The voltage-gated Ca(2+) channel is the Ca(2+) sensor protein of secretion.
Neurotransmitter release involves two consecutive Ca(2+)-dependent steps, an initial Ca(2+) binding to the selectivity filter of voltage-gated Ca(2+) channels (VGCC) followed by Ca(2+) binding to synaptic vesicle protein. The unique Ca(2+)-binding site of the VGCC is located within the alpha(1) subunit of the Ca(2+) channel. The structure of the selectivity filter allows for the binding of Ca(2+), Sr(2+), Ba(2+), and La(3+). Despite its cell impermeability, La(3+) supports secretion, which is in contradistinction to the commonly accepted mechanism in which elevation of cytosolic ion concentrations ([Ca(2+)](i)) and binding to synaptotagmin(s) trigger release. Here we show that a Cav1.2-mutated alpha(1)1.2/L775P subunit which does not conduct Ca(2+) currents supports depolarization-evoked release by means of Ca(2+) 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 Ca(2+) sensor protein. The 25% lower efficiency, however, implied that additional ancillary effects of elevated [Ca(2+)](i) were essential for optimizing the overall release process. Our findings suggest that the rearrangement of Ca(2+) ions within the pore of the channel during membrane depolarization triggers secretion prior to Ca(2+) entry. This allows for a tight temporal coupling between the depolarization event and exocytosis of vesicles tethered to the channel.
Hagalili
Yamit
Y
Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, 91904 Israel.
Bachnoff
Niv
N
Atlas
Daphne
D
D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism*; D002417:Cattle; D019439:Chromaffin Cells; D004563:Electrochemistry; D005089:Exocytosis; D007700:Kinetics; D050505:Mutant Proteins; D011485:Protein Binding; D021122:Protein Subunits; D014162:Transfection
D015220:Calcium Channels; D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D050505:Mutant Proteins; D021122:Protein Subunits; D002118:Calcium