Regulation Of Presynaptic Calcium In A Rod Bipolar Cell

Abstract

Purpose: : Presynaptic Ca²⁺ regulates many aspects of synaptic physiology, including neurotransmitter release. We investigated the roles of the plasma membrane calcium ATPase (PMCA), and the sodium/calcium exchanger (NCX) in presynaptic calcium handling in the synaptic terminals of a mammalian rod bipolar cell.

Methods: : Isolated mouse rod bipolar cells were held under voltage-clamp control via a whole-cell patch pipette positioned on the cell soma. The intracellular calcium concentration in a synaptic terminal or soma was measured with calcium-sensitive fluorescent indicator dyes.

Results: : Under control conditions, the resting calcium concentration in synaptic terminals was ~ 50nM (55 ± 7nM, n=23). Treatment with Na₃VO₄ to block PMCA or removal of Na⁺ from external solution to block NCX resulted in an approximate doubling of resting calcium (Na₃VO₄: 131 ± 20nM, n=14; 0 external Na⁺: 107 ± 12nM, n=33). Thapsigargin, an inhibitor of the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) had no effect on resting calcium. Following a 1s depolarization, recovery of intraterminal Ca²⁺ was prolonged in the presence of Na₃VO₄ (2.8 ± 0.4s, n=5) relative to controls (1.6 ± 0.2s, n=15). Calcium recovery was not significantly prolonged by 0 external Na⁺ (2 ± 0.2s, n=25) or thapsigargin (1.8 ± 0.4s, n=5). When dialyzed with a Ca²⁺-buffered internal solution with a free Ca²⁺ concentration of 50 µM, the calcium concentration in synaptic terminals remained close to resting levels (77 ± 16nM; n=14). Inhibition of PMCA or NCX resulted in a modest elevation of intraterminal Ca²⁺ to ~ 1 µM (n=12), but in the presence of mitochondrial inhibitors, the intraterminal calcium concentration rose to tens of µM (n=4).

Conclusions: : PMCA and NCX are instrumental in setting the resting calcium concentration in synaptic terminals of rod bipolar cells. Restoration of intraterminal calcium levels following depolarization-evoked calcium entry is regulated by PMCA, but not NCX. Under conditions of internal Ca²⁺ dialysis, a dominant role is played by mitochondria, although PMCA and NCX also contribute.

Keywords: bipolar cells • calcium • synapse