The Voltage-Gated Ca2+ Channel Is the Ca2+ Sensor Protein of Secretion

Neurotransmitter release involves two consecutive Ca2+-dependent steps, an initial Ca2+ binding to the selectivity filter of voltage-gated Ca2+ channels (VGCC) followed by Ca2+ binding to synaptic vesicle protein. The unique Ca2+-binding site of the VGCC is located within the alpha(1) subunit of the Ca2+ channel. The structure of the selectivity filter allows for the binding of Ca2+ Sr2+, Ba2+, and La3+. Despite its cell impermeability, La3+ supports secretion, which is in contradistinction to the commonly accepted mechanism in which elevation of cytosolic ion concentrations ([Ca2+](i)) and binding to synaptotagmin(s) trigger release. Here we show that a Cav 1.2-mutated (alpha(1) 1.2/L775P subunit which does not conduct Ca2+ currents supports depolarization-evoked release by means of Ca2+ binding to the pore. Bovine chromaffin cells, which secrete catecholamine almost exclusively via nifedipine-sensitive Cav1.2, were infected with the Semliki Forest Virus, pSFV (alpha(1) 1.2/L775P. This construct also harbored a second mutation that rendered the channel insensitive to nifedipine. Depolarization of cells infected with alpha(1) 1.2/L775P triggered release in the presence of nifedipine. Thus, the initial Ca (2+) binding at the pore of the channel appeared to be sufficient to trigger secretion, indicating that the VGCC could be the primary Ca2+ sensor protein. The 25% lower efficiency, however, implied that additional ancillary effects of elevated [Ca2+](i) were essential for optimizing the overall release process. Our findings suggest that the rearrangement of Ca2+ ions within the pore of the channel during membrane depolarization triggers secretion prior to Ca2+ entry. This allows for a tight temporal coupling between the depolarization event and exocytosis of vesicles tethered to the channel.