Ca2+ Diffusion through Endoplasmic Reticulum Supports Elevated Intraterminal Ca2+ Levels Needed to Sustain Synaptic Release from Rods in Darkness

In addition to vesicle release at synaptic ribbons, rod photoreceptors are capable of substantial slow release at non-ribbon release sites triggered by Ca2+-induced Ca2+ release (CICR) from intracellular stores. To maintain CICR as rods remain depolarized in darkness, we hypothesized that Ca2+ released into the cytoplasm from terminal endoplasmic reticulum (ER) can be replenished continuously by ions diffusing within the ER from the soma. We measured [Ca2+] changes in cytoplasm and ER of rods from Ambystoma tigrinum retina using various dyes. ER [Ca2+] changes were measured by loading ER with fluo-5N and then washing dye from the cytoplasm with a dye-free patch pipette solution. Small dye molecules diffused within ER between soma and terminal showing a single continuous ER compartment. Depolarization of rods to ？40 mV depleted Ca2+ from terminal ER, followed by a decline in somatic ER [Ca2+]. Local activation of ryanodine receptors in terminals with a spatially confined puff of ryanodine caused a decline in terminal ER [Ca2+], followed by a secondary decrease in somatic ER. Localized photolytic uncaging of Ca2+ from o -nitrophenyl-EGTA in somatic ER caused an abrupt Ca2+ increase in somatic ER, followed by a slower Ca2+ increase in terminal ER. These data suggest that, during maintained depolarization, a soma-to-terminal [Ca2+] gradient develops within the ER that promotes diffusion of Ca2+ ions to resupply intraterminal ER Ca2+ stores and thus sustain CICR-mediated synaptic release. The ability of Ca2+ to move freely through the ER may also promote bidirectional communication of Ca2+ changes between soma and terminal. SIGNIFICANCE STATEMENT Vertebrate rod and cone photoreceptors both release vesicles at synaptic ribbons, but rods also exhibit substantial slow release at non-ribbon sites triggered by Ca2+-induced Ca2+ release (CICR). Blocking CICR inhibits >50% of release from rods in darkness. How do rods maintain sufficiently high [Ca2+] in terminal endoplasmic reticulum (ER) to support sustained CICR-driven synaptic transmission? We show that maintained depolarization creates a [Ca2+] gradient within the rod ER lumen that promotes soma-to-terminal diffusion of Ca2+ to replenish intraterminal ER stores. This mechanism allows CICR-triggered synaptic release to be sustained indefinitely while rods remain depolarized in darkness. Free diffusion of Ca2+ within the ER may also communicate synaptic Ca2+ changes back to the soma to influence other critical cell processes