Endogenous Calcium Buffers Regulate Fast Exocytosis in the Synaptic Terminal of Retinal Bipolar Cells

Abstract

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca(2+) buffers have not been tested. We find that 80% of Ca(2+) binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca(2+)-sensitive K(+) channels with an efficiency equivalent to approximately 1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca(2+) tail currents lasting approximately 0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca(2+) channels. We propose that endogenous Ca(2+) buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.

Calcium spike-mediated digital signaling increases glutamate output at the visual threshold of retinal bipolar cells

“…Longer depolarizations transfer larger Q Ca and trigger larger exocytosis from Mbs (Burrone et al 2002;Burrone and Lagnado 2000;Mennerick and Matthews 1996;Neves and Lagnado 1999). Nonetheless, fast "tail" I Ca , associated with repolarization of Mb terminals from ϩ60 mV to Ϫ60 mV, triggered larger exocytosis than depolarization from Ϫ60 to Ϫ10 mV, even if the latter transferred much larger Q Ca (Tachibana 1999;, emphasizing the importance of I Ca activation kinetics over Q Ca (Mennerick and Matthews 1996) and suggesting that Ca 2ϩ spikes might drive exocytosis more efficiently than graded responses (Protti et al 2000;Zenisek and Matthews 1998).…”

“…For voltage-clamp recordings, we used a cesium-based internal solution containing (in mM) 102 cesium gluconate, 10 TEA-Cl, 2 EGTA, 10 phosphocreatine-di(tris), 3 Mg-ATP, 0.5 Na 3 -GTP, 3 L-ascorbic acid, 0.04 Alexa Fluor 488, 13 NaOH (pH 7.25, osmolarity 260 mosM). In both internal solutions, the concentration of EGTA was 2 mM, approximating the buffering capacity of soluble endogenous Ca 2ϩ buffers (Burrone et al 2002). In voltageclamp recordings, Ames medium was supplemented with picrotoxin (PTX, 100 M) to block GABA-gated chloride conductances in Mb terminals (Vigh et al 2011;Vigh and von Gersdorff 2005).…”

Ca2+from One or Two Channels Controls Fusion of a Single Vesicle at the Frog Neuromuscular Junction

“…To test the dependence of our conclusions on endogenous buffer capacity, we assumed the presence of different amounts (0 -0.5 mM) of a buffer with a rather high affinity (dissociation constant, K D ϭ 2 M), fast kinetics (k on ϭ 3 ϫ 10 8 M Ϫ 1 /s), and slow diffusion (27.5 m 2 /s) (Burrone et al, 2002). This is equivalent to buffer capacities of up to 250.…”

Calcium influx selects the fast mode of endocytosis in the synaptic terminal of retinal bipolar cells

“…The final [EGTA] was estimated to be at least at 5 mM because it was sufficient to completely block bulk rises in free Ca 2ϩ in response to long stimuli (24). Terminals were then washed in Ringer's solution lacking CaCl 2 for 10 min.…”