Alpha-Latrotoxin: A Potent Dual Mode Enhancer of Beta Cell Exocytosis
Abstract Number: 2465-PO
Authors: JUNE LIU-GENTRY, AMELIA SILVA, DAVID BARNETT, STANLEY MISLER
Institutions: St. Louis, MO
Results: Alpha-latrotoxin (a-LT), from the black widow spider, is a potent excitatory and secretory toxin at synapses and a host of excitable endocrine cells. At nanomolar levels a-LT provokes massive Ca-dependent exocytosis from quiescent voltage clamped cells, while at lower levels it enhances depolarization-evoked exocytosis. In contrast, in the beta cell a-LT is reported to produce at best a small increase in glucose-induced insulin release, perhaps by a Ca-independent action. By applying a-LT of consistenty high activity in adrenal chromaffin cells (ACCs) to single canine and human beta cells we have reconciled this discrepancy. (i) As in ACCs, a-LT induces large conductance cation selective channels and rises in cytosolic Ca into the nM range, albeit at 5-10 fold higher concentrations than in ACCs. The sustained opening of as few as one or two 100 pS channels in buffered isotonic Ca bath is sufficient to raise cytosolic Ca (Cai) to > 500 nM. The rise in Cai causes concurrent and parallel increases in membrane capacitance (Cm) and quantal release of serotonin, a marker of insulin granule marker contents, is consistent with the exocytosis of at least 50-100 insulin granules over 10-20 s at resting membrane potentials. (ii) As in ACCs, concentrations of a-LT too low to induce sustained channel activity nevertheless increase exocytosis evoked by a train of depolarizations, often in absence of a change in background cytosolic Ca. The majority of the enhancement in Cm occurs as a delayed component appearing during and immediately after a short train of depolarizations and is often correlated with slowed decay of cytosolic Ca. Hence, in beta cells high potency a-LT provides a continuous pathway for Ca entry at nM concentrations as well as modulates the coupling of cytosolic Ca to exocytosis (?by enhancing granule recruitment) beginning at pM concentrations. Thus a-LT can serve as a valuable tool for probing insulin granule exocytosis or examining secretory capacity of beta cells where other features of excitation-secretion coupling have been altered.