for how cells in the retina stay healthy and how and mitochondria respond to energy demands.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.