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 α₁ subunit of the Ca ²⁺ channel. The structure of the selectivity filter allows for the binding of Ca²⁺, Sr²⁺, Ba²⁺, and La ³⁺. 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 Cavl.2-mutated α₁1.2/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 Cavl.2, were infected with the Semliki Forest Virus, pSFV α₁1.2/L775P. This construct also harbored a second mutation that rendered the channel insensitive to nifedipine. Depolarization of cells infected with α₁1.2/L775P. triggered release in the presence of nifedipine. Thus, the initial Ca2+ 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.