Ca2+-dependent inactivation of a neuronal Ca2+ channel is regulated by intracellular Ca2+ handling

Abstract

Intracellular Ca2+ is a critical regulator of gene expression, neuronal excitability, and neurosecretion. Free cytosolic Ca2+ is controlled by numerous channels, pumps, and exchangers in the plasma membrane, endoplasmic reticulum, or mitochondria. In the marine mollusc, Aplysia californica, ovulation is initiated from the central nervous system by bag cell neurons. Upon brief synaptic input, these neuroendocrine cells fire a synchronous afterdischarge, starting with an ~5-Hz, ~1-min fast-phase, then an ~1-Hz, ~30-min slow-phase, that culminates in the Ca2+-dependent neurohaemal secretion of egg-laying hormone. During the fast-phase, Ca2+ rises due to entry through voltage-gated Ca2+ channels. Repetitive opening results in Ca2+-dependent inactivation (CDI) of the channel, aka use-dependent rundown, which serves as a form of negative feedback. Our laboratory previously showed that mitochondria sequester voltage-gated Ca2+ influx, although how this influences Ca2+ channel function is unknown. Here, I test the hypothesis that intracellular Ca2+ handling, by both membrane transport and organelles, controls CDI. Rundown of isolated Ca2+ current was recorded in single cultured bag cell neurons under whole-cell voltage-clamp using recurring step depolarizations (~70% current remaining at the end of a 5-Hz, 5-sec train-stimulus). Rundown was significantly decreased by swapping Ba2+ for Ca2+ in the external solution, or adding the Ca2+ chelator, EGTA, to the intracellular/pipette solution (both ~90% current remaining). However, blocking either the mitochondrial Ca2+ uniporter or the plasma membrane Ca2+-ATPase significantly increased rundown (both ~60% current remaining). To further understand mitochondrial Ca2+ handling, neurons were loaded with the Ca2+ sensitive dye, fura-PE3, and Ca2+ liberated from the mitochondria by collapsing the organelle membrane potential with the protonophore, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP). Compared to control, a prior train-stimulus to elicit Ca2+ entry significantly increased the FCCP-induced percent-change in Ca2+ (from ~175% to ~450%), and this was prevented by inhibiting the mitochondrial Ca2+ uniporter. These findings suggest that both mitochondrial uptake and plasma membrane extrusion play a key role in CDI by buffering Ca2+ during prolonged influx. Overall, Ca2+ dynamics serve as a key regulator of Ca2+ channel function, with the potential to govern Ca2+-dependent processes in general.